Wheat germ agglutinin is a biomarker of whole grain content in wheat flour and pasta.

When consumed as whole grain, wheat has a high nutrient density that contributes to a healthy diet. Yet, products labeled as whole wheat can still contain a substantial amount of refined grain leading to the confusion for consumers, so a method was designed to determine the whole grain status within wheat-based foods. Wheat germ agglutinin (WGA), a lectin found in the germ tissue of wheat kernels, was evaluated as a biomarker of whole grain wheat. WGA content strongly correlated with the percentage of whole wheat within premade mixtures of whole and refined (white) flours. Then, commercial flours labeled as whole wheat were tested for WGA content and found to contain up to 40% less WGA compared to a whole grain standard. Commercial pasta products labeled as whole wheat were also tested for WGA content and found to contain up to 90% less WGA compared to a whole grain standard. The differences in WGA content were not likely due to varietal differences alone, as the WGA content in common varieties used in domestic wheat flour production varied less than 25%. The levels of other constituents in wheat kernels, including starch, mineral, phytate, and total protein, were not different among the commercial whole wheat flours and pasta products. WGA is a unique biomarker that can identify wheat products with the highest whole grain content. PRACTICAL ABSTRACT: Whole grain wheat has a high nutrient density that can be part of a healthy diet, but products labeled as whole wheat can still contain some refined grain. Wheat germ agglutinin (WGA) was tested as a biomarker to measure whole grain status in wheat-based foods and revealed that some commercial whole wheat flour and pasta contained unexpectedly lower levels of the WGA biomarker compared to a whole grain standard. WGA may therefore be a useful way to test for whole grain wheat content.

Electrostatic Assemblies of Single-Walled Carbon Nanotubes and Sequence-Tunable Peptoid Polymers Detect a Lectin Protein and Its Target Sugars.

A primary limitation to real-time imaging of metabolites and proteins has been the selective detection of biomolecules that have no naturally occurring or stable molecular recognition counterparts. We present developments in the design of synthetic near-infrared fluorescent nanosensors based on the fluorescence modulation of single-walled carbon nanotubes (SWNTs) with select sequences of surface-adsorbed N-substituted glycine peptoid polymers. We assess the stability of the peptoid-SWNT nanosensor candidates under variable ionic strengths, protease exposure, and cell culture media conditions and find that the stability of peptoid-SWNTs depends on the composition and length of the peptoid polymer. From our library, we identify a peptoid-SWNT assembly that can detect lectin protein wheat germ agglutinin (WGA) with a sensitivity comparable to the concentration of serum proteins. To demonstrate the retention of nanosensor-bound protein activity, we show that WGA on the nanosensor produces an additional fluorescent signal modulation upon exposure to the lectin's target sugars, suggesting the lectin protein remains active and selectively binds its target sugars through ternary molecular recognition interactions relayed to the nanosensor. Our results inform design considerations for developing synthetic molecular recognition elements by assembling peptoid polymers on SWNTs and also demonstrate these assemblies can serve as optical nanosensors for lectin proteins and their target sugars. Together, these data suggest certain peptoid sequences can be assembled with SWNTs to serve as versatile optical probes to detect proteins and their molecular substrates.

Biotecnología y trigo (y otros cereales) sin gluten: ¿dónde estamos? / No disponible

No disponible

Surface-Enhanced Raman Scattering Based Microfluidics for Single-Cell Analysis.

The integration of surface-enhanced Raman scattering (SERS) with droplet microfluidics has the potential to improve our understanding of cellular systems. Herein, we present the first application of SERS droplet microfluidics for single-cell analysis. A microfluidic device was used to encapsulate single prostate cancer cells and wheat germ agglutin (WGA)-functionalized SERS nanoprobes in water-in-oil droplets that were subsequently locked into a storage droplet array for spectroscopic investigation. The stationary droplets enabled the rapid identification of SERS regions of interest in live cancer cells by allowing collection of "fast" coarse maps over an area of several square millimeters followed by "slower" detailed interrogation of the identified hotspots. We demonstrate SERS at cellular resolution via a proof-of-concept assay that detects glycan expression on the surface of prostate cancer cells using WGA-modified metallic nanoparticles. The data illustrates the potential of SERS optofluidic systems for high-throughput cell screening and illustrates a previously unobserved high degree of cell-to-cell variability in the size and number of glycan islands.

