The surface-located fibrinogen-binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall-anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the alpha- and beta-chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the gamma-chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (i) clumping of exponential-phase cells in a solution of fibrinogen, (ii) adherence of exponential-phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial-related infections. However, in wild-type exponential-phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary-phase cultures with fibrinogen. ClfB-dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca2+ and Mn2+, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low-affinity inhibitory cation binding site.

Three new genes encoding the serine-aspartate (SD) repeat-containing proteins SdrC, SdrD and SdrE were found in Staphy/ococcus aureus strain Newman. The SD repeats had earlier been found in the S. aureus fibrinogenbinding clumping factors ClfA and ClfB. The clfA and clfB genes encode highmolecular-mass f ibrinogen-binding proteins that are anchored to the cell surface of 5. aureus. The sdr genes now reported are closely linked and tandemly arrayed. The putative Sdr proteins have both organizational and sequence similarity to ClfA and ClfB. At the N-terminus, putative secretory signal sequences precede approximately 500 residue A regions. The A regions of the Sdr and Clf proteins exhibit only 20-30% residue identity when aligned with any other member of the family. The only conserved sequence is the consensus motif TYTFTDYVD. The Sdr proteins differ from ClfA and ClfB by having two to five additional 110-1 13 residue repeated sequences (B-motifs) located between region A and the R-region. Each B-motif contains a consensus Ca2+-binding EF-hand loop normally found in eukaryotic proteins. The structural integrity of recombinant SdrD(Bl-B5) protein comprising the five B-repeats of SdrD was shown by bisANS fluorescence analysis to be Ca2+-dependent, suggesting that the EF-hands are functional. When Ca2+ was removed the structure collapsed to an unfolded conformation. The original structure was restored by addition of Ca2? The C-terminal R-domains of the Sdr proteins contain 132-170 SD residues. These are followed by conserved wallanchoring regions characteristic of many surface proteins of Gram-positive bacteria. The sdr locus was present in all 31 5. aureus strains from human and bovine sources tested by Southern hybridization, although in a few strains it contained two rather than three genes.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

Crawling T cell locomotion in which activated lymphocyte function-associated antigen 1 (LFA-1) integrins participate is associated with translocation of the protein kinase C-beta (PKC-beta) isoenzyme to the microtubule cytoskeleton. In normal T cells and T lymphoma cell lines, this type of motility is accompanied by PKC-beta-sensitive cytoskeletal rearrangements and the formation of trailing cell extensions, which are supported by microtubules. Expression of PKC-beta(I) and enhanced green fluorescent protein (EGFP) in nonmotile PKC-beta-deficient T cells restored their locomotory behavior in response to a triggering stimulus delivered via LFA-1 and correlated directly with the degree of cell polarization. We have also shown that PKC-beta(I) is a component of the tubulin-enriched LFA-1-cytoskeletal complex assembled upon LFA-1 cross-linking. These observations may have physiological equivalents at advanced (post-integrin activation) stages of lymphocyte extravasation.

Staphylococcus epidermidis can express three different cell-surface-associated proteins, designated SdrF, SdrG and SdrH, that contain serine-aspartate dipeptide repeats. Proteins SdrF and SdrG are similar in sequence and structural organization to the Sdr proteins of Staphylococcus aureus and comprise unique 625-and 548-residue A regions at their N termini, respectively, followed by 110-119-residue B-repeat regions and SD-repeat regions. The C termini contain LPXTG motifs and hydrophobic amino acid segments characteristic of surface proteins covalently anchored to peptidoglycan. In contrast, SdrH has a short 60-residue A region at its N terminus followed by a SD-repeat region, a unique 277-residue C region and a C-terminal hydrophobic segment. SdrH lacks a LPXTG motif. Recombinant proteins representing the A regions of SdrF, SdrG and SdrH were expressed and purified from Escherichia coli. Antisera specific to these proteins were raised in rabbits and used to identify Sdr proteins expressed by S. epidermidis. Only SdrF was released from lysostaphin-generated protoplasts of cells grown to late-exponential phase. SdrG and SdrH remained associated with the protoplast fraction and thus appear to be ineffectively sorted along the conventional pathway used for cell-wall-anchored proteins. In Southern hybridization analyses, the sdrG and sdrH genes were present in all 16 strains tested, whilst sdrF was present in 12 strains. Antisera from 16 patients who had recovered from S. epidermidis infections contained antibodies that reacted with recombinant A regions of SdrG and SdrH, suggesting that these proteins can be expressed during infection.

The oxidative stability of w3-rich oil from Camelina sativa and the storage stability of a camelina oilbased spread were evaluated. Camelina oil was more stable than fish oil and linseed oil, but less stable than sunflower, corn, sesame, and olive oils, indicated by measuring peroxide values (PV), r-anisidine values (AV), total oxidation values (Totox), thiobarbituric acid reactive substances (TBARS), conjugated diene levels (CD), and conjugated triene levels (CT) during storage at 65 °C for 16 d. The camelina oil-based spread had higher PV, AV, Totox, TBARS, CD, and CT than the sunflower spread but maintained adequate sensory quality for 16 wk of storage at 4 °C or 8 °C.

