Growing Up After Adolescent Bariatric Surgery.

This study investigates the effects of adolescent bariatric surgery among young adults approximately 10 years post-surgery. Participants were recruited from a hospital-based bariatric registry. We used an exploratory, qualitatively-driven mixed methods design. Findings were integrated with medical chart data and the SF-36, Body QoL, and the Transition Readiness Assessment Questionnaire. Of the 22 participants who completed surveys (14 females and 8 males), 20 participants also completed a phone interview. Median participant age was 25 years (range = 19-30). Median weight-loss was 23% (6.0%‒58%). Four themes emerged: taking control, weight loss challenges, body image adjustment, and growing up. Participants reported physical benefits of surgery yet were challenged by eating habits, body image, and interpersonal relationships. Participants were indifferent to preventative healthcare, despite the potential for vitamin deficiencies and the return of weight-related comorbidities. Clinicians can facilitate the transition to young adulthood by providing continued mental support, education, and medical monitoring.

Proteomic analysis of Drosophila CLOCK complexes identifies rhythmic interactions with SAGA and Tip60 complex component NIPPED-A.

Circadian clocks keep time via ~ 24 h transcriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status varies over a circadian cycle. Although changes in the state of activators and repressors has been characterized, how their status is translated to transcriptional activity is not understood. We used mass spectrometry to identify proteins that interact with GFP-tagged CLK (GFP-CLK) in fly heads at different times of day. Many expected and novel interacting proteins were detected, of which several interacted rhythmically and were potential regulators of protein levels, activity or transcriptional output. Genes encoding these proteins were tested to determine if they altered circadian behavior via RNAi knockdown in clock cells. The NIPPED-A protein, a scaffold for the SAGA and Tip60 histone modifying complexes, interacts with GFP-CLK as transcription is activated, and reducing Nipped-A expression lengthens circadian period. RNAi analysis of other SAGA complex components shows that the SAGA histone deubiquitination (DUB) module lengthened period similarly to Nipped-A RNAi knockdown and weakened rhythmicity, whereas reducing Tip60 HAT expression drastically weakened rhythmicity. These results suggest that CLK-CYC binds NIPPED-A early in the day to promote transcription through SAGA DUB and Tip60 HAT activity.

Guidelines for animal exercise and training protocols for cardiovascular studies.

Whole body exercise tolerance is the consummate example of integrative physiological function among the metabolic, neuromuscular, cardiovascular, and respiratory systems. Depending on the animal selected, the energetic demands and flux through the oxygen transport system can increase two orders of magnitude from rest to maximal exercise. Thus, animal models in health and disease present the scientist with flexible, powerful, and, in some instances, purpose-built tools to explore the mechanistic bases for physiological function and help unveil the causes for pathological or age-related exercise intolerance. Elegant experimental designs and analyses of kinetic parameters and steady-state responses permit acute and chronic exercise paradigms to identify therapeutic targets for drug development in disease and also present the opportunity to test the efficacy of pharmacological and behavioral countermeasures during aging, for example. However, for this promise to be fully realized, the correct or optimal animal model must be selected in conjunction with reproducible tests of physiological function (e.g., exercise capacity and maximal oxygen uptake) that can be compared equitably across laboratories, clinics, and other proving grounds. Rigorously controlled animal exercise and training studies constitute the foundation of translational research. This review presents the most commonly selected animal models with guidelines for their use and obtaining reproducible results and, crucially, translates state-of-the-art techniques and procedures developed on humans to those animal models.

Revisiting the Force-Joint Angle Relationship After Eccentric Exercise.

