Effects of delayed NSAID administration after experimental eccentric contraction injury - A cellular and proteomics study.

BACKGROUND:Acute muscle injuries are exceedingly common and non-steroidal anti-inflammatory drugs (NSAIDs) are widely consumed to reduce the associated inflammation, swelling and pain that peak 1-2 days post-injury. While prophylactic use or early administration of NSAIDs has been shown to delay muscle regeneration and contribute to loss of muscle strength after healing, little is known about the effects of delayed NSAID use. Further, NSAID use following non-penetrating injury has been associated with increased risk and severity of infection, including that due to group A streptococcus, though the mechanisms remain to be elucidated. The present study investigated the effects of delayed NSAID administration on muscle repair and sought mechanisms supporting an injury/NSAID/infection axis. METHODS:A murine model of eccentric contraction (EC)-induced injury of the tibialis anterior muscle was used to profile the cellular and molecular changes induced by ketorolac tromethamine administered 47 hr post injury. RESULTS:NSAID administration inhibited several important muscle regeneration processes and down-regulated multiple cytoprotective proteins known to inhibit the intrinsic pathway of programmed cell death. These activities were associated with increased caspase activity in injured muscles but were independent of any NSAID effect on macrophage influx or phenotype switching. CONCLUSIONS:These findings provide new molecular evidence supporting the notion that NSAIDs have a direct negative influence on muscle repair after acute strain injury in mice and thus add to renewed concern about the safety and benefits of NSAIDS in both children and adults, in those with progressive loss of muscle mass such as the elderly or patients with cancer or AIDS, and those at risk of secondary infection after trauma or surgery

Proteomic Investigation of the Time Course Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica▿ †

To investigate the extent to which macrophages respond to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serotype Typhimurium and analyzed macrophage proteins at various time points following infection by using a global proteomic approach. A total of 1,006 macrophage and 115 Salmonella proteins were identified with high confidence. Most of the Salmonella proteins were observed in the late stage of the infection time course, which is consistent with the fact that the bacterial cells proliferate inside RAW 264.7 macrophages. The peptide abundances of most of the identified macrophage proteins remained relatively constant over the time course of infection. Compared to those of the control, the peptide abundances of 244 macrophage proteins (i.e., 24% of the total identified macrophage proteins) changed significantly after infection. The functions of these Salmonella-affected macrophage proteins were diverse, including production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase), production of prostaglandin H2 (i.e., cyclooxygenase 2), and regulation of intracellular traffic (e.g., sorting nexin 5 [SNX5], SNX6, and SNX9). Diverse functions of the Salmonella-affected macrophage proteins demonstrate a global macrophage response to Salmonella infection. Western blot analysis not only confirmed the proteomic results for a selected set of proteins but also revealed that (i) the protein abundance of mitochondrial superoxide dismutase increased following macrophage infection, indicating an infection-induced oxidative stress in mitochondria, and (ii) in contrast to infection of macrophages by wild-type Salmonella, infection by the sopB deletion mutant had no negative impact on the abundance of SNX6, suggesting a role for SopB in regulating the abundance of SNX6

Automated Data Extraction from <i>In Situ</i> Protein-Stable Isotope Probing Studies

Protein-stable isotope probing (protein-SIP) has strong potential for revealing key metabolizing taxa in complex microbial communities. While most protein-SIP work to date has been performed under controlled laboratory conditions to allow extensive isotope labeling of the target organism(s), a key application will be <i>in situ</i> studies of microbial communities for short periods of time under natural conditions that result in small degrees of partial labeling. One hurdle restricting large-scale <i>in situ</i> protein-SIP studies is the lack of algorithms and software for automated data processing of the massive data sets resulting from such studies. In response, we developed Stable Isotope Probing Protein Extraction Resources software (SIPPER) and applied it for large-scale extraction and visualization of data from short-term (3 h) protein-SIP experiments performed <i>in situ</i> on phototrophic bacterial mats isolated from Yellowstone National Park. Several metrics incorporated into the software allow it to support exhaustive analysis of the complex composite isotopic envelope observed as a result of low amounts of partial label incorporation. SIPPER also enables the detection of labeled molecular species without the need for any prior identification

Correction to Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

Correction to Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virion

Correction to Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

Correction to Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virion

NSAID-induced decrease in anti-apoptosis proteins is associated with increased caspase activity.

<p>Caspase 3/7activity in muscle homogenates from saline- and NSAID-treated animals (4/group) was measured at 54 hrs after injury (7 hrs post-treatment) by commercial ELISA. Data are given as the fold-change for each animal relative to its own non-injured TA muscle. Responses between saline versus NSAID-treated animals were statistically significant as determined by ANOVA using a mixed model with random effect of animal within treatment with the level of significance set at p < .05.</p

Experimental muscle injury disrupts myofiber architecture and causes marked influx of inflammatory cells.

<p>Mice underwent the eccentric contraction (EC) exercise regimen as described in Methods. At 24 hr post-EC, animals were euthanized and both left and right tibialis anterior (TA) muscles were harvested post-mortem for routine histopathology. One representative animal of 3 is depicted. The exercised muscle, but not the control TA, displays destruction of normal muscle architecture and a marked inflammatory cell influx. These pathologies, and the corresponding reduction in muscle force, are accepted criteria that define muscle injury.</p

IPA predicts an NSAID-induced Increase in Cell Death and a Reduction in Proliferation/Survival in Regenerating TA Muscles.

<p>IPA predicts an NSAID-induced Increase in Cell Death and a Reduction in Proliferation/Survival in Regenerating TA Muscles.</p