Role of lysozyme inhibitors in the virulence of avian pathogenic Escherichia coli

Lysozymes are key effectors of the animal innate immunity system that kill bacteria by hydrolyzing peptidoglycan, their major cell wall constituent. Recently, specific inhibitors of the three major lysozyme families occuring in the animal kingdom (c-, g- and i-type) have been discovered in Gram-negative bacteria, and it has been proposed that these may help bacteria to evade lysozyme mediated lysis during interaction with an animal host. Escherichia coli produces two inhibitors that are specific for c-type lysozyme (Ivy, Inhibitor of vertebrate lysozyme; MliC, membrane bound lysozyme inhibitor of c-type lysozyme), and one specific for g-type lysozyme (PliG, periplasmic lysozyme inhibitor of g-type lysozyme). Here, we investigated the role of these lysozyme inhibitors in virulence of Avian Pathogenic E. coli (APEC) using a serum resistance test and a subcutaneous chicken infection model. Knock-out of mliC caused a strong reduction in serum resistance and in in vivo virulence that could be fully restored by genetic complementation, whereas ivy and pliG could be knocked out without effect on serum resistance and virulence. This is the first in vivo evidence for the involvement of lysozyme inhibitors in bacterial virulence. Remarkably, the virulence of a ivy mliC double knock-out strain was restored to almost wild-type level, and this strain also had a substantial residual periplasmic lysozyme inhibitory activity that was higher than that of the single knock-out strains. This suggests the existence of an additional periplasmic lysozyme inhibitor in this strain, and indicates a regulatory interaction in the expression of the different inhibitors

Protein glycosylation as a diagnostic and prognostic marker of chronic inflammatory gastrointestinal and liver diseases

Glycans are sequences of carbohydrates that are added to proteins or lipids to modulate their structure and function. Glycans modify proteins required for regulation of immune cells, and alterations have been associated with inflammatory conditions. For example, specific glycans regulate T-cell activation, structures, and functions of immunoglobulins; interactions between microbes and immune and epithelial cells; and malignant transformation in the intestine and liver. We review the effects of protein glycosylation in regulation of gastrointestinal and liver functions, and how alterations in glycosylation serve as diagnostic or prognostic factors, or as targets for therapy

Hormone specific regulation of natural killer cells by cortisol Direct inactivation of the cytotoxic function of cloned human NK cells without an effect on cellular proliferation

AbstractCorticosteroids have previously been reported to partially inhibit the natural cytotoxic activity of peripheral blood lymphocytes. However, since only a few percent of peripheral lymphocytes are natural killer (NK) cells, it has not been possible to determine whether corticosteroids directly inhibit NK cells or mediate this effect via other cell types. This report documents direct functional inactivation, but unimpeded proliferation, of cloned human NK cells by subphysiologic levels of cortisol. In contrast, high concentrations of testosterone, progesterone or estradiol had no significant effect on proliferation or cytotoxic activity of the cloned NK cells. The kinetics of inhibition of NK function by cortisol are consistent with a transcription-dependent mechanism

3D Microstructured Carbon Nanotube Electrodes for Trapping and Recording Electrogenic Cells

Electrogenic cells such as cardiomyocytes and neurons rely mainly on electrical signals for intercellular communication. Microelectrode arrays (MEAs) have been developed for long-term recording of cell signals and stimulation of electrogenic cells under low-cell-stress conditions, providing new insights in the behavior of electrogenic cells and the operation of the brain. To date, MEAs are relying on flat or needle-shaped electrode surfaces, mainly due to limitations in the lithographic processes. This paper relies on a previously reported elasto-capillary aggregation process to create 3D carbon nanotube (CNT) MEAs. This study shows that CNTs aggregate in well-shaped structures of similar size as cardiomyocytes are particularly interesting for MEA applications. This is because i) CNT microwells of the right diameter preferentially trap individual cardiomyocytes, which facilitates single cell recording without the need for clamping cells or signal deconvolution, and ii) once the cells are trapped inside of the CNT wells, this 3D CNT structure is used as an electrode surrounding the cell, which increases the cell-electrode contact area. As a result, this study finds that the recorded output voltages increase significantly (more than 200%). This fabrication process paves the way for future study of complex interactions between electrogenic cells and 3D recording electrodes.This work was supported by the Research Foundation—Flanders (FWO, Belgium) under Project No. 11S1214N. Michael De Volder was supported by the ERC Starting Grant (337739)—HIENA and the Marie Curie Grant CANA (618250). Davor Copic was supported by the Marie Curie Grant EmuCam (660351)

Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems

The PgIB oligosaccharyltransferase (OTase) of Campylobacter jejuni can be functionally expressed in Escherichia coli, and its relaxed oligosaccharide substrate specificity allows the transfer of different glycans from the lipid carrier undecaprenyl pyrophosphate to an acceptor protein. To investigate the substrate specificity of PgIB, we tested the transfer of a set of lipid-linked polysaccharides in E. coli and Salmonella enterica serovar Typhimurium. A hexose linked to the C-6 of the monosaccharide at the reducing end did not inhibit the transfer of the O antigen to the acceptor protein. However, PgIB required an acetamido group at the C-2. A model for the mechanism of PgIB involving this functional group was proposed. Previous experiments have shown that eukaryotic OTases have the same requirement, suggesting that eukaryotic and prokaryotic OTases catalyze the transfer of oligosaccharides by a conserved mechanism. Moreover, we demonstrated the functional transfer of the C. jejuni glycosylation system into S. enterica. The elucidation of the mechanism of action and the substrate specificity of PgIB represents the foundation for engineering glycoproteins that will have an impact on biotechnology

Fed-batch fermentation of GM-CSF-producing glycoengineered Pichia pastoris under controlled specific growth rate

<p>Abstract</p> <p>Background</p> <p>Yeast expression systems with altered N-glycosylation are now available to produce glycoproteins with homogenous, defined N-glycans. However, data on the behaviour of these strains in high cell density cultivation are scarce.</p> <p>Results</p> <p>Here, we report on cultivations under controlled specific growth rate of a GlycoSwitch-Man5 <it>Pichia pastoris </it>strain producing Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) at high levels (hundreds of milligrams per liter). We demonstrate that homogenous Man₅GlcNAc₂N-glycosylation of the secreted proteins is achieved at all specific growth rates tested.</p> <p>Conclusions</p> <p>Together, these data illustrate that the GlycoSwitch-Man5 <it>P. pastoris </it>is a robust production strain for homogenously N-glycosylated proteins.</p

Performance evaluation of CHP with heat storage in buildings

Paper presented at the 5th International Conference on Heat Transfer, Fluid Mechanics and Thermodynamics, South Africa, 1-4 July, 2007.Combined heat and power (CHP) production gains more and more attention. Offices and public buildings often have a large thermal power demand in combination with a fairly large electrical power demand. On the other hand they are seldom occupied by night and in weekends, reducing the actual operational time of the heating system. This in turn brings down the financial benefits of investing in CHP. A second problem is that electrical and thermal demands are often shifted in time. The running time of the engine is again limited this way, as it is often not allowed to deliver electricity to the power grid. A possible solution is using heat storage. This way the CHP-engine can run when the electricity demand is high. In the paper a simulation model of CHP with gas engine and heat storage by means of a hot water vessel is developed. The model is validated through experiments on an engine and a vessel. This model is used to analyze the design, control and performance of cogeneration plants. It is shown that storage is marginal beneficial and the design has to be done with great care.cs201

Sexually dimorphic tibia shape is linked to natural osteoarthritis in STR/Ort mice

Human osteoarthritis (OA) is detected only at late stages. Male STR/Ort mice develop knee OA spontaneously with known longitudinal trajectory, offering scope to identify OA predisposing factors. We exploit the lack of overt OA in female STR/Ort and in both sexes of parental, control CBA mice to explore whether early divergence in tibial bone mass or shape are linked to emergent OA

Oximetry and Glucose Sensors A SMART SENSOR FOR BIOMEDICAL APPLICATIONS

Abstract Monolithic smart sensors for the voltammetric measurement of glucose or oxygen concentrations have been developed. Each smart sensor consists of a planar voltammetric sensor, a CMOS interface circuit and temperature sensor. The interface circuit and the temperature sensor are realised in a standard CMOS process. The sensor specific layers are added afterwards on the same chip with the CMOS compatible sensor technology developed for this purpose. Two versions of this smart sensor have been realised. The two electrode configuration with a Au working electrode has been implemented for the measurement of p02. The three electrode configuration with a Pt working electrode can measure glucose when an additional glucose oxidase membrane is applied on the electrodes. The interface circuit can apply voltages from +lV to -lV to the sensor and can handle sensor current ranges from 30 nA full scale to 1 pA full scale with a +/-2.5 V supply voltage. The temperature sensor has a sensitivity of 154 pV/K. The total dimensions of this smart sensor are 0.75 mm by 5 mm