The purpose of this study was to evaluate force-angle curve fitting techniques pre-eccentric exercise, quantify changes in curve characteristics postexercise, and examine the relationship between curve changes and markers of muscle damage. Fourteen males unaccustomed to eccentric exercise performed 60 eccentric muscle actions of the elbow flexors. Maximal voluntary isometric force was measured throughout a range of angles pre- (Pre1 and Pre2), immediately post (IP), and 1, 2, 4, and 7 days postexercise. Force-angle curves for each visit were constructed using second-order polynomials. Changes in curve characteristics (optimal angle, peak force, curve height), range of motion, soreness, and creatine kinase activity were quantified. Optimal joint angle and force at optimal angle were significantly correlated from Pre1 to Pre2 (ICC = 0.821 and 0.979, respectively). Optimal angle was significantly right shifted (p = 0.035) by 10.4 ± 12.9° from Pre2 to IP and was restored by 1 day post exercise. Interestingly, the r value for curve fit was significantly decreased (p < 0.001) from Pre2 (r = 0.896) to IP (r = 0.802) and 1 day post exercise (r = 0.750). Curve height was significantly decreased (39%) IP and restored to pre-exercise height by 4 days postexercise. There was no correlation between optimal angle or curve height and other damage markers. In conclusion, force-angle relationships can be accurately described using second-order polynomials. After eccentric exercise, the force-angle curve is flattened and shifted (downward and rightward), but these changes are not correlated to other markers of muscle damage. Changes in the force-angle relationship are multifaceted, but determining the physiological significance of these changes requires further investigation.

Phosphorylation of the transcription activator CLOCK regulates progression through a ∼ 24-h feedback loop to influence the circadian period in Drosophila.

Circadian (≅ 24 h) clocks control daily rhythms in metabolism, physiology, and behavior in animals, plants, and microbes. In Drosophila, these clocks keep circadian time via transcriptional feedback loops in which clock-cycle (CLK-CYC) initiates transcription of period (per) and timeless (tim), accumulating levels of PER and TIM proteins feed back to inhibit CLK-CYC, and degradation of PER and TIM allows CLK-CYC to initiate the next cycle of transcription. The timing of key events in this feedback loop are controlled by, or coincide with, rhythms in PER and CLK phosphorylation, where PER and CLK phosphorylation is high during transcriptional repression. PER phosphorylation at specific sites controls its subcellular localization, activity, and stability, but comparatively little is known about the identity and function of CLK phosphorylation sites. Here we identify eight CLK phosphorylation sites via mass spectrometry and determine how phosphorylation at these sites impacts behavioral and molecular rhythms by transgenic rescue of a new Clk null mutant. Eliminating phosphorylation at four of these sites accelerates the feedback loop to shorten the circadian period, whereas loss of CLK phosphorylation at serine 859 increases CLK activity, thereby increasing PER levels and accelerating transcriptional repression. These results demonstrate that CLK phosphorylation influences the circadian period by regulating CLK activity and progression through the feedback loop.

Human platelet antigen 1a epitopes are dependent on the cation-regulated conformation of integrin α(IIb)ß(3) (GPIIb/IIIa).

BACKGROUND: The HPA-1a (Leu(33)) polymorphism of platelet integrin αIIbß3 is the target of alloantibodies in 70-80% cases of neonatal alloimmune thrombocytopenia (NAIT) in Caucasians and reliable detection of these antibodies is essential for appropriate clinical management. However, the ability to detect such antibodies is highly variable between laboratories and, in a number of clinical cases where there is a HPA-1 genotype mismatch between mother and neonate, HPA-1a antibodies are undetectable. Furthermore, some studies have not shown a consistent relationship between maternal anti-HPA-1a level and neonatal platelet count. Since the integrity and conformation of the αIIbß3 complex are dependent on divalent cations, we investigated whether HPA-1a epitope integrity and/or conformation might be affected by the presence of the cation chelator EDTA in patient samples or in assay buffers, thus providing a possible explanation for the variable sensitivity of current assays. PRINCIPLE FINDINGS: Exposure of the αIIbß3 complex to EDTA resulted in reduced reactivity of three anti-HPA-1a mAbs (B2, 19-7 and 23-15). More significantly, cation chelation adversely affected detection of polyclonal anti-HPA-1a, not only in the platelet immunofluorescence assay, where alloantibody binding was reduced compared to control platelets (mean MFI reduction 44.5%, range 17.3-69.7%, n=4), but also in the commonly used monoclonal antibody specific immobilisation of platelet antigens assay (MAIPA) where both alloantibody and monoclonal capture antibody binding were reduced (mean OD reduction 82.8%, range 68.3-96.6%, n=9). CONCLUSIONS: These data show that HPA-1a antibodies recognise epitopes on αIIbß3 that are sensitive to EDTA treatment and that cation chelation grossly reduces the sensitivity of the MAIPA assay by diminishing not only HPA-1a alloantibody binding but also 'capture' monoclonal antibody binding. These findings may, in part, explain the current variability in antibody measurement and will guide the development of more sensitive tests for anti-integrin antibodies in NAIT and other conditions.

Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.

Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA) and vancomycin-intermediate ST239 S. aureus (VISA); each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.

IIb or not IIb? Regulation of myosin heavy chain gene expression in mice and men.

BACKGROUND: While the myosin heavy chain IIb isoform (MyHC-IIb) is the predominant motor protein in most skeletal muscles of rats and mice, the messenger RNA (mRNA) for this isoform is only expressed in a very small subset of specialized muscles in adult large mammals, including humans. RESULTS: We identify the DNA sequences limiting MyHC-IIb expression in humans and explore the activation of this gene in human skeletal muscle. We demonstrate that the transcriptional activity of ~1.0 kb of the human MyHC-IIb promoter is greatly reduced compared to that of the corresponding mouse sequence in both mouse and human myotubes in vitro and show that nucleotide differences that eliminate binding sites for myocyte enhancer factor 2 (MEF2) and serum response factor (SRF) account for this difference. Despite these differences, we show that MyHC-IIb mRNA is expressed in fetal human muscle cells and that MyHC-IIb mRNA is significantly up-regulated in the skeletal muscle of Duchene muscular dystrophy patients. CONCLUSIONS: These data identify the genetic basis for a key phenotypic difference between the muscles of large and small mammals, and demonstrate that mRNA expression of the MyHC-IIb gene can be re-activated in human limb muscle undergoing profound degeneration/regeneration.

Expression and function of myostatin in obesity, diabetes, and exercise adaptation.

Myostatin is a member of the transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) superfamily of secreted factors that functions as a potent inhibitor of skeletal muscle growth. Moreover, considerable evidence has accumulated that myostatin also regulates metabolism and that its inhibition can significantly attenuate the progression of obesity and diabetes. Although at least part of these effects on metabolism can be attributable to myostatin's influence over skeletal muscle growth and therefore on the total volume of metabolically active lean body mass, there is mounting evidence that myostatin affects the growth and metabolic state of other tissues, including the adipose and the liver. In addition, recent work has explored the role of myostatin in substrate mobilization, uptake, and/or utilization of muscle independent of its effects on body composition. Finally, the effects of both endurance and resistance exercise on myostatin expression, as well as the potential role of myostatin in the beneficial metabolic adaptations occurring in response to exercise, have also begun to be delineated in greater detail. The purpose of this review was to summarize the work to date on the expression and function of myostatin in obesity, diabetes, and exercise adaptation.

Matrix metalloproteinase-9 deficiency results in decreased fiber cross-sectional area and alters fiber type distribution in mouse hindlimb skeletal muscle.

