Investigating the Roles of Heparan Sulfate Structures in Alpha-Synuclein Aggregation in Cell Culture Models.

Glycosaminoglycans (GAGs), belonging to a family of negatively charged linear polysaccharides, have been found in the cores of amyloid inclusions such as Lewy bodies, which are the central pathological features in Parkinson's disease (PD), a neurodegenerative disease. Lewy bodies/neurites are mostly composed of α-synuclein protein (α-syn) aggregates. Recent studies have shown that α-syn aggregates can propagate via neurons in a prion-like fashion by seeding the endogenous cellular α-syn.

Applications of Xylosides in the Manipulation of Stem Cell Niche to Regulate Human Neural Stem Cell Differentiation and Neurite Outgrowth.

The extracellular matrix (ECM) plays a pivotal role in the regulation of neural stem cell differentiation, axon guidance and growth, and neural plasticity. Glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, are significant components of brain ECM that dictates neurogenesis and neural repair. Herein, we describe a simple method to assess the effect of xylsoides, which serve as primers and inhibitors of GAG biosynthesis, on human neural stem cell differentiation and neurite outgrowth in in vitro culture conditions.

Xyloside Derivatives as Molecular Tools to Selectively Inhibit Heparan Sulfate and Chondroitin Sulfate Proteoglycan Biosynthesis.

Glycosaminoglycan (GAG) side chains of proteoglycans are involved in a wide variety of developmental and pathophysiological functions. Similar to a gene knockout, the ability to inhibit GAG biosynthesis would allow us to examine the function of endogenous GAG chains. However, ubiquitously and irreversibly knocking out all GAG biosynthesis would cause multiple effects, making it difficult to attribute a specific biological role to a specific GAG structure in spatiotemporal manner.

Preparation of Nanosensors for Detecting the Activity of Glycosaminoglycan Cleaving Enzymes.

Glycosaminoglycans (GAGs) play crucial roles in several biological processes including cell division, angiogenesis, anticoagulation, neurogenesis, axon guidance and growth, and viral and bacterial infections among others. The GAG cleaving hydrolases/lyases play a major role in the control of GAG structures, functions, and turn over. Dysregulation of GAG cleaving enzymes in vivo are linked to a number of human diseases including cancer, diabetes, atherosclerosis, arthritis, inflammation, and cardiovascular diseases.

Manipulation of Glycosaminoglycans Using Synthetic Xylosides to Study Their Roles in Lung Branching Morphogenesis in Ex Vivo Lung Bud Culture System.

The primary left and right bronchial buds grow and sprout secondary bronchi, which in turn develop tertiary bronchi, and so on. Branching continues for a total of 6-8 generations in the mouse and for about 23 generations in humans, forming the estimated 50 million branches of the human lung. Thus, patterns of branching are incalculably complex.

Methods for Assessing the Effects of Xylosides on Angiogenesis.

Xylosides are small synthetic molecules consisting of a xylose molecule attached to an aglycone group and serve as primers in the assembly of core protein free glycosaminoglycans using cellular machinery. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. Recent advances in the study of xylosides have opened up the possibility of developing xylosides as therapeutics to achieve a desirable biological outcome through their selective priming and inhibitory activities toward glycosaminoglycan biosynthesis.

Procedures to Evaluate the Role of Heparan Sulfate on the Reactivity of Resistance and Conductance Arteries Ex Vivo.

Evidence is emerging that disruption of the endothelial glycocalyx might contribute importantly to arterial dysfunction in the context of diabetes. One approach to assess the integrity of the endothelium and the vascular smooth muscle cell layer, in the absence of neural, humoral, and mechanical influences, is by measuring arterial vasomotion ex vivo. Here we describe a procedure to assess non-receptor-mediated vasoconstriction, receptor-mediated vasoconstriction, and endothelium-dependent and -independent vasodilation, in resistance and conductance arteries pressurized to 60 mmHg.

Enzymatic Alteration of ECM to Explore Muscle Function and Motor Control of a Learned Behavior.

Nerves and muscle interact to perform learned motor behavior such as birdsong. Glycosaminoglycans play a major role in the function of muscle as well as the formation and function of the neuromuscular junction. The alteration of GAG chains provides a unique opportunity to alter muscle behavior and thus motor control of a behavior.

