Analysis of copper-induced protein precipitation across the E. coli proteome.

Metal cations have been exploited for their precipitation properties in a wide variety of studies, ranging from differentiating proteins from serum and blood to identifying the protein targets of drugs. Despite widespread recognition of this phenomenon, the mechanisms of metal-induced protein aggregation have not been fully elucidated. Recent studies have suggested that copper's (Cu) ability to induce protein aggregation may be a main contributor to Cu-induced cell death. Here, we provide the first proteome-wide analysis of the relative sensitivities of proteins across the Escherichia coli proteome to Cu-induced aggregation. We utilize a metal-induced protein precipitation (MiPP) methodology that relies on quantitative bottom-up proteomics to define the metal concentration-dependent precipitation properties of proteins on a proteomic scale. Our results establish that Cu far surpasses other metals in promoting protein aggregation and that the protein aggregation is reversible upon metal chelation. The bulk of the Cu bound in the protein aggregates is Cu1+, regardless of the Cu2+ source. Analysis of our MiPP data allows us to investigate underlying biophysical characteristics that determine a protein's sensitivity to Cu-induced aggregation, which is independent of the relative concentration of protein in the lysate. Overall, this analysis provides new insights into the mechanism behind Cu cytotoxicity, as well as metal cation-induced protein aggregation.

Membrane Transporters Involved in the Antimicrobial Activities of Pyrithione in Escherichia coli.

Pyrithione (2-mercaptopyridine-N-oxide) is a metal binding modified pyridine, the antibacterial activity of which was described over 60 years ago. The formulation of zinc-pyrithione is commonly used in the topical treatment of certain dermatological conditions. However, the characterisation of the cellular uptake of pyrithione has not been elucidated, although an unsubstantiated assumption has persisted that pyrithione and/or its metal complexes undergo a passive diffusion through cell membranes. Here, we have profiled specific membrane transporters from an unbiased interrogation of 532 E. coli strains of knockouts of genes encoding membrane proteins from the Keio collection. Two membrane transporters, FepC and MetQ, seemed involved in the uptake of pyrithione and its cognate metal complexes with copper, iron, and zinc. Additionally, the phenotypes displayed by CopA and ZntA knockouts suggested that these two metal effluxers drive the extrusion from the bacterial cell of potentially toxic levels of copper, and perhaps zinc, which hyperaccumulate as a function of pyrithione. The involvement of these distinct membrane transporters contributes to the understanding of the mechanisms of action of pyrithione specifically and highlights, more generally, the important role that membrane transporters play in facilitating the uptake of drugs, including metal-drug compounds.

Apple polyphenols require T cells to ameliorate dextran sulfate sodium-induced colitis and dampen proinflammatory cytokine expression.

Human IBD, including UC and Crohn's disease, is characterized by a chronic, relapsing, and remitting condition that exhibits various features of immunological inflammation and affects at least one/1000 people in Western countries. Polyphenol extracts from a variety of plants have been shown to have immunomodulatory and anti-inflammatory effects. In this study, treatment with APP was investigated to ameliorate chemically induced colitis. Oral but not peritoneal administration of APP during colitis induction significantly protected C57BL/6 mice against disease, as evidenced by the lack of weight loss, colonic inflammation, and shortening of the colon. APP administration dampened the mRNA expression of IL-1ß, TNF-α, IL-6, IL-17, IL-22, CXCL9, CXCL10, CXCL11, and IFN-γ in the colons of mice with colitis. APP-mediated protection requires T cells, as protection was abated in Rag-1(-/-) or TCRα(-/-) mice but not in IL-10(-/-), IRF-1(-/-), µMT, or TCRδ(-/-) mice. Administration of APP during colitis to TCRα(-/-) mice actually enhanced proinflammatory cytokine expression, further demonstrating a requirement for TCRαß cells in APP-mediated protection. APP treatment also inhibited CXCR3 expression by TCRαß cells, but not B or NK cells, in the colons of mice with colitis; however, depletion of CD4(+) or CD8(+) T cells alone did not abolish APP-mediated protection. Collectively, these results show that oral administration of APP protects against experimental colitis and diminishes proinflammatory cytokine expression via T cells.

Validation of the bispectral index monitor for measuring the depth of sedation in children.

The Bispectral Index (BIS) is an empirically calibrated number derived from adult electroencephalograph data that correlates with the depth of sedation in adults. We tested the hypothesis that the BIS score is a valid measure of the depth of pediatric sedation in a study designed to avoid limitations of a previously published report. BIS values from 96 healthy ASA physical status I-II children aged 1-12 yr undergoing sedation were continually recorded and electronically transferred to a computer. Two independent observers blinded as to BIS score evaluated sedation using the Observer's Assessment of Alertness/Sedation (OAA/S) and the University of Michigan Sedation Scale (UMSS) at 3-5 min intervals. There was a significant correlation between BIS and UMSS and between BIS and OAA/S by both the Spearman's rank correlation test and by prediction probability (P < 0.001). In children <6 yr, there was a significant correlation between BIS and the clinical sedation scores for subgroups undergoing invasive and noninvasive procedures (P < 0.001). There was also good agreement between the 2 independent observers who assessed clinical sedation scores (kappa = 0.51, P < 0.001). We conclude that the BIS monitor is a quantitative, nondisruptive and easy to use depth of sedation monitor in children.