Matrix metalloproteinases (MMPs) play a major role in the degradation of the extracellular matrix (ECM) of skeletal muscle, and the inducible gelatinase MMP-9 in particular appears to be critical for the remodeling of muscle ECM during growth and repair. Here we determined the effects of MMP-9 gene inactivation on fiber type and size in the tibialis anterior (TA), gastrocnemius (GAST), and soleus (SOL) muscles in female mice. In the TA, the cross-sectional area (CSA) of the myosin heavy chain (MyHC) IIb-expressing fibers was significantly smaller in MMP-9 null mice while in the GAST, CSA of all three fast fiber types was decreased. In the SOL, MyHC type I-expressing fibers were significantly smaller in the MMP-9 null mice. The percentage of MyHC type IIb-expressing fibers was significantly increased in the TA and GAST of MMP-9 null mice, while the percentage of MyHC IId-expressing fibers significantly decreased in the GAST of MMP-9 null mice. Fiber percentages in the SOL were not significantly different between the two lines. Despite these changes in fiber size and type, in vivo hindlimb force production was not changed in MMP-9 null mice. Meanwhile, neither expression of the constitutive gelatinase MMP-2 nor immunohistochemical staining for type IV collagen was significantly altered by MMP-9 inactivation in any muscles examined. The present study demonstrates that MMP-9 inactivation results in changes in fiber size and type in adult mouse hindlimb muscles that may depend on indirect mechanisms involving reduced bone growth or nerve changes in response to MMP-9 inactivation.

Plasma matrix metalloproteinase-9 response to eccentric exercise of the elbow flexors.

Recent efforts to establish a role for plasma matrix metalloproteinase-9 (MMP-9) as a marker of exercise-induced muscle damage have been inconsistent. Methodological and experimental design issues have contributed to confusion in this area. The purpose of this study was to use a damaging eccentric arm task to evaluate the relationship between activity-induced muscle damage and plasma MMP-9 levels in humans while controlling for physical activity history and quantifying day-to-day variability of the dependent variables. Fourteen physically inactive males performed 6 sets of 10 eccentric contractions of the elbow flexors at 120% of their voluntary concentric maximum. Soreness ratings, maximum voluntary isometric strength, range of motion (ROM), limb circumference, and plasma creatine kinase (CK) and MMP-9 levels were measured at 2 time points before, immediately after, and 1, 2, 4, and 7 days post-exercise. Changes in traditional markers of muscle damage mirrored patterns previously reported in the literature, but plasma MMP-9 concentration and activity measured by ELISA and gelatin zymography were unchanged at all time points examined. Plasma levels of the MMP-9 inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), were also unchanged post-exercise. Finally, although mean MMP-9 levels were not significantly different between the two pre-exercise timepoints, the high total error of measurement and low day-to-day correlation suggest substantial within and between subject variability. Plasma MMP-9 levels are not a robust or reliable marker for eccentric exercise-induced damage of the elbow flexor musculature, though this may not preclude a role for MMPs in skeletal muscle remodeling in response to injury.

Posttranscriptional mechanisms involving microRNA-27a and b contribute to fast-specific and glucocorticoid-mediated myostatin expression in skeletal muscle.

Expression of the antigrowth factor myostatin (MSTN) differs between fast and slow skeletal muscles and is increased in nearly every form of muscle atrophy, but the contribution of transcriptional vs. posttranscriptional mechanisms to its differing expression in these states has not been defined. We show here that levels of mature MSTN mRNA were sixfold greater in fast vs. slow muscle and were increased twofold in fast muscle in response to dexamethasone (Dex) injection in vivo and in C2C12 myotubes following Dex treatment in vitro, but that levels of MSTN pre-mRNA, a readout of transcription, only minimally and nonsignificantly differed in these states. Moreover, Dex treatment with or without cotransfection with a glucocorticoid receptor expression construct had only modest effects on mouse MSTN promoter activity in C2C12 myotubes. We therefore explored the potential contribution of posttranscriptional mechanisms, and the role of the microRNAs miR-27a and b in particular, on MSTN expression. The MSTN 3'-untranslated region (UTR) contains a putative recognition sequence for miR-27a and b that is conserved across a wide range of vertebrate species. Cotransfection of a MSTN 3'-UTR-luciferase construct with a miR-27b expression construct significantly attenuated by approximately half while mutation of the miR-27 recognition sequence significantly increased by approximately twofold the activity of a MSTN 3'-UTR construct and decreased mRNA degradation of a luciferase reporter construct in C2C12 myotubes. Expression of miR-27a and b was almost sixfold greater in slow-twitch than in fast-twitch muscle in vivo, and miR-27a expression was significantly decreased by nearly half by glucocorticoid treatment in vitro. Finally, the miR-27a and b promoters were activated by cotransfection with the slow-specific signaling molecules calcineurin and peroxisome proliferator-activated receptor-γ coactivator-1α. The present data represent the first demonstration that posttranscriptional mechanisms involving miR-27a and b may contribute to fast-specific and glucocorticoid-dependent myostatin expression in muscle.