Methods to Analyze the Effect of Diet-Derived Metabolites on Endothelial Inflammation and Cell Surface Glycosaminoglycans.

The glycocalyx is a biologically active barrier that covers the luminal side of the vascular endothelium and it is comprised of proteoglycans [core proteins with glycosaminoglycans (GAG) side chains], glycoproteins, and plasma proteins. Evidence shows that the disruption in the structure and function of the endothelial glycocalyx exacerbates vascular inflammation and atherosclerosis. The GAG components of the glycocalyx undergo remodeling in the setting of diabetes and these alterations in endothelial GAGs negatively impact the vascular function.

anexVis: A Transcriptome Tool to Visualize Organ/Tissue-Specific Glycosaminoglycan Biosynthetic and Catabolic Pathways in Human Health and Diseases.

Our lab has developed a new visualization tool, anexVis, for transcriptome analysis of glycosaminoglycan-related genes. This tool allows one to analyze a large number of genes that are related to biosynthetic and catabolic pathways of all glycosaminoglycans, such as heparan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid, in parallel across various human tissues/organs. Such visual analyses have not been accessible to the broad research community despite the accumulation of a large amount of RNA-seq data.

Methods for the Production of Recombinant Heparosan, a Critical Heparin Precursor, from Nonpathogenic E. coli Strains.

Heparin is an essential anticoagulant drug discovered over a century ago. Heparin is the second most highly used natural drug and remains a mainstay of therapy with an expected global market share of more than $14 billion in the next 10 years. However, it is still naturally derived from unsustainable animal sources, such as bovine lungs and porcine intestines, as an unfractionated, heterogeneous complex mixture with unpredictable pharmacokinetic properties.

Visualizing antithrombin-binding 3--sulfated heparan sulfate motifs on cell surfaces.

To map the cellular topography of the rare 3-O-sulfated structural motif of heparan sulfate (HS), we constructed quantum dot-based probes for antithrombin and FGF2, which reveal widely different distribution of the targeted HS motifs. The technology helps show that old and young aortic endothelia display widely different levels of the antithrombin-binding 3-O-sulfated HS motif.

Metabolic engineering of non-pathogenic Escherichia coli strains for the controlled production of low molecular weight heparosan and size-specific heparosan oligosaccharides.

Background: Heparin, a lifesaving blood thinner used in over 100 million surgical procedures worldwide annually, is currently isolated from over 700 million pigs and ~200 million cattle in slaughterhouses worldwide. Though animal-derived heparin has been in use over eight decades, it is a complex mixture that poses a risk for chemical adulteration, and its availability is highly vulnerable. Therefore, there is an urgent need in devising bioengineering approaches for the production of heparin polymers, especially low molecular weight heparin (LMWH), and thus, relying less on animal sources.

Preparation and Application of Nanosensor in Safeguarding Heparin Supply Chain.

Heparin has been in clinical use as an anticoagulant for the last eight decades and used worldwide in more than 100 million medical procedures every year. This lifesaving drug is predominantly obtained from ~700 million pig intestines or bovine organs through millions of small and medium-sized slaughterhouses. However, the preparations from animal sources have raised many safety concerns, including the contamination of heparin with potential pathogens, proteins, and other impurities.

Combinatorial virtual library screening analysis of antithrombin binding oligosaccharide motif generation by heparan sulfate 3--Sulfotransferase 1.

Pharmaceutical heparin's activity arises from a key high affinity and high selectivity antithrombin binding motif, which forms the basis for its use as an anticoagulant. The current problems with the supply of pig heparin raises the emphasis of understanding heparin biosynthesis so as to control and advance recombinantly expressed agent that could bypass the need for animals. Unfortunately, much remains to be understood about the generation of the antithrombin-binding motif by the key enzyme involved in its biosynthesis, 3--sulfotransferase-1 (3OST-1).

A graph-based algorithm for RNA-seq data normalization.

The use of RNA-sequencing has garnered much attention in recent years for characterizing and understanding various biological systems. However, it remains a major challenge to gain insights from a large number of RNA-seq experiments collectively, due to the normalization problem. Normalization has been challenging due to an inherent circularity, requiring that RNA-seq data be normalized before any pattern of differential (or non-differential) expression can be ascertained; meanwhile, the prior knowledge of non-differential transcripts is crucial to the normalization process.

