Modular glycosphere assays for high-throughput functional characterization of influenza viruses

Background: The ongoing global efforts to control influenza epidemics and pandemics require high-throughput technologies to detect, quantify, and functionally characterize viral isolates. The 2009 influenza pandemic as well as the recent in-vitro selection of highly transmissible H5N1 variants have only increased existing concerns about emerging influenza strains with significantly enhanced human-to-human transmissibility. High-affinity binding of the virus hemagglutinin to human receptor glycans is a highly sensitive and stringent indicator of host adaptation and virus transmissibility. The surveillance of receptor-binding characteristics can therefore provide a strong additional indicator for the relative hazard imposed by circulating and newly emerging influenza strains. Results: Streptavidin-coated microspheres were coated with selected biotinylated glycans to mimic either human or avian influenza host-cell receptors. Such glycospheres were used to selectively capture influenza virus of diverse subtypes from a variety of samples. Bound virus was then detected by fluorescently labelled antibodies and analyzed by quantitative flow cytometry. Recombinant hemagglutinin, inactivated virus, and influenza virions were captured and analyzed with regards to receptor specificity over a wide range of analyte concentration. High-throughput analyses of influenza virus produced dose-response curves that allow for functional assessment of relative receptor affinity and thus transmissibility. Conclusions: Modular glycosphere assays for high-throughput functional characterization of influenza viruses introduce an important tool to augment the surveillance of clinical and veterinarian influenza isolates with regards to receptor specificity, host adaptation, and virus transmissibility.Singapore-MIT Alliance for Research and Technolog

Disaccharide Analysis of Glycosaminoglycans Using Hydrophilic Interaction Chromatography and Mass Spectrometry

Heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) participate in many important biological processes. Quantitative disaccharide analysis of HS and CS/DS is essential for the characterization of GAGs and enables modeling of the GAG domain structure. Methods involving enzymatic digestion and chemical depolymerization have been developed to determine the type and location of sulfation/acetylation modifications as well as uronic acid epimerization. Enzymatic digestion generates disaccharides with Δ-4,5-unsaturation at the nonreducing end. Chemical depolymerization with nitrous acid retains the uronic acid epimerization. This work shows the use of hydrophilic interaction liquid chromatography mass spectrometry (HILIC–MS) for quantification of both enzyme-derived and nitrous acid depolymerization products for structural analysis of HS and CS/DS. This method enables biomedical researchers to determine complete disaccharide profiles on GAG samples using a single LC–MS platform

Biochemical and gene expression analysis of MSCs undergoing chondrogenesis (absence of ascorbic acid) in the presence of GalNAc-a or dexamethasone in 3D hydrogels.

<p>Quantification of biochemical composition of (A) DNA normalized to construct dry weight and (B) sGAG normalized to DNA content (n = 4, **P<0.01, *** P<0.001 versus no analog exposure). (C) Histological staining for proteoglycans using Safranin-O (scale bar: 50 µm). Gene expression analysis of markers for (D-G) chondrogenesis and matrix production with (H) MMP13 were evaluated (presented as described in Figure Legend 6).</p

Differential Response of Chondrocytes and Chondrogenic-Induced Mesenchymal Stem Cells to C1-OH Tributanoylated <em>N</em>-Acetylhexosamines

<div><p>Articular cartilage has a limited ability to self-repair because of its avascular nature and the low mitotic activity of the residing chondrocytes. There remains a significant need to develop therapeutic strategies to increase the regenerative capacity of cells that could repair cartilage. Multiple cell types, including chondrocytes and mesenchymal stem cells, have roles in articular cartilage regeneration. In this study, we evaluated a platform technology of multiple functionalized hexosamines, namely 3,4,6-<i>O</i>-tributanoylated-<i>N</i>-acetylgalactosamine (3,4,6-<i>O</i>-Bu₃GalNAc), 3,4,6-<i>O</i>-tributanoylated-<i>N</i>-acetylmannosamine (3,4,6-<i>O</i>-Bu₃ManNAc) and 3,4,6-<i>O</i>-Bu₃GlcNAc, with the potential ability to reduce NFκB activity. Exposure of IL-1β-stimulated chondrocytes to the hexosamine analogs resulted in increased expression of ECM molecules and a corresponding improvement in cartilage-specific ECM accumulation. The greatest ECM accumulation was observed with 3,4,6-<i>O</i>-Bu₃GalNAc. In contrast, mesenchymal stem cells (MSCs) exposed to 3,4,6-<i>O</i>-Bu₃GalNAc exhibited a dose dependent decrease in chondrogenic differentation as indicated by decreased ECM accumulation. These studies established the disease modification potential of a hexosamine analog platform on IL-1β-stimulated chondrocytes. We determined that the modified hexosamine with the greatest potential for disease modification is 3,4,6-<i>O</i>-Bu₃GalNAc. This effect was distinctly different with 3,4,6-<i>O</i>-Bu₃GalNAc exposure to chondrogenic-induced MSCs, where a decrease in ECM accumulation and differentiation was observed. Furthermore, these studies suggest that NFκB pathway plays a complex role cartilage repair.</p> </div

Gene expression analysis of MSCs undergoing chondrogenesis in the presence of ascorbic acid and GalNAc-a or dexamethasone in 3D hydrogels.

<p>Markers for (A-D) chondrogenesis and matrix production and (E) MMP13 were evaluated. All data were normalized to individual β-actin levels and presented relative to untreated controls (no analog, no dexamethasone).</p

Biochemical analysis of MSCs undergoing chondrogenesis in the presence of ascorbic acid and GalNAc-a or dexamethasone in 3D hydrogels.

<p>Quantification of biochemical composition of (A) DNA normalized to construct dry weight, (B) sGAG normalized to DNA content and (C) total collagen normalized to DNA content (n = 4, * P<0.05, ** P<0.01, * P<0.001 versus no analog exposure). (D) Histological staining for proteoglycans using Safranin-O and (E,F) immunohistochemical staining for (E) type II collagen and (F) type I collagen (scale bar: 50 µm).</p

Biochemical analysis of MSCs undergoing chondrogenesis under IL-1β stimulation and GalNAc-a exposure in 3D hydrogels.

<p>(A) DNA normalized to construct dry weight and (B) sGAG normalized to DNA content (n = 3, **P<0.01 versus no analog exposure). (C) Histological staining for proteoglycans using Safranin-O (scale bar: 50 µm).</p