Identification of heparin-binding amino acid residues in antibody HS4C3 with the potential to design antibodies against heparan sulfate domains.

Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.

Evidence for Multiple Binding Modes in the Initial Contact Between SARS-CoV-2 Spike S1 Protein and Cell Surface Glycans.

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the 'open' conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required.

Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity.

The opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments. ExoU is a phospholipase which causes destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitrothan other antimicrobial classes. Their overuse over the past decade, however, has resulted in the emergence of antibiotic resistance. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered that tobramycin disrupted T3SS expression and reduced both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-t cells at concentrations below the minimal inhibitory concentration (MIC). The fluoroquinolones moxifloxacin and ciprofloxacin, however, up-regulated the T3SS and did not inhibit and may have increased the cytotoxic effects of ExoS and ExoU.

Anion binding to a cationic europium(III) probe enables the first real-time assay of heparan sulfotransferase activity.

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3'-phosphate-5'-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3',5'-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.

Glycosaminoglycans from Litopenaeus vannamei Inhibit the Alzheimer's Disease ß Secretase, BACE1.

Only palliative therapeutic options exist for the treatment of Alzheimer's Disease; no new successful drug candidates have been developed in over 15 years. The widely used clinical anticoagulant heparin has been reported to exert beneficial effects through multiple pathophysiological pathways involved in the aetiology of Alzheimer's Disease, for example, amyloid peptide production and clearance, tau phosphorylation, inflammation and oxidative stress. Despite the therapeutic potential of heparin as a multi-target drug for Alzheimer's disease, the repurposing of pharmaceutical heparin is proscribed owing to the potent anticoagulant activity of this drug. Here, a heterogenous non-anticoagulant glycosaminoglycan extract, obtained from the shrimp Litopenaeus vannamei, was found to inhibit the key neuronal ß-secretase, BACE1, displaying a more favorable therapeutic ratio compared to pharmaceutical heparin when anticoagulant activity is considered.

New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors.

Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein-protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.

New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

A Glycosaminoglycan Extract from Portunus pelagicus Inhibits BACE1, the ß Secretase Implicated in Alzheimer's Disease.

Therapeutic options for Alzheimer's disease, the most common form of dementia, are currently restricted to palliative treatments. The glycosaminoglycan heparin, widely used as a clinical anticoagulant, has previously been shown to inhibit the Alzheimer's disease-relevant ß-secretase 1 (BACE1). Despite this, the deployment of pharmaceutical heparin for the treatment of Alzheimer's disease is largely precluded by its potent anticoagulant activity. Furthermore, ongoing concerns regarding the use of mammalian-sourced heparins, primarily due to prion diseases and religious beliefs hinder the deployment of alternative heparin-based therapeutics. A marine-derived, heparan sulphate-containing glycosaminoglycan extract, isolated from the crab Portunus pelagicus, was identified to inhibit human BACE1 with comparable bioactivity to that of mammalian heparin (IC50 = 1.85 µg mL-1 (R2 = 0.94) and 2.43 µg mL-1 (R2 = 0.93), respectively), while possessing highly attenuated anticoagulant activities. The results from several structural techniques suggest that the interactions between BACE1 and the extract from P. pelagicus are complex and distinct from those of heparin.

Expression and purification of an FGF9 fusion protein in E. coli, and the effects of the FGF9 subfamily on human hepatocellular carcinoma cell proliferation and migration.

Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.

High production in E. coli of biologically active recombinant human fibroblast growth factor 20 and its neuroprotective effects.

Fibroblast growth factor 20 (FGF20) has a wide range of biological activities; its expression is most pronounced in neural tissues where it has functions in development and neuroprotection. Given these activities, interest in the clinical applications of FGF20 is rising, which will lead to increasing demand for active recombinant human FGF20 (rhFGF20). To improve the production of rhFGF20, an artificial gene encoding fgf20 was cloned into pET3a and expressed in E. coli BL21(DE3)pLysS. By optimizing induction conditions, we successfully induced large amounts of insoluble rhFGF20. Following solubilization and refolding of the rhFGF20 from inclusion bodies, it was purified by HiTrap heparin affinity chromatography to a purity of over 96% with a yield of 218 mg rhFGF20/100 g wet cells. The purified rhFGF20 could stimulate proliferation of both NIH 3T3 cells and PC-12 cells, measured by the MTT assay. In a model of Aß25-35-induced apoptosis on PC-12 cells, rhFGF20 had a clear protective effect. RT-PCR and Western blot analysis of apoptosis-related genes and proteins revealed that the FGF20-derived protective mechanism was likely due to the relief of endoplasmic reticulum stress (ER stress). In conclusion, the approach described here may be a better means to produce active rhFGF20 in good quantity, thereby allowing for its future pharmacological and clinical use.

Structural determinants of heparin-transforming growth factor-ß1 interactions and their effects on signaling.

