Identification of Potentially Diarrheagenic Atypical Enteropathogenic Escherichia coli Strains Present in Canadian Food Animals at Slaughter and in Retail Meats

This study identified and characterized enteropathogenic Escherichia coli (EPEC) in the Canadian food supply. Eighteen of 450 E. coli isolates from food animal sources were identified as atypical EPEC (aEPEC). Several of the aEPEC isolates identified in this study possessed multiple virulence genes, exhibited adherence and attaching and effacing (A/E) lesion formation, disrupted tight junctions, and were coclassified with the extraintestinal pathogenic E. coli (ExPEC) and enterotoxigenic E. coli (ETEC) pathotypes.     

Matrix-bound PAI-1 supports cell blebbing via RhoA/ROCK1 signaling

The microenvironment of a tumor can influence both the morphology and the behavior of cancer cells which, in turn, can rapidly adapt to environmental changes. Increasing evidence points to the involvement of amoeboid cell migration and thus of cell blebbing in the metastatic process; however, the cues that promote amoeboid cell behavior in physiological and pathological conditions have not yet been clearly identified. Plasminogen Activator Inhibitor type-1 (PAI-1) is found in high amount in the microenvironment of aggressive tumors and is considered as an independent marker of bad prognosis. Here we show by immunoblotting, activity assay and immunofluorescence that, in SW620 human colorectal cancer cells, matrix-associated PAI-1 plays a role in the cell behavior needed for amoeboid migration by maintaining cell blebbing, localizing PDK1 and ROCK1 at the cell membrane and maintaining the RhoA/ROCK1/MLC-P pathway activation. The results obtained by modeling PAI-1 deposition around tumors indicate that matrix-bound PAI-1 is heterogeneously distributed at the tumor periphery and that, at certain spots, the elevated concentrations of matrix-bound PAI-1 needed for cancer cells to undergo the mesenchymal-amoeboid transition can be observed. Matrix-bound PAI-1, as a matricellular protein, could thus represent one of the physiopathological requirements to support metastatic formation.     

Extracellular urease from Arthrobacter creatinolyticus MTCC 5604: scale up,purification and its cytotoxic effect thereof

Molecular Biology Reports - Urease is a potent metalloenzyme with diverse applications. This paper describes the scale up and purification of an extracellular urease from Arthrobacter...     

Meeting report: SMART timing—principles of single molecule techniques course at the University of Michigan 2014

Four days after the announcement of the 2014 Nobel Prize in Chemistry for “the development of super‐resolved fluorescence microscopy” based on single molecule detection, the Single Molecule Analysis in Real‐Time (SMART) Center at the University of Michigan hosted a “Principles of Single Molecule Techniques 2014” course. Through a combination of plenary lectures and an Open House at the SMART Center, the course took a snapshot of a technology with an especially broad and rapidly expanding range of applications in the biomedical and materials sciences. Highlighting the continued rapid emergence of technical and scientific advances, the course underscored just how brightly the future of the single molecule field shines. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 296–302, 2015.     

Metal‐dependent assembly of a protein nano‐cage

Short, alpha‐helical coiled coils provide a simple, modular method to direct the assembly of proteins into higher order structures. We previously demonstrated that by genetically fusing de novo–designed coiled coils of the appropriate oligomerization state to a natural trimeric protein, we could direct the assembly of this protein into various geometrical cages. Here, we have extended this approach by appending a coiled coil designed to trimerize in response to binding divalent transition metal ions and thereby achieve metal ion‐dependent assembly of a tetrahedral protein cage. Ni²⁺, Co²⁺, Cu²⁺, and Zn²⁺ ions were evaluated, with Ni²⁺ proving the most effective at mediating protein assembly. Characterization of the assembled protein indicated that the metal ion–protein complex formed discrete globular structures of the diameter expected for a complex containing 12 copies of the protein monomer. Protein assembly could be reversed by removing metal ions with ethylenediaminetetraacetic acid or under mildly acidic conditions.     

Effect of covalent links on the structure, spectra, and redox properties of myeloperoxidase--a density functional study

The enzyme myeloperoxidase shows several unusual properties compared to other peroxidases, e.g. a red-shifted absorption spectrum and a peroxidase activity towards chloride. It has been suggested that this is caused by the unusual covalent links between the heme group and the surrounding protein, but whether it is caused by the two ester links to Glu-242 and Asp-94 or the sulfonium ion linkage to Met-243 is unclear. To investigate these suggestions, we have used density functional theory to study the structure, spectra, and reduction potential of 25 models of myeloperoxidase in the reduced (Fe^II) and oxidized (Fe^III) states, as well as in the compound I (formally Fe^VO) and II (Fe^IVO or Fe^IVOH) states, using appropriate models of the linkages to the Asp, Glu, and Met residues (including the back-bone connection between Glu-242 and Met-243) in varying combinations. The calculated spectral shifts indicate that both the ester and sulfonium linkages play a role in the spectral shift. On the other hand, the sulfonium linkage seems to be mainly responsible for the high positive reduction potential for the both ferric/ferrous and compound I/II couples of myeloperoxidase.     

Cardiobutanolide,a styryllactone from Goniothalamus cardiopetalus

A styryllactone namely cardiobutanolide was isolated from the stem bark of Goniothalamus cardiopetalus together with four known styryllactones goniothalamin, goniodiol, goniofufurone, goniofupyrone and known acetogenins squamocin and an epimeric mixture of goniodonin and 34-epi-goniodonin. The structure of the new compound was elucidated on the basis of 1D and 2D NMR experiments and mass spectroscopic techniques.     

Sec24 interaction is essential for localization and virulence-associated function of the bacterial effector protein NleA

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that cause severe diarrhoeal disease in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium and is required for bacterial virulence. NleA localizes to the host cell secretory pathway and inhibits vesicle trafficking by interacting with the Sec24 subunit of mammalian coatamer protein II complex (COPII). Mammalian cells express four paralogues of Sec24 (Sec24A-D), which mediate selection of cargo proteins for transport and possess distinct, but overlapping cargo specificities. Here, we show that NleA binds Sec24A-D with two distinct mechanisms. An NleA protein variant with greatly diminished interaction with all Sec24 paralogues does not properly localize, does not inhibit COPII-mediated vesicle budding, and does not confer virulence in the mouse infection model. Together, this work provides strong evidence that the interaction and inhibition of COPII by NleA is an important aspect of EPEC- and EHEC-mediated disease.     

Neutrophil Extracellular Traps Confine Pseudomonas aeruginosa Ocular Biofilms and Restrict Brain Invasion

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