Glycosylated Peptoid Nanosheets as a Multivalent Scaffold for Protein Recognition.

Glycoproteins adhered on the cellular membrane play a pivotal role in a wide range of cellular functions. Their importance is particularly relevant in the recognition process between infectious pathogens (such as viruses, bacteria, toxins) and their host cells. Multivalent interactions at the pathogen-cell interfaces govern binding events and can result in a strong and specific interaction. Here we report an approach to mimic the cell surface presentation of carbohydrate ligands by the multivalent display of sugars on the surface of peptoid nanosheets. The constructs provide a highly organized 2D platform for recognition of carbohydrate-binding proteins. The sugars were displayed using different linker lengths or within loops containing 2-6 hydrophilic peptoid monomers. Both the linkers and the loops contained one alkyne-bearing monomer, to which different saccharides were attached by copper-catalyzed azide-alkyne cycloaddition reactions. Peptoid nanosheets functionalized with different saccharide groups were able to selectively bind multivalent lectins, Concanavalin A and Wheat Germ Agglutinin, as observed by fluorescence microscopy and a homogeneous Förster resonance energy transfer (FRET)-based binding assay. To evaluate the potential of this system as sensor for threat agents, the ability of functionalized peptoid nanosheets to bind Shiga toxin was also studied. Peptoid nanosheets were functionalized with globotriose, the natural ligand of Shiga toxin, and the effective binding of the nanomaterial was verified by the FRET-based binding assay. In all cases, evidence for multivalent binding was observed by systematic variation of the ligand display density on the nanosheet surface. These cell surface mimetic nanomaterials may find utility in the inactivation of pathogens or as selective molecular recognition elements.

La capacitación y la reacción acrosómica se asocian con cambios en la localización del ácido siálico y en la morfometría de la región cefálica del espermatozoide humano / Capacitation and acrosome reaction are associated with changes in sialic acid location and head morphometry in human sperm

Objetivo. Evaluar los cambios de distribución del ácido siálico durante el proceso de capacitación y reacción acrosómica in vitro y estudiar las modificaciones morfométricas en estas condiciones fisiológicas en la región cefálica del espermatozoide humano. Material y método. En este estudio prospectivo, evaluamos 6 muestras espermáticas normozoospérmicas. La distribución de ácido siálico se evaluó mediante la lectina Wheat germ agglutinin en diferentes condiciones fisiológicas: antes, después de la capacitación y tras la reacción acrosómica. La forma y el tamaño cefálico de cada estadio se estudiaron mediante métodos de morfometría geométrica. Resultados. Tras la capacitación, un 73,07±21,43% de espermatozoides presentaron ácido siálico en la región acrosomal en relación directa con una expansión del acrosoma y una contracción del segmento ecuatorial. Por otra parte, después de la reacción acrosómica se registra un mayor efecto alométrico entre los estadios debido a que los espermatozoides experimentaron una marcada expansión del segmento ecuatorial. En relación con la localización de Wheat germ agglutinin, encontramos una disminución significativa en el porcentaje de espermatozoides con fluorescencia en la región acrosomal, así como un incremento del marcaje en la banda ecuatorial. Conclusiones. Nuestros resultados demuestran que la distribución de la lectina Wheat germ agglutinin covaría con importantes cambios en la morfometría de la cabeza del espermatozoide y evidencia importantes implicaciones en los procesos de capacitación y reacción acrosómica (AU)

