Smyd1: Implications for novel approaches in rhabdomyosarcoma therapy.

Rhabdomyosarcoma (RMS), a tumor that consists of poorly differentiated skeletal muscle cells, is the most common soft-tissue sarcoma in children. Despite considerable progress within the last decades, therapeutic options are still limited, warranting the need for novel approaches. Recent data suggest deregulation of the Smyd1 protein, a sumoylation target as well as H3K4me2/3 methyltransferase and transcriptional regulator in myogenesis, and its binding partner skNAC, in RMS cells. Here, we show that despite the fact that most RMS cells express at least low levels of Smyd1 and skNAC, failure to upregulate expression of these genes in reaction to differentiation-promoting signals can always be observed. While overexpression of the Smyd1 gene enhances many aspects of RMS cell differentiation and inhibits proliferation rate and metastatic potential of these cells, functional integrity of the putative Smyd1 sumoylation motif and its SET domain, the latter being crucial for HMT activity, appear to be prerequisites for most of these effects. Based on these findings, we explored the potential for novel RMS therapeutic strategies, employing small-molecule compounds to enhance Smyd1 activity. In particular, we tested manipulation of (a) Smyd1 sumoylation, (b) stability of H3K4me2/3 marks, and (c) calpain activity, with calpains being important targets of Smyd1 in myogenesis. We found that specifically the last strategy might represent a promising approach, given that suitable small-molecule compounds will be available for clinical use in the future.

Stability of Smyd1 in endothelial cells is controlled by PML-dependent SUMOylation upon cytokine stimulation.

Smyd1 is an epigenetic modulator of gene expression that has been well-characterized in muscle cells. It was recently reported that Smyd1 levels are modulated by inflammatory processes. Since inflammation affects the vascular endothelium, this study aimed to characterize Smyd1 expression in endothelial cells. We detected Smyd1 in human endothelial cells (HUVEC and EA.hy926 cells), where the protein was largely localized in PML nuclear bodies (PML-NBs). By transfection of EA.hy926 cells with expression vectors encoding Smyd1, PML, SUMO1, active or mutant forms of the SUMO protease SuPr1 and/or the SUMO-conjugation enzyme UBC9, as well as Smyd1- or PML-specific siRNAs, in the presence or absence of the translation blocker cycloheximide or the proteasome-inhibitor MG132, and supported by computational modeling, we show that Smyd1 is SUMOylated in a PML-dependent manner and thereby addressed for degradation in proteasomes. Furthermore, transfection with Smyd1-encoding vectors led to PML up-regulation at the mRNA level, while PML transfection lowered Smyd1 protein stability. Incubation of EA.hy926 cells with the pro-inflammatory cytokine TNF-α resulted in a constant increase in Smyd1 mRNA and protein over 24 h, while incubation with IFN-γ induced a transient increase in Smyd1 expression, which peaked at 6 h and decreased to control values within 24 h. The IFN-γ-induced increase in Smyd1 was accompanied by more Smyd1 SUMOylation and more/larger PML-NBs. In conclusion, our data indicate that in endothelial cells, Smyd1 levels are regulated through a negative feedback mechanism based on SUMOylation and PML availability. This molecular control loop is stimulated by various cytokines.

Implementation and evaluation of an individualized physical exercise promotion program in people with manifested risk factors for multimorbidity (MultiPill-Exercise): a study protocol for a pragmatic randomized controlled trial.