Transforming growth factor-ß1 (TGF-ß1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-ß1's activity on hMSCs. Using a proteomics "Protect and Label" approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin (Data are available via ProteomeXchange with identifier PXD002772). Competition assays in an optical biosensor demonstrated that TGF-ß1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-ß1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14-24 saccharides (dp14-24) in length all increased the phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) after 6 h of stimulation with TGF-ß1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-ß1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-ß1 in hMSCs.

Transport of fibroblast growth factor 2 in the pericellular matrix is controlled by the spatial distribution of its binding sites in heparan sulfate.

The heparan sulfate (HS) chains of proteoglycans are a key regulatory component of the extracellular matrices of animal cells, including the pericellular matrix around the plasma membrane. In these matrices they regulate transport, gradient formation, and effector functions of over 400 proteins central to cell communication. HS from different matrices differs in its selectivity for its protein partners. However, there has been no direct test of how HS in the matrix regulates the transport of its partner proteins. We address this issue by single molecule imaging and tracking in fibroblast pericellular matrix of fibroblast growth factor 2 (FGF2), stoichiometrically labelled with small gold nanoparticles. Transmission electron microscopy and photothermal heterodyne imaging (PHI) show that the spatial distribution of the HS-binding sites for FGF2 in the pericellular matrix is heterogeneous over length scales ranging from 22 nm to several µm. Tracking of individual FGF2 by PHI in the pericellular matrix of living cells demonstrates that they undergo five distinct types of motion. They spend much of their time in confined motion (∼110 nm diameter), but they are not trapped and can escape by simple diffusion, which may be slow, fast, or directed. These substantial translocations (µm) cover distances far greater than the length of a single HS chain. Similar molecular motion persists in fixed cells, where the movement of membrane PGs is impeded. We conclude that FGF2 moves within the pericellular matrix by translocating from one HS-binding site to another. The binding sites on HS chains form non-random, heterogeneous networks. These promote FGF2 confinement or substantial translocation depending on their spatial organisation. We propose that this spatial organisation, coupled to the relative selectivity and the availability of HS-binding sites, determines the transport of FGF2 in matrices. Similar mechanisms are likely to underpin the movement of many other HS-binding effectors.

Polysaccharide sulfotransferases: the identification of putative sequences and respective functional characterisation.

The vast structural diversity of sulfated polysaccharides demands an equally diverse array of enzymes known as polysaccharide sulfotransferases (PSTs). PSTs are present across all kingdoms of life, including algae, fungi and archaea, and their sulfation pathways are relatively unexplored. Sulfated polysaccharides possess anti-inflammatory, anticoagulant and anti-cancer properties and have great therapeutic potential. Current identification of PSTs using Pfam has been predominantly focused on the identification of glycosaminoglycan (GAG) sulfotransferases because of their pivotal roles in cell communication, extracellular matrix formation and coagulation. As a result, our knowledge of non-GAG PSTs structure and function remains limited. The major sulfotransferase families, Sulfotransfer_1 and Sulfotransfer_2, display broad homology and should enable the capture of a wide assortment of sulfotransferases but are limited in non-GAG PST sequence annotation. In addition, sequence annotation is further restricted by the paucity of biochemical analyses of PSTs. There are now high-throughput and robust assays for sulfotransferases such as colorimetric PAPS (3'-phosphoadenosine 5'-phosphosulfate) coupled assays, Europium-based fluorescent probes for ratiometric PAP (3'-phosphoadenosine-5'-phosphate) detection, and NMR methods for activity and product analysis. These techniques provide real-time and direct measurements to enhance the functional annotation and subsequent analysis of sulfated polysaccharides across the tree of life to improve putative PST identification and characterisation of function. Improved annotation and biochemical analysis of PST sequences will enhance the utility of PSTs across biomedical and biotechnological sectors.

Interactions of proteins with heparan sulfate.

Heparan sulfate (HS) is a glycosaminoglycan, polysaccharides that are considered to have arisen in the last common unicellular ancestor of multicellular animals. In this light, the large interactome of HS and its myriad functions in relation to the regulation of cell communication are not surprising. The binding of proteins to HS determines their localisation and diffusion, essential for embryonic development and homeostasis. Following the biosynthesis of the initial heparosan polymer, the subsequent modifications comprise an established canonical pathway and a minor pathway. The more frequent former starts with N-deacetylation and N-sulfation of GlcNAc residues, the latter with C-5 epimerisation of a GlcA residue adjacent to a GlcNAc. The binding of proteins to HS is driven by ionic interactions. The multivalent effect arising from the many individual ionic bonds between a single protein and a polysaccharide chain results in a far stronger interaction than would be expected from an ion-exchange process. In many instances, upon binding, both parties undergo substantial conformational change, the resulting hydrogen and van der Waal bonds contributing significant free energy to the binding reaction. Nevertheless, ionic bonds dominate the protein-polysaccharide interaction kinetically. Together with the multivalent effect, this provides an explanation for the observed trapping of HS-binding proteins in extracellular matrix. Importantly, individual ionic bonds have been observed to be dynamic; breaking and reforming, while the protein remains bound to the polysaccharide. These considerations lead to a model for 1D diffusion of proteins in extracellular matrix on HS, involving mechanisms such as sliding, chain switching and rolling.