Objective. Assess changes in sialic acid distribution during capacitation and acrosome reaction processes, and evaluate head sperm morphometrics modifications in these physiological conditions in human sperm. Material and method. In this prospective study, we included 6 normozoospermics sperm samples. Sialic acid distribution was evaluated by Wheat germ agglutinin lectin in different physiological conditions: before, after capacitation and after acrosome reaction. Head shape and size of each stage were analyzed by means of geometric morphometric methods. Results. After capacitation, 73.07±21.43% of sperm showed sialic acid in acrosomal region, linked with an acrosome expansion and equatorial segment contraction. Otherwise, after acrosome reaction higher allometric effect between stages was recorded since sperm undergo further expansion of equatorial segment. Regarding Wheat germ agglutinin location, we found that sperm percentage significant decline in acrosomal fluorescence and an increase of equatorial band labeling. Conclusions. Our findings demonstrate that modifications in Wheat germ agglutinin expression covariate with dramatic changes in sperm head morphometry, suggesting important implications in capacitation and acrosome reaction processes (AU)

Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH-π Stacking Interactions by Different Fluorination Patterns.

We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar-protein interactions by simple 1 H and/or 19 F NMR spectroscopy methods. In this process, we have chosen the binding of N,N'-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2 CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2 FCONH- group and the natural molecule (with the CH3 CONH- fragment). In contrast, the molecule with the CF3 CONH- isoster displayed the weakest intermolecular interaction (one order of magnitude weaker). Because sugar-aromatic CH-π interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.

WGA-Alexa transsynaptic labeling in the phrenic motor system of adult rats: Intrapleural injection versus intradiaphragmatic injection.

BACKGROUND: Intrapleural injection of CTB-Alexa 488, a retrograde tracer, provides an alternative labeling technique to the surgically invasive laparotomy required for intradiaphragmatic injection. However, CTB-Alexa 488 is incapable of crossing synapses restricting the tracer to the phrenic nuclei and the intercostal motor nuclei in the spinal cord. NEW METHOD: Intrapleural injection of WGA-Alexa 488, a transsynaptic tracer, provides a method to label the respiratory motor pathway in both the spinal cord and medulla. Intradiaphragmatic injection of WGA-Alexa 594 and vagal nerve injections of True blue were used to confirm the phrenic nuclei and to differentiate between the rVRG and the NA in the medulla. RESULTS: Following intrapleural injection, WGA-Alexa 488 was retrogradely transported to the phrenic nuclei and to the intercostal motor nuclei. Subsequently WGA-Alexa 488 was transsynaptically transported from the phrenic motoneurons to the pre-motor neurons in the rVRG that provide the descending drive to the phrenic neurons during inspiration. In addition WGA-Alexa 488 was identified in select cells of the NA confirmed by a dual label of both WGA-Alexa 488 and True blue. COMPARISON WITH EXISTING METHOD: WGA-Alexa 488 demonstrates retrograde transsynaptic labeling following intrapleural injection whereas the previous method of injecting CTB-Alexa 488 only demonstrates retrograde labeling. CONCLUSIONS: Intrapleural injection of WGA-Alexa fluor conjugates is an effective method to transsynaptically label the phrenic motor system providing an alternative for the invasive laparotomy required for intradiaphragmatic injections. Furthermore, the study provides the first anatomical evidence of a direct synaptic relationship between rVRG and select NA cells.

Evaluation of WGA and Concanavalin A (Con A) lectin as biomarkers of hepatosplenic schistosomiasis in human biopsies with no evidence of egg-granuloma system.

INTRODUCTION: Colonic lesions are predominant in patients with schistosomiasis. However, carbohydrate alterations in colonic schistosomiasis remain unclear. Lectin-ligands allow us to identify changes in the saccharide patterns of cells. METHODS: Biopsies of descending and rectosigmoid colon of patients were submitted to WGA and Con A lectin histochemistry. RESULTS: WGA stained stroma and gland cells of descending colon and rectosigmoid tissues in a granular strong cytoplasmatic pattern in schistosomiasis specimens differing from normal control and Con A failing to recognize all samples analyzed. CONCLUSIONS: WGA ligands are expressed differently in patients with hepatosplenic schistosomiasis and no evidence of egg-granuloma system.

