Role of Monoubiquitylation on the Control of IκBα Degradation and NF-κB Activity

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Biocompatible Bacterial Cellulose-Poly(2-hydroxyethyl methacrylate) Nanocomposite Films

A series of bacterial cellulose-poly(2-hydroxyethyl methacrylate) nanocomposite films was prepared by in situ radical polymerization of 2-hydroxyethyl methacrylate (HEMA), using variable amounts of poly(ethylene glycol) diacrylate (PEGDA) as crosslinker. Thin films were obtained, and their physical, chemical, thermal, and mechanical properties were evaluated. The films showed improved translucency compared to BC and enhanced thermal stability and mechanical performance when compared to poly(2-hydroxyethyl methacrylate) (PHEMA). Finally, BC/PHEMA nanocomposites proved to be nontoxic to human adipose-derived mesenchymal stem cells (ADSCs) and thus are pointed as potential dry dressings for biomedical applications.

A Computational Module Assembled from Different Protease Family Motifs Identifies PI PLC from Bacillus cereus as a Putative Prolyl Peptidase with a Serine Protease Scaffold

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a beta-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

In Vitro Degradation of Poly(caprolactone)/nHA Composites

The degradation behavior and mechanical properties of polycaprolactone/nanohydroxyapatite composite scaffolds are studied in phosphate buffered solution (PBS), at 37 degrees C, over 16 weeks. Under scanning electron microscopy (SEM), it was observed that the longer the porous scaffolds remained in the PBS, the more significant the thickening of the pore walls of the scaffold morphology was. A decrease in the compressive properties, such as the modulus and the strength of the PCL/nHA composite scaffolds, was observed as the degradation experiment progressed. Samples with high nHA concentrations degraded more significantly in comparison to those with a lower content. Pure PCL retained its mechanical properties comparatively well in the study over the period of degradation. After the twelfth week, the results obtained by GPC analysis indicated a significant reduction in their molecular weight. The addition of nHA particles to the scaffolds accelerated the weight loss of the composites and increased their capacity to absorb water during the initial degradation process. The addition of these particles also affected the degradation behavior of the composite scaffolds, although they were not effective at compensating the decrease in pH prompted by the degradation products of the PCL.

Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.