S100P dissociates myosin IIA filaments and focal adhesion sites to reduce cell adhesion and enhance cell migration.

S100 proteins promote cancer cell migration and metastasis. To investigate their roles in the process of migration we have constructed inducible systems for S100P in rat mammary and human HeLa cells that show a linear relationship between its intracellular levels and cell migration. S100P, like S100A4, differentially interacts with the isoforms of nonmuscle myosin II (NMIIA, K(d) = 0.5 µM; IIB, K(d) = 8 µM; IIC, K(d) = 1.0 µM). Accordingly, S100P dissociates NMIIA and IIC filaments but not IIB in vitro. NMIIA knockdown increases migration in non-induced cells and there is no further increase upon induction of S100P, whereas NMIIB knockdown reduces cell migration whether or not S100P is induced. NMIIC knockdown does not affect S100P-enhanced cell migration. Further study shows that NMIIA physically interacts with S100P in living cells. In the cytoplasm, S100P occurs in discrete nodules along NMIIA-containing filaments. Induction of S100P causes more peripheral distribution of NMIIA filaments. This change is paralleled by a significant drop in vinculin-containing, actin-terminating focal adhesion sites (FAS) per cell. The induction of S100P, consequently, causes significant reduction in cellular adhesion. Addition of a focal adhesion kinase (FAK) inhibitor reduces disassembly of FAS and thereby suppresses S100P-enhanced cell migration. In conclusion, this work has demonstrated a mechanism whereby the S100P-induced dissociation of NMIIA filaments leads to a weakening of FAS, reduced cell adhesion, and enhanced cell migration, the first major step in the metastatic cascade.

Diversification of the structural determinants of fibroblast growth factor-heparin interactions: implications for binding specificity.

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of fibroblast growth factors (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-21) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K(D) values varying between 38 nM (FGF-18) and 620 nM (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 M(-1) s(-1) and FGF-9, 130,000 M(-1) s(-1)). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.

Long-term tracking of cells using inorganic nanoparticles as contrast agents: are we there yet?

The use of inorganic nanoparticles as probes to label and track cells in vivo is already a reality. While superparamagnetic nanoparticles have been the subject of clinical studies involving magnetic resonance imaging, quantum dots and gold nanoparticles are starting to be explored for similar goals in pre-clinical studies involving fluorescence and photoacoustic imaging. Although exciting results have been obtained from in vivo investigations, there appears to be a general lack of understanding on the effects of physicochemical properties on the labelling efficiency and toxicity of those nanoparticles, as well as on their stability in the intracellular microenvironment; essential requirements for using them as probes for cellular tracking. In this tutorial review, we look at what the current literature can teach us in respect to cell interactions with these nanoparticles, with the perspective of using them as probes for cell labelling. We also examine the findings obtained in pre-clinical studies that expose potential misinterpretation that can occur when using inorganic nanoparticles for in vivo imaging.

A sulphated glycosaminoglycan extract from Placopecten magellanicus inhibits the Alzheimer's disease ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1).

The clinically important anticoagulant heparin, a member of the glycosaminoglycan family of carbohydrates that is extracted predominantly from porcine and bovine tissue sources, has previously been shown to inhibit the ß-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a key drug target in Alzheimer's Disease. In addition, heparin has been shown to exert favourable bioactivities through a number of pathophysiological pathways involved in the disease processes of Alzheimer's Disease including inflammation, oxidative stress, tau phosphorylation and amyloid peptide generation. Despite the multi-target potential of heparin as a therapeutic option for Alzheimer's disease, the repurposing of this medically important biomolecule has to-date been precluded by its high anticoagulant potential. An alternative source to mammalian-derived glycosaminoglycans are those extracted from marine environments and these have been shown to display an expanded repertoire of sequence-space and heterogeneity compared to their mammalian counterparts. Furthermore, many marine-derived glycosaminoglycans appear to retain favourable bioactivities, whilst lacking the high anticoagulant potential of their mammalian counterparts. Here we describe a sulphated, marine-derived glycosaminoglycan extract from the Atlantic Sea Scallop, Placopecten magellanicus that displays high inhibitory potential against BACE-1 (IC50 = 4.8 µg.mL-1) combined with low anticoagulant activity; 25-fold less than that of heparin. This extract possesses a more favourable therapeutic profile compared to pharmaceutical heparin of mammalian provenance and is composed of a mixture of heparan sulphate (HS), with a high content of 6-sulphated N-acetyl glucosamine (64%), and chondroitin sulphate.