Coordinated regulation of acid resistance in Escherichia coli.

BACKGROUND: Enteric Escherichia coli survives the highly acidic environment of the stomach through multiple acid resistance (AR) mechanisms. The most effective system, AR2, decarboxylates externally-derived glutamate to remove cytoplasmic protons and excrete GABA. The first described system, AR1, does not require an external amino acid. Its mechanism has not been determined. The regulation of the multiple AR systems and their coordination with broader cellular metabolism has not been fully explored. RESULTS: We utilized a combination of ChIP-Seq and gene expression analysis to experimentally map the regulatory interactions of four TFs: nac, ntrC, ompR, and csiR. Our data identified all previously in vivo confirmed direct interactions and revealed several others previously inferred from gene expression data. Our data demonstrate that nac and csiR directly modulate AR, and leads to a regulatory network model in which all four TFs participate in coordinating acid resistance, glutamate metabolism, and nitrogen metabolism. This model predicts a novel mechanism for AR1 by which the decarboxylation enzymes of AR2 are used with internally derived glutamate. This hypothesis makes several testable predictions that we confirmed experimentally. CONCLUSIONS: Our data suggest that the regulatory network underlying AR is complex and deeply interconnected with the regulation of GABA and glutamate metabolism, nitrogen metabolism. These connections underlie and experimentally validated model of AR1 in which the decarboxylation enzymes of AR2 are used with internally derived glutamate.

Rapid Detection of Bacteria from Blood with Surface-Enhanced Raman Spectroscopy.

Traditional methods for identifying pathogens in bacteremic patients are slow (24-48+ h). This can lead to physicians making treatment decisions based on an incomplete diagnosis and potentially increasing the patient's mortality risk. To decrease time to diagnosis, we have developed a novel technology that can recover viable bacteria directly from whole blood and identify them in less than 7 h. Our technology combines a sample preparation process with surface-enhanced Raman spectroscopy (SERS). The sample preparation process enriches viable microorganisms from 10 mL of whole blood into a 200 µL aliquot. After a short incubation period, SERS is used to identify the microorganisms. We further demonstrated that SERS can be used as a broad detection method, as it identified a model set of 17 clinical blood culture isolates and microbial reference strains with 100% identification agreement. By applying the integrated technology of sample preparation and SERS to spiked whole blood samples, we were able to correctly identify both Staphylococcus aureus and Escherichia coli 97% of the time with 97% specificity and 88% sensitivity.

A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples.

Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps into a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings.

Energy weighted x-ray dark-field imaging.

The dark-field image obtained in grating-based x-ray phase-contrast imaging can provide information about the objects' microstructures on a scale smaller than the pixel size even with low geometric magnification. In this publication we demonstrate that the dark-field image quality can be enhanced with an energy-resolving pixel detector. Energy-resolved x-ray dark-field images were acquired with a 16-energy-channel photon-counting pixel detector with a 1 mm thick CdTe sensor in a Talbot-Lau x-ray interferometer. A method for contrast-noise-ratio (CNR) enhancement is proposed and validated experimentally. In measurements, a CNR improvement by a factor of 1.14 was obtained. This is equivalent to a possible radiation dose reduction of 23%.

Grating-based x-ray phase-contrast imaging with a multi energy-channel photon-counting pixel detector.

We have carried out grating-based x-ray differential phase-contrast measurements with a hybrid pixel detector in 16 energy channels simultaneously. A method for combining the energy resolved phase-contrast images based on energy weighting is presented. An improvement in contrast-to-noise ratio by 58.2% with respect to an emulated integrating detector could be observed in the final image. The same image quality could thus be achieved with this detector and with energy weighting at 60.0% reduced dose compared to an integrating detector. The benefit of the method depends on the object, spectrum, interferometer design and the detector efficiency.