CCAAT/enhancer binding protein-delta expression is increased in fast skeletal muscle by food deprivation and regulates myostatin transcription in vitro.

We recently demonstrated that mRNA levels of three members of the CCAAT/enhancer binding factor (C/EBP) family of transcription factors are increased in skeletal muscle following 12 days of spaceflight. In the present study, we further explored the expression of C/EBP-δ in atrophying fast skeletal muscle by examining its expression in muscle from food-deprived (FD) mice, and investigated its role in regulating the expression of the secreted antigrowth factor myostatin. C/EBP-δ mRNA and protein levels were significantly increased by 2 days of food deprivation in the tibialis anterior (TA) muscle, and expression of both myostatin and C/EBP-δ mRNA during food deprivation was attenuated by injection with the glucocorticoid inhibitor RU486. The increase in myostatin mRNA levels with food deprivation appears to be at least partially transcriptionally driven, since levels of myostatin pre-mRNA were significantly increased in the TA muscle. C/EBP-δ mRNA levels and promoter activity were significantly increased by transfection of C(2)C(12) myotubes with a glucocorticoid receptor construct and 24 h of treatment with the synthetic glucocorticoid dexamethasone. Furthermore, activity of the C/EBP-δ promoter was significantly increased with as little as 1 h of dexamethasone treatment, while activity of the mouse myostatin promoter was only significantly increased with longer treatment periods of 24 h or more. Activity of the myostatin promoter-reporter construct was significantly increased in C(2)C(12) myotubes by cotransfection with expression constructs for C/EBP-α, -ß, and -δ, with C/EBP-δ having the greatest effect. The myostatin promoter contains two potential C/EBP binding sequences, a CCAAT box, and a C/EBP binding element (CBE). Mutation of the CCAAT box attenuated basal myostatin promoter activity but potentiated C/EBP-δ-activated myostatin promoter activity in C(2)C(12) myotubes in vitro, while mutation of the CBE abolished glucocorticoid receptor and C/EBP-δ responsiveness. The present results support a model in which glucocorticoid-induced increases in C/EBP-δ expression may contribute to myostatin transcription during atrophic states.

Myostatin expression is increased by food deprivation in a muscle-specific manner and contributes to muscle atrophy during prolonged food deprivation in mice.

During food deprivation (FD), skeletal muscle protein is broken down to produce amino acids for hepatic gluconeogenesis to maintain blood glucose levels. However, it is unclear what role, if any, the secreted antigrowth factor myostatin (MSTN) plays in the muscle atrophy induced by FD. We therefore examined expression and function of MSTN in FD in mice. Two days of FD significantly decreased muscle mass and protein content and increased mRNA levels of ubiquitin ligases MuRF-1 and atrogin-1 in fast-twitch tibialis anterior (TA) muscle but not slow-twitch soleus (Sol) muscle, while 2 days of refeeding returned these to fed values in TA. MSTN mRNA levels were significantly increased approximately threefold by 2 days, but not 1 day, of FD and returned to fed levels with 2 days of refeeding in TA but were not significantly affected by FD or refeeding in Sol. TA mass decreased to a similar amount after 1 day of FD in wild-type mice and mice null for the MSTN gene but was decreased to a greater amount in wild-type than MSTN-null mice by 2 days of FD. In addition, blood glucose levels decreased and corticosterone levels increased to a greater extent in MSTN-null mice after 2 days of FD, but surprisingly muscle MuRF-1 and atrogin-1 mRNA levels were not affected by the lack of MSTN during FD. Similarly, changes in hepatic enzyme expression in response to FD were identical between wild-type and MSTN-null mice. Our data are consistent with the hypothesis that MSTN is dispensable for the initial atrophy occurring in response to FD but attenuates the decrease in fast-twitch muscle mass during prolonged FD.

