No abstract

Polymeric materials have emerged as appealing alternatives to conventional inorganic substrates for the fabrication of microscale analytical systems; however, native polymeric surfaces typically require covalent modification to ensure optimum biocompatibility. 2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA microcapillary electrophoresis (μCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins on microchannel surfaces. Experiments using fluorescein isothiocyanate-conjugated bovine serum albumin indicated that both column efficiency and migration time reproducibility were 1 order of magnitude better with derivatized compared to untreated PMMA μCE chips. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA μCE chips. All analyses were completed in less than 60 s, and separation efficiencies as high as 5.2 × 104 plates for a 3.5-cm-long separation channel were obtained. These results demonstrate the general applicability of surface-grafted PMMA microdevices for a broad range of protein analyses.

A new method for solvent bonding polymeric substrates to form microfluidic systems has been demonstrated. Prior to device sealing, channels in an embossed poly(methyl methacrylate) (PMMA) piece are filled with a heated liquid (paraffin wax) that forms a solid sacrificial layer at room temperature. The sacrificial material prevents the bonding solvent (acetonitrile) and softened PMMA from filling the channels. Once the sealing step is complete, the sacrificial layer is melted and removed, leaving enclosed microfluidic channels. We found that PMMA substrates welded together using this method could withstand internal pressures of >2250 psi, more than 1 order of magnitude higher than their thermally bonded counterparts. To demonstrate the usefulness of this method, microchip capillary electrophoresis (CE) devices in PMMA were created and tested. Amino acid and peptide mixtures were separated in <15 s, with >40,000 theoretical plates in a 2.5-cm separation distance. Electric fields as high as 1.5 kV/cm were applied in these microchips, and >300 CE runs were performed on a single device with no degradation of separation performance. The simplicity of the methods presented here and the improved robustness of the resulting devices should facilitate the broader implementation of polymer microchips in microfluidic analyses.

Exercise is known to enhance tendon size and strength, but the stem cell-based mechanisms for such exercise-induced effects are largely unknown. This study aims to explore these mechanisms by using a mouse treadmill running model to examine the effects of exercise on newly discovered tendon stem cells (TSCs). After treadmill running, patellar TSCs (PTSCs) and Achilles TSCs (ATSCs) were isolated from the mice, and their proliferation was measured in vitro. We found that treadmill running nearly doubled proliferation rates of both PTSCs and ATSCs compared to cage control mice. Moreover, using a mixed tendon cell culture consisting of TSCs and tenocytes, cellular production of collagen was found to increase by 70% and 200% in PTSCs and ATSCs, respectively, from the treadmill running group over cells from the cage control group. These findings suggest that exercise exerts its anabolic effects on tendons at least in part by increasing proliferation to expand the pool of TSCs and also by increasing TSC-related cellular production of collagen, the predominant component of tendons. ß

Geldanamycin (GA) is an antibiotic targeting the ADP/ATP binding site of heat shock protein 90 (Hsp90).In screening for anti-herpes simplex virus type 1 (HSV-1) candidates, we found GA active against HSV-1. HSV-1 replication in vitro was significantly inhibited by GA with an 50% inhibitory concentration of 0.093 M and a concentration that inhibited cellular growth 50% in comparison with the results seen with untreated controls of 350 M. The therapeutic index of GA was over 3,700 (comparable to the results seen with acyclovir). GA did not inhibit HSV-1 thymidine kinase. Cells infected with HSV-1 demonstrated cell cycle arrest at the G 1 /S transition; however, treatment with GA resulted in a cell cycle distribution pattern identical to that of untreated cells, indicating a restoration of cell growth in HSV-1-infected cells by GA treatment. Accordingly, HSV-1 DNA synthesis was suppressed in HSV-1 ؉ cells treated with GA. The antiviral mechanism of GA appears to be associated with Hsp90 inactivation and cell cycle restoration, which indicates that GA exhibits broad-spectrum antiviral activity. Indeed, GA exhibited activities in vitro against other viruses, including severe acute respiratory syndrome coronavirus. Since GA inhibits HSV-1 through a cellular mechanism unique among HSV-1 agents, we consider it a new candidate agent for HSV-1.Geldanamycin (GA) is a benzoquinone ansamycin and was first isolated as a new entity from the fermentation of Streptomyces hygroscopicus (3). GA binds with a high level of specificity within the ADP/ATP binding pocket of heat shock protein 90 (Hsp90) and inhibits the function of this chaperone (27,36), resulting in inappropriately functioning and rapid degradation of Hsp90-associated client proteins (2, 28). The client proteins are mainly short-lived proteins, including several protein kinases (Raf-1, ErbB-2, and Bcr-Abl), p53, and pRb as well as cyclins and cyclin-dependent kinases (2, 25), which are degraded through the ubiquitin-proteasome pathway but protected by Hsp90 (4,19). As a specific inhibitor of Hsp90 function, GA demonstrated antitumor activity in a multitude of animal models (23) and is now in clinical trial (phase I) in the United States (26). Interference with the function of Hsp90 seems to be the major mechanism of action of GA (29).In a large-scale screening for novel candidates exhibiting activity against herpes simplex virus type 1 (HSV-1), we found (from the fermentation of S. hygroscopicus) a component active against HSV-1 replication. Chemical analysis of the purified compound showed a structure identical to that of GA. A study was then initiated to evaluate the potential of this compound in the treatment of HSV-1 infection. In the present study, the anti-HSV-1 effect of GA was examined in cell cultures. The possible molecular mechanism responsible for its activity against viral infection was also explored. Our investigation showed that the mode of action of GA was closely related to the inactivation of cellular Hsp90 and different from that seen with the vira...