Chronic vascular disease in diabetes isC hronic vascular disease is the major cause of morbidity and mortality in diabetes (1). This is associated with dysfunction of endothelial cells in hyperglycemia (2) and damage to the endothelium; the latter is indicated in vivo by increased detachment and premature death of endothelial cells by apoptosis (including anoikis) (3,4). A cellular marker of damage to the endothelium, increased number of circulating endothelial cells, in diabetes was not linked directly to glycemic control (HbA 1c [A1C]) (5). Extracellular matrix (ECM) interactions with endothelial cells maintain cell survival (6) and support angiogenesis driven by vascular endothelial-derived growth factor and other angiogenic factors (7). Early stages of microangiopathy and wound healing are characterized by development of acellular capillaries and decreased angiogenesis with consequent ischemia (8). A metabolic link to ECM disengagement of endothelial cells and impaired angiogenesis has not been identified.Most cell adhesion and signaling occur via integrins, which mediate a variety of cell-cell and cell-matrix interactions. The ␣ 1 ␤ 1 and ␣ 2 ␤ 1 integrins recognize the GFOGER sequence found in collagens (9,10) (Fig. 1A). Several integrins recognize the RGD sequence within ECM proteins (6,11) where the RGD moiety binds astride the integrin ␣-and ␤-subunits with the Arg residue making electrostatic interaction with one or two Asp residues of the ␣-subunit (12,13).Methylglyoxal is a potent arginine-directed glycating agent formed mainly by the degradation of triosephosphates (14,15) with increased flux of formation in hyperglycemia associated with diabetes (16). It reacts with arginine residues to form a hydroimidazolone derivative, N␦-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1)residues, an advanced glycation end product (AGE) (17,18), with loss of associated side chain positive charge (19) (Fig. 1B). MG-H1 residues are a major type of protein damage by glycation in diabetes, occurring on both cellular and extracellular proteins (20,21). Increased concentration of MG-H1 residues in plasma protein of diabetic patients was not linked directly to A1C (22), probably because methylglyoxal formation is increased in both fasting and postprandial hyperglycemia (16,23) and influenced by factors other than hyperglycemia (low glyceraldehyde-3-phosphate dehydrogenase activity [24]). MG-H1 residue formation occurred at susceptible hotspot sites in proteins with loss of functional activity (19). The surface sheath network of type IV collagen in blood vessels (25) binds integrins of vascular endothelial cells, anchoring and sustaining the vascular endothelium by interaction with integrins at GFOGER and RGD sites of the triple helical domain (9,26). These integrin binding sites are potential targets for methylglyoxal modification.We report here that modification by methylglyoxal of GFOGER and RGD sites in type IV collagen in hyperglycemia impairs ECM attachment, viability, and angiogenic activity of endothelial ce...

The aim of this study was twofold: first, to characterize the free extracellular polymeric substances (EPS) and bound EPS produced by Escherichia coli during different growth phases in different media, and then to investigate the role of the free EPS in promoting aggregation. EPS was extracted from a population of E. coli MG1655 cells grown in different media composition (Luria-Bertani (LB) and Luria-Bertani with the addition of 0.5 w/v% glucose at the beginning of the growth phase (LBG)) and at different growth phases (6 and 24 h). The extracted EPS was characterized using Fourier transform infrared spectroscopy and further identified using one-dimensional gel-based electrophoresis and tandem mass spectrometry. E. coli MG1655 was found to produce significantly lower amounts of bound EPS compared to free EPS under all conditions. The protein content of free EPS increased as the cells progressed from the exponential to stationary phase when grown in LB or LBG, while the carbohydrate content only increased across the growth phases for cells grown in LBG. FTIR revealed a variation in the different functional groups such as amines, carboxyl, and phosphoryl groups for free EPS extracted at the different growth conditions. Over 500 proteins were identified in the free EPS, with 40 proteins common in all growth conditions. Proteins with functionality related to amino acid and carbohydrate metabolism, as well as cell wall and membrane biogenesis were among the highest proteins identified in the free EPS extracted from E. coli MG1655 under all growth and media conditions. The role of bound and free EPS was investigated using a standardized aggregation assay. Bound EPS did not contribute to aggregation of E. coli MG1655. The readdition of free EPS to E. coli MG1655 resulted in aggregation of the cells in all growth conditions. Free EPS extracted from the 24 h E. coli MG1655 cultures grown in LB had the greatest effect on aggregation of cells grow in LBG, with a 30% increase in aggregation observed.

Bacteria exist as aggregates or in biofilms to help with adaptation and protection from environmental stresses. The hypothesis that is tested in this paper is that the relative presence of glucose in the media, at the beginning of the growth phase, influences the surface chemistry of the cell, which as a consequence reduces the tendency for the cells to interact and form aggregates. In this study, we used Escherichia coli (E. coli) MG1655 as a model organism and measured the change in the surface chemistry of cells harvested at different growth phases, which had been cultured in Luria-Bertani media with and without the addition of glucose, using potentiometric titration and infrared spectroscopy. Cells, cultivated with the additional supplement of glucose at the beginning of the growth phase, displayed a higher concentration of bacterial surface functional groups and a variation in outer membrane proteins. As a consequence, the tendency for cell-to-cell attachment was significantly reduced. Our findings therefore revealed that glucose limits aggregation in E. coli MG1655 by altering the concentration of functional groups from macromolecules present on the bacterial surface.

Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA). SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO) into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2′-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

Bacteria exhibit properties similar to those of nonbiological colloids and can display pairwise attractions when in close proximity. This interaction is governed by the surface chemistry of the cells. We seek to understand bacterial aggregation at the cellular level using Escherichia coli (E. coli) AB1157. Aggregation studies were carried out using 0.5 to 2.5 wt% E. coli AB1157 harvested in different growth phases with varying concentrations of a nonadsorbing polymer, sodium polystyrene sulfonate (SPS). The electrophoretic mobility of E. coli AB1157 in different growth phases was determined using phase-amplitude light scattering. E. coli AB1157 was found to be negatively charged, and the cell surface properties changed in different growth phases. The electrokinetic results correlated well with the different concentrations of nonadsorbing polymer needed to induce depletion aggregation. This shows that a difference in aggregation properties is due to changes in the bacteria electrokinetic properties during their growth.