A simple method for comparing fungal biomass in infected plant tissues.

Plant phenotypes resistant and susceptible to fungal pathogens are usually scored using qualitative, subjective methods that are based upon disease symptoms or by an estimation of the amount of visible fungal growth. Given that plant resistance genes often confer partial resistance to fungal pathogens, a simple, sensitive, nonsubjective quantitative method for measuring pathogen growth would be highly advantageous. This report describes an in planta quantitative assay for fungal biomass based upon detection of chitin using wheat germ agglutinin conjugated to a fluorophore. Using this assay, the growth of wheat rust pathogens on wheat was assayed and the additivity of several adult plant and seedling resistance genes to Puccinia striiformis, P. graminis, and P. triticina was assayed on both glasshouse- and field-grown material. The assay can discriminate between individual rust pustules on a leaf segment or, alternatively, compare fungal growth on field plots. The quantification of Erysiphe necator (powdery mildew) growth on Vitis vinifera (grapevine) is also demonstrated, with resistant and susceptible cultivars readily distinguished. Given that chitin is a major cell wall component of many plant fungal pathogens, this robust assay will enable simple and accurate measurement of biomass accumulation in many plant-fungus interactions.

Untypical connectivity from olfactory sensory neurons expressing OR37 into higher brain centers visualized by genetic tracing.

The OR37 subfamily of odorant receptors(ORs) exists exclusively in mammals. In contrast to ORs in general, they are highly conserved within and across species.These unique features raise the question, whether olfactory information gathered by the OR37 sensory cells is processed in specially designated brain areas. To elucidate the wiring of projection neurons from OR37 glomeruli into higher brain areas, tracing experiments were performed.The application of DiI onto the ventral area of the olfactory bulb, which harbors the OR37 glomeruli, led to the labeling of fibers not only in the typical olfactory cortical regions,but also in the medial amygdala and the hypothalamus. To visualize the projections from a defined OR37 glomerulus more precisely, transgenic mice were studied in which olfactory sensory neurons co-express the receptor subtype OR37C and the transsynaptic tracer wheat germ agglutinin(WGA). WGA became visible not only in the OR37C sensory neurons and the corresponding OR37C glomerulus,but also in cell somata located in the mitral/tufted cell layer adjacent to the OR37C glomerulus, indicating a transfer of WGA onto projection neurons. In the brain, WGA immunoreactivity was not detectable in typical olfactory cortical areas, but instead in distinct areas of the medial amygdala.Detailed mapping revealed that the WGA immunoreactivity was restricted to the posterior-dorsal subnucleus of the medial amygdala. In addition, WGA immunoreactivity was visible in some well-circumscribed areas of the hypothalamus.These results are indicative for a unique connectivity from OR37C sensory cells into higher brain centers.

Nose-to-brain transport pathways of wheat germ agglutinin conjugated PEG-PLA nanoparticles.

PURPOSE: To investigate the possible pathways for transport of wheat germ agglutinin conjugated PEG-PLA nanoparticles (WGA-NP) into the brain after nasal administration. METHODS: The nose-to-brain pathways were investigated using WGA-NP containing 6-coumarin (as a fluorescent marker) and (125)I-labeled WGA-NP. Ex vivo imaging analysis was also employed to visualize the transport process. RESULTS: Nasal administration of WGA-NP to rats resulted in transcellular absorption across the olfactory epithelium and transfer to the olfactory bulb within 5 min. After entering the lamina propria, a proportion of WGA-NP were transferred from the olfactory nerve bundles and their surrounding connective tissue to the olfactory bulb. The trigeminal nerves also contributed to WGA-NP brain transfer, especially to WGA-NP distribution in the caudal brain areas. However, cerebrospinal fluid pathway may have little contribution to the process of transferring WGA-NP into the central nervous system (CNS) after intranasal administration. CONCLUSIONS: These results demonstrated that intranasally administered WGA-NP reach the CNS via olfactory pathway and trigeminal nerve pathway, and extracellular transport along these nerves is the most possible mechanism.

