Antimicrobial and antifouling polymeric coating mitigates persistence of Pseudomonas aeruginosa biofilm.

Food wasted due to food spoilage remains a global challenge to the environmental sustainability and security of food supply. In food manufacturing, post-processing contamination of food can occur due to persistent bacterial biofilms, which can be resistant to conventional cleaning and sanitization. The objective was to characterize the efficacy of a polymeric coating in reducing Pseudomonas aeruginosa biofilm establishment and facilitating its removal. Viable cell density of a 48 h biofilm was reduced by 2.10 log cfu cm-2 on the coated surface, compared to native polypropylene. Confocal laser scanning and electron microscopy indicated reductions in mature biofilm viability and thickness on the coated material. The antifouling coating improved cleanability, with ∼2.5 log cfu cm-2 of viable cells remaining after 105 min cleaning by water at 65 °C, compared to 4.5 log cfu cm-2 remaining on native polypropylene. Such coatings may reduce the persistence of biofilms in food processing environments, in support of reducing food spoilage and waste.

Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak.

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein µ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed µ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of µ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for µ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of µ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing µ1, indicating that caspases were not required. Additionally, µ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.

In Vivo Capsid Engineering of Bacteriophages for Oriented Surface Conjugation.

The current state-of-the-art in bacteriophage (phage) immobilization onto magnetic particles is limited to techniques that are less expensive and/or facile but nonspecific or those that are more expensive and/or complicated but ensure capsid-down orientation of the phages, as necessary to preserve infectivity and performance in subsequent applications (e.g., therapeutics, detection). These cost, complexity, and effectiveness limitations constitute the major hurdles that limit the scale-up of phage-based strategies and thus their accessibility in low-resource settings. Here, we report a plasmid-based technique that incorporates a silica-binding protein, L2, into the T7 phage capsid, during viral assembly, with and without inclusion of a flexible linker peptide, allowing for targeted binding of the phage capsid to silica without requiring the direct modification of the phage genome. L2-tagged phages were then immobilized onto silica-coated magnetic nanoparticles. Inclusion of the flexible linker between the phage capsid protein and the L2 protein improved immobilization density compared to both wild type T7 phages and L2-tagged phages without the flexible linker. Taken together, this work demonstrates phage capsid modification without engineering the phage genome, which provides an important step toward reducing the cost and increasing the specificity/directionality of phage immobilization methods and could be more broadly applied in the future for other phages for a range of other capsid tags and nanomaterials.

Two-dimensional protein electrophoresis: from molecular pathway discovery to biomarker discovery in neurological disorders.

Two-dimensional protein electrophoresis (2-DE) has undergone many technical improvements in the past 30 years, resulting in an analytical method that is unparalleled in the resolution of complex protein mixtures and capable of quantifying changes in protein expression from a wide variety of tissues and samples. The technique has been applied in many studies of neurologic disease to identify changes in spot patterns that correlate with disease. The true power of the technique emerges when it is coupled to state-of-the-art methods in mass spectrometry, which enable identification of the protein or proteins contained within a spot of interest on a 2-DE map. Investigators have successfully applied the technique to gain improved understanding of neurologic disease mechanisms in humans and in animal models and to discover biomarkers that are useful in the clinical setting. An important extension to these efforts that has not been realized thus far is the desire to profile changes in protein expression that result from therapy to help relate disease-modifying effects at the molecular level with clinical outcomes. Here we review the major advances in 2-DE methods and discuss specific examples of its application in the study of neurologic diseases.