Detection of Cell Surface Ligands for Human Synovial γδ T Cells.

Lack of understanding of the nature and physiological regulation of γδ T cell ligands has considerably hampered full understanding of the function of these cells. We developed an unbiased approach to identify human γδ T cells ligands by the production of a soluble TCR-γδ (sTCR-γδ) tetramer from a synovial Vδ1 γδ T cell clone from a Lyme arthritis patient. The sTCR-γδ was used in flow cytometry to initially define the spectrum of ligand expression by both human tumor cell lines and certain human primary cells.

Blueberry metabolites restore cell surface glycosaminoglycans and attenuate endothelial inflammation in diabetic human aortic endothelial cells.

Background: Glycosaminoglycan (GAG), a major component of the endothelial glycocalyx, is severely perturbed in diabetic vasculature leading to endothelial inflammation and vascular disease in diabetes. We tested the hypothesis that blueberry metabolites (BBM) ameliorate endothelial inflammation in diabetic endothelial cells (ECs) by restoring cell surface GAGs.

Methods: ECs isolated from healthy individuals [human aortic ECs (HAECs)] and diabetic patients (diabetic HAECs) were treated with ±BBM (benzoic acid-4-sulfate, hippuric acid, hydroxyhippuric acid, isovanillic acid-3-sulfate, and vanillic acid-4-sulfate at concentrations known to circulate in human plasma following blueberry consumption) for 3 days, and indices for endothelial inflammation were measured.

anexVis: visual analytics framework for analysis of RNA expression.

Summary: Although RNA expression data are accumulating at a remarkable speed, gaining insights from them still requires laborious analyses, which hinder many biological and biomedical researchers. This report introduces a visual analytics framework that applies several well-known visualization techniques to leverage understanding of an RNA expression dataset. Our analyses on glycosaminoglycan-related genes have demonstrated the broad application of this tool, anexVis (analysis of RNA expression), to advance the understanding of tissue-specific glycosaminoglycan regulation and functions, and potentially other biological pathways.

Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic Machinery for Biomedical Applications.

Glycosaminoglycans (GAGs) are polysaccharides ubiquitously found on cell surfaces and in the extracellular matrix (ECM). They regulate numerous cellular signaling events involved in many developmental and pathophysiological processes. GAGs are composed of complex sequences of repeating disaccharide units, each of which can carry many different modifications.

Regulation of glycosaminoglycan biogenesis is critical for sensitive-period-dependent vocal ontogeny.

In the brain, the extracellular matrix (ECM) plays a central role during neural development and thus modulates critical-period regulated behavioral ontogeny. The major components of the ECM are glycosaminoglycans (GAGs) including chondroitin sulfate (CS). However, the specific roles of GAGs in behavioral development are largely unknown.

Ruthenium(II)- and copper(I)-catalyzed synthesis of click-xylosides and assessment of their glycosaminoglycan priming activity.

Xylosides are small molecules that serve as primers of glycosaminoglycan biosynthesis. Xyloside mediated modulation of biological functions depends on the extent of priming activity and fine structures of primed GAG chains. In earlier studies, copper (Cu) catalyzed synthesis of click-xylosides and their priming activity were extensively documented.

Immobilization alters heparin cleaving properties of heparinase I.

We report here a novel observation that immobilization of heparinase I on CNBr-activated Sepharose results in heparin degradation properties that are different from heparinase I in the free solution form. Studies over a range of pHs (5-8) and temperatures (5-50°C) as well as under batch and flow conditions show that immobilized heparinase 1 displays altered pH and temperature optima, and a higher propensity for generation of longer chains (hexa- and octa-) with variable sulfation as compared to that in the free form, which is known to yield disaccharides. The immobilized enzyme retained good eliminase activity over at least five cycles of reuse.

A glycan-based approach to therapeutic angiogenesis.

Angiogenesis, the sprouting of new blood vessels from existing vasculature, involves multiple complex biological processes, and it is an essential step for hemostasis, tissue healing and regeneration. Angiogenesis stimulants can ameliorate human disease conditions including limb ischemia, chronic wounds, heart disease, and stroke. The current strategies to improve the bioavailability of pro-angiogenic growth factors, including VEGF and FGF2, have remained largely unsuccessful.