Nanoparticle-based biocompatible and targeted drug delivery: characterization and in vitro studies.

Paclitaxel nanoparticles (PAX NPs) prepared with the size of 110 ± 10 nm and ζ potential of -40 ± 3 mV were encapsulated in synthetic/biomacromolecule shell chitosan, dextran-sulfate using a layer-by-layer self-assembly technique. Zeta potential measurements, analysis of X-ray photoelectron spectroscopy, and scanning electron microscopy confirmed the successful adsorption of each layer. Surface modifications of these core-shell NPs were performed by covalently conjugating with poly(ethylene glycol) (H(2)N-PEG-carboxymethyl, M(w) 3400) and fluorescence labeled wheat germ agglutinin (F-WGA) to build a biocompatible and targeted drug delivery system. 32% of PAX was released from four bilayers of biomacromolecule assembled NPs within 8 h as compared with >85% of the drug released from the bare NPs. Moreover, high cell viability with PEG conjugation and high binding capacity of WGA-modified NPs with Caco-2 cells were observed. This biocompatible and targeted NP-based drug delivery system, therefore, may be considered as a potential candidate for the treatment of colonic cancer and other diseases.

Visualizing the endocytic and exocytic processes of wheat germ agglutinin by quantum dot-based single-particle tracking.

Wheat germ agglutinin (WGA) is a paradigm for understanding intracellular transport of lectins. As a protein exploiting the receptor-mediated endocytosis for internalization, WGA is also a valuable model system for exploring the endocytic and exocytic pathway. In this study, quantum dot-based single-particle tracking was performed to investigate the transport of WGA in live cells, revealing firstly that the endocytic and exocytic processes of WGA were both actin- and microtubule-dependent, each including five stages. The vesicle fusion event occurred near the cytomembrane, followed by two destinies with WGA: shedding to the extracellular or reversing to the cytoplasm. These findings suggest a distinct and dynamic scenario for the transport of lectins following a receptor-mediated endo/exocytic pathway in live cells. This is important for the application of lectins as drug carriers and antineoplastic drugs in medicine, and also offers insights into the pathway of endocytosis and exocytosis.

Application of WGA lectin staining for visualization of the connective tissue in skeletal muscle, bone, and ligament/tendon studies.

During immunostaining of specific proteins in tissue sections using monoclonal and polyclonal antibodies, visualization of general tissue staining/background or major structural features is helpful to pinpoint precise localization of the protein of interest. Often in skeletal muscle research, immunostaining with antibodies against connective tissue or plasma membrane proteins (collagen 1, laminin, and caveolin 3) are used for this purpose. Although immunostaining for these proteins works well, it is time consuming, costly, limits the number of antibodies against protein of interest that can be used on a single section, and is not applicable to some staining techniques. Lectins were frequently used in earlier publications for skeletal muscle fiber boundaries and connective tissue visualization, but are not common in the current research studies. This work investigates costaining of muscle, bone, ligament, and tendon tissue sections with fluorescently tagged wheat germ agglutinin (WGA) lectin as a tool for the visualization of connective tissue. The results of this study show that fluorescent WGA lectin costaining is a cost-effective, fast, and convenient method for connective tissue visualization, especially in the studies where extensive washes reduce staining of the structures that are the primary interest of the investigation.

Doença do armazenamento lisossomal induzida pelo consumo de Sida carpinifolia em bovinos do Rio Grande do Sul / Lysosomal storage disease caused by Sida carpinifolia in cattle in Rio Grande do Sul

This paper reports the natural poisoning by Sida carpinifolia (guanxuma, chá-da-índia) in cattle in Rio Grande do Sul, Brazil. Five cattle were affected in the period 2001-2008. Clinical signs included weight loss, incoordination, walking difficulty, generalized tremors, frequent falls, and death. Microscopically, the main changes were vacuolation of Purkinje neurons in the cerebellum, pancreatic acinar cells, and thyroid follicular cells. Transmission electron microscopy revealed vacuoles bordered by membrane containing finely granular material. Lectin histochemistry showed positive staining in neurons with the lectins Concanavalia ensiformis (Con-A), Triticum vulgaris (WGA), and Succinyl Triticum vulgaris (sWGA).