Skeletal muscle expression of bone morphogenetic protein-1 and tolloid-like-1 extracellular proteases in different fiber types and in response to unloading, food deprivation and differentiation.

Members of the bone morphogenetic protein-1/mammalian tolloid (BMP-1/mTLD) family of proteases cleave diverse extracellular proteins, including the growth inhibitor myostatin. The purpose of this work was to examine the expression of BMP-1/mTLD, tolloid-like-1 and -2 (TLL1 and TLL2) in hindlimb muscles of the mouse in vivo and in C(2)C(12) muscle cells in vitro. Quantitative real-time polymerase chain reaction revealed that neither BMP-1/mTLD nor TLL1 mRNA levels differed between the predominantly fast-twitch tibialis anterior (TA) and gastrocnemius (GAST) muscles and the more slow-twitch soleus (SOL) muscle; TLL2 mRNA levels were not detectable in any of the muscles examined. Interestingly, however, immunohistochemical analysis revealed that BMP-1 protein was expressed in type I and IIa but not in IIb fibers. TLL1 mRNA levels significantly increased in the TA but not the SOL with 3 days of hindlimb suspension and significantly decreased in both TA and SOL in response to 2 days of food deprivation. In contrast, BMP-1/mTLD mRNA levels were unaffected in either muscle by either condition. In addition, BMP-1/mTLD and TLL1 mRNA levels significantly decreased during C(2)C(12) myoblast differentiation in vitro, and activity of a 1,200-bp mouse TLL1 promoter construct was significantly decreased in C(2)C(12) myotubes by differentiation, by mutation of an nuclear factor kappa-beta (NF-kappaB) site, or deletion of a sma/mothers against decapentaplegic (SMAD) site. Together, these data demonstrate that TLL1 mRNA levels are altered by loading, energy status, and differentiation, and thus its expression may be regulated so as to modulate activity of myostatin or other extracellular substrates during these adaptive states.

Acute daily psychological stress causes increased atrophic gene expression and myostatin-dependent muscle atrophy.

Psychological stress is known to attenuate body size and lean body mass. We tested the effects of 1, 3, or 7 days of two different models of psychological stress, 1 h of daily restraint stress (RS) or daily cage-switching stress (CS), on skeletal muscle size and atrophy-associated gene expression in mice. Thymus weights decreased in both RS and CS mice compared with unstressed controls, suggesting that both models activated the hypothalamic-pituitary-adrenal axis. Body mass was significantly decreased at all time points for both models of stress but was greater for RS than CS. Mass of the tibialis anterior (TA) and soleus (SOL) muscles was significantly decreased after 3 and 7 days of RS, but CS only significantly decreased SOL mass after 7 days. TA mRNA levels of the atrophy-associated genes myostatin (MSTN), atrogin-1, and the phosphatidylinositol 3-kinase inhibitory subunit p85alpha were all significantly increased relative to unstressed mice after 1 and 3 days of RS, and expression of MSTN and p85alpha mRNA remained elevated after 7 days of RS. Expression of muscle ring finger 1 was increased after 1 day of RS but returned to baseline at 3 and 7 days of RS. MSTN, atrogin-1, and p85alpha mRNA levels also significantly increased after 1 and 3 days of CS but atrogen-1 mRNA levels had resolved back to normal levels by 3 days and p85alpha with 7 days of CS. p21CIP mRNA levels were significantly decreased by 3 days of CS or RS. Finally, body mass was minimally affected, and muscle mass was completely unaffected by 3 days of RS in mice null for the MSTN gene, and MSTN inactivation attenuated the increase in atrogin-1 mRNA levels with 4 days of RS compared with wild-type mice. Together these data suggest that acute daily psychological stress induces atrophic gene expression and loss of muscle mass that appears to be MSTN dependent.

