ABSTRACT
Metabolomics is a mainstream strategy for investigating microbial metabolism. One emerging application of metabolomics is the systematic quantification of metabolic boundary fluxes - the rates at which metabolites flow into and out of cultured cells. Metabolic boundary fluxes can capture complex metabolic phenotypes in a rapid assay, allow computational models to be built that predict the behavior of cultured organisms, and are an emerging strategy for clinical diagnostics. One advantage of quantifying metabolic boundary fluxes rather than intracellular metabolite levels is that it requires minimal sample processing. Whereas traditional intracellular analyses require a multi-step process involving extraction, centrifugation, and solvent exchange, boundary fluxes can be measured by simply analyzing the soluble components of the culture medium. To further simplify boundary flux analyses, we developed a custom 96-well sampling system-the Microbial Containment Device (MCD)-that allows water-soluble metabolites to diffuse from a microbial culture well into a bacteria-free analytical well via a semi-permeable membrane. The MCD was designed to be compatible with the autosamplers present in commercial liquid chromatography-mass spectrometry systems, allowing metabolic fluxes to be analyzed with minimal sample handling. Herein, we describe the design, evaluation, and performance testing of the MCD relative to traditional culture methods. We illustrate the utility of this platform, by quantifying the unique boundary fluxes of four bacterial species and demonstrate antibiotic-induced perturbations in their metabolic activity. We propose the use of the MCD for enabling single-step metabolomics sample preparation for microbial identification, antimicrobial susceptibility testing, and other metabolic boundary flux applications where traditional sample preparation methods are impractical.
ABSTRACT
Despite significant evidence regarding the increased risk of cartilage degeneration due to traumatic injuries to joints, there is still a lack of understanding of the mechanisms underlying osteoarthritis development following a joint injury. Injuries in knee cartilage are often characterized by lesions or tears. In addition to acute traumatic joint injuries, microscale damages, which may form because of wear, are thought to be a contributing factor in the development of osteoarthritis. While the overall function of a joint may not be affected by the presence of microcracks, we hypothesized that strain magnification in the vicinity of microcracks might be significant. We tested this hypothesis by creating partial cuts in articular cartilages and measuring the strain within 20 µm from the edge of these cuts. Measurements were made in the superficial and middle zones of articular cartilage extract samples. We found that local strain in the vicinity of cuts is magnified by a factor of 1.2-1.6 compared to strains in intact regions for nominal compressions exceeding 5%. For nominal compressions of less than 5%, no strain magnification was detected in the vicinity of the cracks. We concluded that articular cartilage cracks magnify local strains by damaging the structural integrity and decreasing the fluid pressure in the matrix.