The stability of typical vanadium flow battery (VFB) catholytes was investigated at temperatures in the range 30-60 • C for V V concentrations of 1.4-2.2 mol dm −3 and sulfate concentrations of 3.6-5.4 mol dm −3 . In all cases, V 2 O 5 precipitates after an induction time, which decreases with increasing temperature. Plots of the logarithm of induction time versus the inverse of temperature (equivalent to Arrhenius plots) show excellent linearity and all have similar slopes. The logarithm of induction time also increases linearly with sulfate concentration and decreases linearly with V V concentration. The slopes of these plots give values of concentration coefficients β S and β V5 which were used to normalize induction times to reference concentrations of sulfate and V V . An Arrhenius plot of the normalized induction times gives a good straight line, the slope of which yields a value of 1.791 ± 0.020 eV for the activation energy. Combining the Arrhenius equation with the observed variation with sulfate and V V concentrations, an equation was derived for the induction time for any catholyte at any temperature in the range investigated. Although the mechanism of precipitation of V V from catholytes is not yet well understood, a precise activation energy can now be assigned to the induction process. The rapid growth of renewable electricity generation from intermittent sources such as solar photovoltaic and wind is driving a need for advanced, cost-effective, electrical energy storage (EES) technologies.1-3 Redox flow batteries 4-11 (RFBs) have attracted much interest for large-scale energy storage due to advantages over other EES technologies, and research activities in this area have grown exponentially in recent years.12,13 The energy storage capability and power output of a flow battery, unlike conventional batteries, can be scaled independently to suit the desired application. 7 Other advantages 14 include a high degree of safety, long lifetime, potentially low capital costs, high reliability and relatively high energy efficiency.Among the numerous systems that have been studied, the vanadium flow battery (VFB), also known as the vanadium redox flow battery (VRFB), is commonly regarded as one of the most promising. [5][6][7][15][16][17] The chemistry of this system is perhaps the most thoroughly characterized and the cell design has been considerably optimized. 2,18,19 It has seen the widest commercial deployment 17 and systems as large as 250-1000 kWh have been demonstrated. 20 Compared to other flow battery systems, VFBs have the additional advantage that crosscontamination due to transport through the separating membrane is effectively eliminated because the anolyte and catholyte differ only in the oxidation state of the vanadium. 21 As a result, electrolyte maintenance issues are reduced; in theory, the electrolyte is indefinitely reuseable. Furthermore, if rebalancing of the system is required the electrolytes in the two reservoirs can be mixed with each other. Since aqueous vanadium species are highly ...

The ultraviolet-visible spectra of catholytes for vanadium flow batteries (VFBs) were measured and analyzed for a range of V IV :V V ratios and vanadium concentrations. Using a model of V 2 O 3 3+ in equilibrium with VO 2+ and VO 2 + , the spectra were characterized in terms of an excess absorbance parameter p and the molar extinction coefficients ε 4 and ε 5 of VO 2+ and VO 2 + , respectively. The results showed that p varies weakly with the vanadium concentration C and this variation was quantified relative to a reference concentration C r by means of a concentration coefficient φ r . Experimental data showed that plots of φ r versus Cφ r and plots of 1/φ r versus C are linear and, based on this linearity, φ r was expressed as a simple function of C in terms of its reference concentration C r and a single parameter M that is independent of the choice of C r . Standard spectra of p at a concentration C 0 = 1 mol dm −3 and of ε 4 and ε 5 were generated from which the spectrum of any catholyte may be simulated using the measured value of M in a governing equation. This enables determination of the state of charge for any VFB catholyte using absorbance measurements at a small number of wavelengths. The use of non-dispatchable power sources such as solar, wind and ocean energy is increasing.1 Due to the intermittency of these sources, their use is restricted unless there is a means of storing the energy they produce in periods of high availability for utilization in periods of limited availability. 2,3 There is considerable interest in flow batteries for storing energy from such sources and for other large and medium scale energy storage applications. 4,5 Vanadium flow batteries (VFB), 5-13 also known as vanadium redox flow batteries (VRFB or VRB), are particularly attractive because, in addition to having long cycle life, they are essentially immune to cross-contamination problems due to mass transfer across the membrane that can limit the service life of the electrolyte in other systems. 3,4,7,[14][15][16][17][18][19] This is because both the positive and negative sides of a VFB are based on vanadium species, eliminating the need for costly re-purification processes. 1,12 Typical cells have carbon felt electrodes; both cell design and the electrochemical behavior of electrodes are active areas of research. 20-31The cells can operate at coulombic efficiencies of over 90% 32,33 and their carbon electrodes have very good stability as long as the positive half-cell is not overcharged. 34,35 Accurate monitoring of state of charge (SoC) is intrinsically important for the reliability of energy storage systems, particularly large systems in critical applications. Furthermore, independent monitoring of the SoC of both electrolytes is important for effective operation of flow battery technology. For example in a VFB, transfer of vanadium ions across the membrane [36][37][38] and side reactions such as hydrogen formation 12,39-44 at the negative electrode can cause the battery to become unbalanced (e.g. more V V on the posi...