Relata-se a intoxicação natural por Sida carpinifolia (guanxuma, chá-da-índia) em bovinos no Rio Grande do Sul. Foram afetados cinco bovinos no período 2001-2008. O quadro clínico foi caracterizado por emagrecimento, incoordenação, dificuldade de locomoção, tremores generalizados, quedas frequentes e morte. Microscopicamente, as principais alterações foram vacuolização dos neurônios de Purkinje do cerebelo, das células acinares do pâncreas e das células foliculares da tireoide. A microscopia eletrônica evidenciou vacúolos com conteúdo finamente granulado e delimitado por membrana. Na lectina-histoquímica, observou-se marcação em neurônios com as lectinas Concanavalia ensiformis (Con-A), Triticum vulgaris (WGA) e Succinyl Triticum vulgaris (sWGA).

Comparison of methods of detecting bacterial biofilm.

Fresh bacterial biofilm produced by Staphylococcus epidermidis was sensitively detected by using a chemiluminescent probe. Its lower detection limit was around 10(-5) - 10(-4) dilution (equivalent to 10(-5) - 10(-4) cm2 biofilm) - 10(2) - 10(3) times as sensitive as ordinary colorimetric methods. The linearity and dynamic range were slightly better than those of fluorometry.

Electrochemical monitoring of binding between wheat germ agglutinin and cellohexose-modified magnetic microbeads.

An electrochemical method that uses glucose labeled with an electroactive compound was developed to evaluate the binding of wheat germ agglutinin (WGA) to cellohexose-modified magnetic microbeads. Cellohexose was attached to amino groups on the magnetic bead surface via formation of a Schiff's base. The labeled glucose acts as an electrochemical probe to monitor binding events between WGA and the cellohexose-modified beads. For a known quantity of cellohexose-modified beads, binding of WGA with cellohexose-modified beads was evaluated based on changes in electrochemical response of the labeled glucose. In particular, the peak current decreased as the concentration of WGA increased. Furthermore, the binding affinities of WGA for beads modified with four different cello-oligosaccharides were systematically compared using a voltammetric method.

Direct observation of transitions between surface-dominated and bulk diffusion regimes in nanochannels.

The diffusion of charged proteins in liquid-filled nanometer-sized apertures with charged surfaces has been investigated with fluorescence correlation spectroscopy (FCS). Based on a two-dimensional (2D) multicomponent diffusion model, key parameters such as the number of molecules diffusing freely inside the nanochannel or interacting with the surfaces, together with the specific diffusion parameters, could be extracted. Different regimes of diffusion have been observed and described by a model, which takes into account the steric exclusion, the reversible surface adsorption of the biomolecules, and the exclusion-enrichment effect that is due to the charge of the proteins and the ionic strength of the solution. Conditions where the diffusion of proteins through nanoconfined spaces can be of the same magnitude as in the bulk were both predicted and experimentally verified.

Voltammetric behaviors of wheat-germ agglutinin on a chitin-modified carbon-paste electrode.

The voltammetric behavior of wheat-germ agglutinin (WGA) on a chitin-modified carbon-paste electrode (CPE) was investigated using glucose labeled with an electroactive compound. WGA usually consists of two subunits, each with two binding sites for sugars. WGA was immobilized on the electrode surface by selective binding to a N-acetylglucosamine residue of chitin. Because glucose also combines with WGA, the glucose was coupled with electroactive daunomycin to evaluate the binding. When a WGA-labeled glucose complex was formed, the electroactive moiety became electroinactive. The binding caused a decrease in the peak current of the labeled glucose. In a measurement of only daunomycin used as a label, the peak current in a solution with WGA was similar to that in a solution without WGA. Therefore, it is clear that the labeled glucose was held in the remaining binding site of WGA on the electrode surface. Thus, a CPE modified with chitin would be powerful as a reaction field between sugar and lectin.