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SugarBindDB lists pathogen and biotoxin lectins and their carbohydrate ligands in a searchable format. Information is collected from articles published in peer-reviewed scientific journals. Help files guide the user through the search process and provide a review of structures and names of sugars that appear in human oligosaccharides. Glycans are written in the condensed form of the carbohydrate nomenclature system developed by the International Union of Pure and Applied Chemistry (IUPAC). Since its online publication by The MITRE Corporation in 2005, the database has served as a resource for research on the glycobiology of infectious disease. SugarBindDB is currently hosted by the Swiss Institute of Bioinformatics on the ExPASy server and will be enhanced and linked to related resources as part of the wider UniCarbKB initiative. Enhancements will include the option to display glycans in a variety of formats, including modified 2D condensed IUPAC and symbolic nomenclature. Copyright © 2013 John Wiley & Sons, Ltd.

An immunosensor for rapid and low level detection of the bacterial pathogen Salmonella enterica Serovar Typhimurium was designed and developed based upon label-free electrochemical impedance spectroscopy and correlated to viable cell counts. The immunosensor was fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode by immobilizing monoclonal antibody (MAb) specific against Salmonella typhimurium cell surface lipopolysaccharide (LPS) onto the surface of the electrode. Use of mass-fabricated and electroplated PCB electrodes allowed for disposable, highly sensitive, and rapid detection of Salmonella in an aqueous environment. Results demonstrate that in purified solution, Salmonella can be detected as low as 10 CFU in a 100 mL volume and label-free and rapid manner in fewer than 90 s. The cost effective approach described here can be used for detection of pathogens with relevance for healthcare, food, and environmental applications.

Advancements in molecular and cell biology have led to the development of cell-based therapies to treat injured or degenerated tissues. [1] The rationale of this concept is that functional regeneration can be achieved best by using the innate capacity of cells to create their own tissue-specific extracellular matrix (ECM) avoiding the shortfalls of man-made devices. Although direct cell injections have demonstrated very promising preclinical and clinical outcomes, [2] the mode of administration offers little control over local retention and distribution of the injected cell suspensions[3] leading to scattered therapeutic efficiency. This deficiency has led to the development of living substitutes for skin[4] and blood vessel[5] composed of cells seeded on a collagen scaffold. Notwithstanding the efficacious results in preclinical models and clinical trials, it soon became apparent that the presence of the scaffold hinders tissue remodelling and function. [6] These drawbacks led to the development of the scaffold-free cell-sheet tissue engineering (CSTE)[7] or tissue engineering by self-assembly (TESA), [8] a therapy that offers the fabrication of a contiguous cell sheet that is stabilised by cell-cell contacts and endogenously produced ECM. Despite the documented, in preclinical and clinical setting, positive outcomes for skin, [9] blood vessel, [10, 11] cornea, [12, 13] heart, [14] lung, [15] liver[16] and bone[17] replacement, only Epicel® (Genzyme, USA) for skin and LifeLine™ for blood vessel (Cytograft, USA) have been commercialised so far. This limited technology transfer from bench-top to clinic has been attributed to the substantial long period of time required for ex vivo culture (e.g. 14-35 days for corneal epithelium; [13] 84 days for corneal stromal; [18] 28 days for corneal endothelium;[19] 70 days for lung cell-sheet; [15] and 196 days for blood vessel[11] ) that often leads to loss of native phenotype and cell senescence.[20] Here, we propose a biophysical approach, termed macromolecular crowding (MMC), that increases thermodynamic activities and biological processes by several orders of magnitude,[21] as means to create ECM-rich tissue equivalents. The principle of MMC is derived from the notion that in vivo cells reside in a highly crowded/dense extracellular space and therefore the conversion of the de novo synthesised procollagen to collagen I is rapid. [22] However, in the even substantially more dilute than body fluids (e.g. urine: 36-50g/l; blood: 80g/l) culture conditions (e.g. HAM F10 nutrient medium: 16.55g/l; DMEM/F12 medium: 16.78g/l; DMEM high glucose and L-glutamine medium: 17.22g/l), the rate limiting conversion of procollagen to collagen I is very slow (Figure 1a). We propose that the addition of inert polydispersed macromolecules (presented as spherical objects of variable diameter in Figure 1b) in the culture media will facilitate amplified production of ECM-rich living substitutes. We thank Dr Oliver Carroll for laboratory management; and Mr Maciek Doczyk (http://doczykdesign.com) for his support in the preparation of Figure 1 of this manuscript. This work is supported by Science Foundation Ireland, Research Frontiers Programme, Project Number: SFI‐09‐RFP‐ENM2483 to D.Z.; Science Foundation Ireland, E.T.S. Walton Visitor Awards Programme, Project Number: 08/W.1/B2568 to M.R., A.P. and D.Z.; Health Research Board, Project Number: HRA_POR/2011/84 to D.Z.; and College of Engineering and Informatics, Postgraduate Scholarship Scheme, NUI Galway to P.K. and D.Z. peer-reviewed

Under High Aspect Ratio Vessel (HARV) bioreactor culture conditions designed to simulate the microgravity of orbital space flight, insect tissue culture cells infected with a baculovirus expression vector produced a human glycoprotein with tri- and tetra-antennary complex N-linked oligosaccharides containing terminal sialic acid residues. The oligosaccharide structures were similar to those produced in human placental cells. Insect cells cultured in T-flasks only performed incomplete oligosaccharide processing. The mechanism of HARV-mediated changes in the eukaryotic N-linked glycosylation pathway was investigated and could be mimicked under T-flask growth conditions with the addition of N-acetylmannosamine to the culture medium. The significance of these investigations is discussed with respect to the production of recombinant therapeutic glycoproteins, insect physiology, and orbital space flight.

The conditions required for mammalian-type complex N-linked glycosylation of human proteins produced in insect cells with the baculovirus expression vector system were investigated. Marked alterations to N-linked glycosylation of human placental secreted alkaline phosphatase (SEAP) were observed with different baculovirus species, insect cell lines, and cell culture media. When a recombinant Autographa californica nucleopolyhedrovirus (AcMNPV) was used to produce SEAP in Trichoplusia ni (Tn-4h) cells cultured in serum-free medium, structural analyses indicated1% hybrid and no complex oligosaccharides attached to SEAP, a typical result with the baculovirus expression vector system. However, when fetal bovine serum was added to the culture medium, 48 +/- 4% of the oligosaccharides were hybrid or complex (but asialylated) glycans. When a recombinant T. ni nucleopolyhedrovirus (TnSNPV) was similarly used to express SEAP in Tn-4h cells cultured in serum-containing medium, only 24 +/- 3% of the glycans contained terminal N-acetylglucosamine and/or galactose residues. In contrast, SEAP produced in Sf9 cells grown in serum-containing medium with AcMNPV contained1% hybrid oligosaccharides and no complex oligosaccharides. The results illustrate that baculovirus type, host cell type, and the growth medium all have a strong influence on the glycosylation pathway in insect cells, resulting in significant alterations in structures and relative abundance of N-linked glycoforms. Although the addition of sialic acid residues to the SEAP glycans was not detected, possible approaches to obtain sialylated glycans are discussed.

Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)-PTD or FITC-PTD-HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD-phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.