Calcineurin activates interleukin-6 transcription in mouse skeletal muscle in vivo and in C2C12 myotubes in vitro.

Expression of the cytokine interleukin-6 (IL-6) by skeletal muscle is hugely increased in response to a single bout of endurance exercise, and this appears to be mediated by increases in intracellular calcium. We examined the effects of endurance exercise on IL-6 mRNA levels and promoter activity in skeletal muscle in vivo, and the role of the calcium-activated calcineurin signaling pathway on muscle IL-6 expression in vivo and in vitro. IL-6 mRNA levels in the mouse tibialis anterior (TA) were increased 2-10-fold by a single bout of treadmill exercise or by 3 days of voluntary wheel running. Moreover, an IL-6 promoter-driven luciferase transgene was activated in TA by both treadmill and wheel-running exercise and by injection with a calcineurin plasmid. Exercise also increased muscle mRNA expression of the calcineurin regulatory gene MCIP1, as did treatment of C(2)C(12) myotubes with the calcium ionophore A23187. Cotransfection of C(2)C(12) myotubes with a constitutively active calcineurin construct significantly increased while cotransfection with the calcineurin inhibitor CAIN inhibited activity of a mouse IL-6 promoter-reporter construct. Cotransfection with a myocyte enhancer-factor-2 (MEF-2) expression construct increased basal IL-6 promoter activity and augmented the effects of calcineurin cotransfection, while cotransfection with the MEF-2 antagonist MITR repressed calcineurin-activated IL-6 promoter activity in vitro. Surprisingly, cotransfection with a dominant-negative form of another calcineurin-activated transcription factor, nuclear factor activator of T cells (NFAT), greatly potentiated both basal and calcineurin-stimulated IL-6 promoter activity in C(2)C(12) myotubes. Mutation of the MEF-2 DNA binding sites attenuated, while mutation of the NFAT DNA binding sites potentiated basal and calcineurin-activated IL-6 promoter activity. Finally, CREB and C/EBP were necessary for basal IL-6 promoter activity and sufficient to increase IL-6 promoter activity but had minimal roles in calcineurin-activated IL-6 promoter activity. Together, these results suggest that IL-6 transcription in skeletal muscle cells can be activated by a calcineurin-MEF-2 axis which is antagonized by NFAT.

The oxazolidinone derivative locostatin induces cytokine appeasement.

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF-alpha production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

Effects of spaceflight on murine skeletal muscle gene expression.

Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85alpha, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.

Genomic analysis reveals a point mutation in the two-component sensor gene graS that leads to intermediate vancomycin resistance in clinical Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), once restricted to hospitals, is spreading rapidly through the wider community. Resistance to vancomycin, the principal drug used to treat MRSA infections, has only recently emerged, is mainly low level, and characteristically appears during vancomycin therapy (vancomycin-intermediate S. aureus [VISA] and hetero-resistant VISA). This phenomenon suggests the adaptation of MRSA through mutation, although defining the mutations leading to resistance in clinical isolates has been difficult. We studied a vancomycin-susceptible clinical MRSA isolate (MIC of 1 microg/ml) and compared it with an isogenic blood culture isolate from the same patient, despite 42 days of vancomycin treatment (MIC of 4 microg/ml). A whole-genome sequencing approach allowed the nearly complete assembly of the genome sequences of the two isolates and revealed only six nucleotide substitutions in the VISA strain compared with the parent strain. One mutation occurred in graS, encoding a putative two-component regulatory sensor, leading to a change from a polar to a nonpolar amino acid (T136I) in the conserved histidine region of the predicted protein. Replacing the graS allele of the vancomycin-susceptible parent strain with the graS allele from the VISA derivative resulted in increased vancomycin resistance at a level between those of the vancomycin-susceptible S. aureus and VISA clinical isolates, confirming a role for graRS in VISA. Our study suggests that MRSA is able to develop clinically significant vancomycin resistance via a single point mutation, and the two-component regulatory system graRS is a key mediator of this resistance. However, additional mutations are likely required to express the full VISA phenotype.