Selective sorting and destruction of mitochondrial membrane proteins in aged yeast.

Mitochondrial dysfunction is a hallmark of aging, and underlies the development of many diseases. Cells maintain mitochondrial homeostasis through a number of pathways that remodel the mitochondrial proteome or alter mitochondrial content during times of stress or metabolic adaptation. Here, using yeast as a model system, we identify a new mitochondrial degradation system that remodels the mitochondrial proteome of aged cells. Unlike many common mitochondrial degradation pathways, this system selectively removes a subset of membrane proteins from the mitochondrial inner and outer membranes, while leaving the remainder of the organelle intact. Selective removal of preexisting proteins is achieved by sorting into a mitochondrial-derived compartment, or MDC, followed by release through mitochondrial fission and elimination by autophagy. Formation of MDCs requires the import receptors Tom70/71, and failure to form these structures exacerbates preexisting mitochondrial dysfunction, suggesting that the MDC pathway provides protection to mitochondria in times of stress.

Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR.

An alternative to qPCR was developed for nucleic acid assays, involving signal rather than target amplification. The new technology, Restriction Cascade Exponential Amplification (RCEA), relies on specific cleavage of probe-target hybrids by restriction endonucleases (REase). Two mutant REases for amplification (Ramp), S17C BamHI and K249C EcoRI, were conjugated to oligonucleotides, and immobilized on a solid surface. The signal generation was based on: (i) hybridization of a target DNA to a Ramp-oligonucleotide probe conjugate, followed by (ii) specific cleavage of the probe-target hybrid using a non-immobilized recognition REase. The amount of Ramp released into solution upon cleavage was proportionate to the DNA target amount. Signal amplification was achieved through catalysis, by the free Ramp, of a restriction cascade containing additional oligonucleotide-conjugated Ramp and horseradish peroxidase (HRP). Colorimetric quantification of free HRP indicated that the RCEA achieved a detection limit of 10 aM (10(-17) M) target concentration, or approximately 200 molecules, comparable to the sensitivity of qPCR-based assays. The RCEA assay had high specificity, it was insensitive to non-specific binding, and detected target sequences in the presence of foreign DNA. RCEA is an inexpensive isothermal assay that allows coupling of the restriction cascade signal amplification with any DNA target of interest.

Targeted deposition of antibodies on a multiplex CMOS microarray and optimization of a sensitive immunoassay using electrochemical detection.

BACKGROUND: The CombiMatrix ElectraSense microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray; and, in this configuration, it has also been used to tether antibodies (Abs) specifically on electrodes using complementary DNA sequences conjugated to the Abs. METHODOLOGY/PRINCIPAL FINDINGS: An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSense microarray based upon targeted deposition of polypyrrole (Ppy) and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical (ECD) reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B (SEB) is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different. CONCLUSIONS/SIGNIFICANCE: Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pg/ml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.

Multiplexed electrochemical detection of Yersinia pestis and staphylococcal enterotoxin B using an antibody microarray.

The CombiMatrix antibody microarray is a versatile, sensitive detection platform based on the generation and transduction of electrochemical signals following antigen binding to surface antibodies. The sensor chip described herein is comprised of microelectrodes coupled to an adjacent bio-friendly matrix coated with antibodies to the biological pathogens Yersinia pestis and Bacillus anthracis, and the bacterial toxin staphylococcal enterotoxin B (SEB). Using this system, we were able to detect SEB and inactivated Y. pestis individually as well as in two-plex assays at concentrations as low as 5 pg/mL and 10(6) CFU/mL, respectively. We also introduce super avidin-biotin system (SABS) as a viable and effective means to enhance assay signal responses and lower detection limits. Together these technologies represent substantial advances in point-of-care and point-of-use detection applications.

Use of a multiplexed CMOS microarray to optimize and compare oligonucleotide binding to DNA probes synthesized or immobilized on individual electrodes.

The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode.

Adaptive mutations in the signal peptide of the type 1 fimbrial adhesin of uropathogenic Escherichia coli.

Signal peptides (SPs) are critical for protein transport across cellular membranes, have a highly conserved structure, and are cleaved from the mature protein upon translocation. Here, we report that naturally occurring mutations in the SP of the adhesive, tip-associated subunit of type 1 fimbriae (FimH) are positively selected in uropathogenic Escherichia coli. On the one hand, these mutations have a detrimental effect, with reduced FimH transport across the inner membrane, fewer FimH and fimbriae expressed on the bacterial surface, and decreased bacterial adhesion under flow conditions. On the other hand, the fimbriae expressed by the mutants are significantly longer on average, with many fimbriae able to stretch to >20 microm in length. More surprisingly, the SP mutant bacteria display an increased ability to resist detachment from the surface upon a switch from high to low flow. This functional effect of longer fimbriae highlights the importance of the nonadhesive fimbrial rod for adhesive function. Also, whereas bacterial adhesion to bladder epithelial cells was preserved in most mutants, binding to and killing by human neutrophils was decreased, providing an additional reason the SP mutations are relatively common among uropathogenic strains. Thus, this study demonstrates how mutations in an SP, while decreasing transport function and not affecting the final structure of the translocated protein, can lead to functional gains of the extracellular organelles that incorporate the protein and overall adaptive changes in the organism's fitness.

Escherichia coli DraE adhesin-associated bacterial internalization by epithelial cells is promoted independently by decay-accelerating factor and carcinoembryonic antigen-related cell adhesion molecule binding and does not require the DraD invasin.

The Dr family of Escherichia coli adhesins are virulence factors associated with diarrhea and urinary tract infections. Dr fimbriae are comprised of two subunits. DraE/AfaE represents the major structural, antigenic, and adhesive subunit, which recognizes decay-accelerating factor (DAF) and carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) CEA, CEACAM1, CEACAM3, and CEACAM6 as binding receptors. The DraD/AfaD subunit caps fimbriae and has been implicated in the entry of Dr-fimbriated E. coli into host cells. In this study, we demonstrate that DAF or CEACAM receptors independently promote DraE-mediated internalization of E. coli by CHO cell transfectants expressing these receptors. We also found that DraE-positive recombinant bacteria adhere to and are internalized by primary human bladder epithelial cells which express DAF and CEACAMs. DraE-mediated bacterial internalization by bladder cells was inhibited by agents which disrupt lipid rafts, microtubules, and phosphatidylinositol 3-kinase (PI3K) activity. Immunofluorescence confocal microscopic examination of epithelial cells detected considerable recruitment of caveolin, beta(1) integrin, phosphorylated ezrin, phosphorylated PI3K, and tubulin, but not F-actin, by cell-associated bacteria. Finally, we demonstrate that the DraD subunit, previously implicated as an "invasin," is not required for beta(1) integrin recruitment or bacterial internalization.