BACKGROUND: Multimorbidity is a major problem in Europe, increasing the need for prevention and rehabilitation programs. In Germany no guidelines have been developed that focus on patients with multiple chronic non-communicable diseases (NCDs). Benefits of physical activity (PA) and exercise in NCDs have been proven, but most interventions focus on single conditions. The evaluation of the effectiveness, efficiency and safety of PA programs in patients suffering from multiple NCDs and the feasibility of the implementation within the health care service remain open research questions. METHODS: The multi-site randomized controlled pragmatic trial includes 320 sedentary subjects with at least two of the following NCDs, either manifested or in a pre-stage with evident risk factors: Cardio-vascular disease, Diabetes mellitus type 2, knee/ hip osteoarthritis and obesity. Participants will be recruited from general practitioners and medical specialists and randomized to standard care of a statutory health insurance or MultiPill-Exercise. Standard care includes a choice of one or a maximum of two 8- to 12-week health programs, including nutrition, exercise, relaxation or special disease management programs. MultiPill-Exercise is based on the bio-psycho-social health model, considering a person-oriented perspective in light of given individual characteristics and context factors. The 24-weeks intervention focuses on aerobic and strengthening exercises in line with the WHO PA recommendations. Psychological and pedagogical elements along with behavior change techniques are implemented to ease the initiation and maintenance of exercise participation and lifestyle change, including nutrition. Primary outcome will be short- and long-term PA measured with the European Health Interview Survey-Physical Activity Questionnaire (EHIS-PAQ). Secondly, the effectiveness of the program on generic, disease specific, economic, and exercise behavioral parameters, as well as program adherence and safety will be evaluated. DISCUSSION: Results of this trial evaluate the PA intervention program in people with multiple NCDs in a real-life scenario. It will serve as a proof of concept with the opportunity of translation into routine practice. This approach, as a multi-site RCT with its rigorous methods and standardized operating procedures for the conduction of the intervention, will allow valid conclusions for the implementation of PA interventions in people with multimorbidity. TRIAL REGISTRATION: The trial was registered at www.drks.de (ID: DRKS00025033 ) on 30th September 2021.

The ROS scavenger PDTC affects adaptation to treadmill running in mice: distinct effects on murine body mass, resting heart rate and skeletal muscle fiber type composition.

Regular exercise induces a broad spectrum of adaptation reactions in a variety of tissues and organs. However, the respective mechanisms are incompletely understood. In the context of their analysis, animal model systems, specifically rodent treadmill running protocols, play an important role. However, few researchers have studied different aspects of adaptation, such as cardiorespiratory and skeletal muscle training effects, within one set of experiments. Here, we analyzed physiological adaptation to 10 weeks of regular, moderate-intensity, uphill treadmill running in mice, a widely used model for endurance exercise training. To study the effects of reactive oxygen species (ROS), which have been suggested to be major regulators of training adaptation, a subgroup of mice was treated with the ROS scavenger PDTC (pyrrolidine dithiocarbamate). We found that mass gain in mice that exercised under PDTC treatment lagged behind that of all other experimental groups. In addition, both exercise and PDTC significantly and additively decreased resting heart rate. Furthermore, there was a trend towards an enhanced proportion of type 2A skeletal muscle fibers and differential expression of metabolism-associated genes, indicating metabolic and functional adaptation of skeletal muscle fibers. By contrast, there were no effects on grip strength and relative mass of individual muscles, suggesting that our protocol of uphill running did not increase skeletal muscle hypertrophy and strength. Taken together, our data suggest that a standard protocol of moderate-intensity uphill running induces adaptation reactions at multiple levels, part of which might be modulated by ROS, but does not enhance skeletal muscle hypertrophy and force.

Individual cardiovascular responsiveness to work-matched exercise within the moderate- and severe-intensity domains.

PURPOSE: We investigated the cardiovascular individual response to 6 weeks (3×/week) of work-matched within the severe-intensity domain (high-intensity interval training, HIIT) or moderate-intensity domain (moderate-intensity continuous training, MICT). In addition, we analyzed the cardiovascular factors at baseline underlying the response variability. METHODS: 42 healthy sedentary participants were randomly assigned to HIIT or MICT. We applied the region of practical equivalence-method for identifying the levels of responders to the maximal oxygen uptake (V̇O2max) response. For investigating the influence of cardiovascular markers, we trained a Bayesian machine learning model on cardiovascular markers. RESULTS: Despite that HIIT and MICT induced significant increases in V̇O2max, HIIT had greater improvements than MICT (p < 0.001). Greater variability was observed in MICT, with approximately 50% classified as "non-responder" and "undecided". 20 "responders", one "undecided" and no "non-responders" were observed in HIIT. The variability in the ∆V̇O2max was associated with initial cardiorespiratory fitness, arterial stiffness, and left-ventricular (LV) mass and LV end-diastolic diameter in HIIT; whereas, microvascular responsiveness and right-ventricular (RV) excursion velocity showed a significant association in MICT. CONCLUSION: Our findings highlight the critical influence of exercise-intensity domains and biological variability on the individual V̇O2max response. The incidence of "non-responders" in MICT was one third of the group; whereas, no "non-responders" were observed in HIIT. The incidence of "responders" was 11 out of 21 participants in MICT, and 20 out of 21 participants in HIIT. The response in HIIT showed associations with baseline fitness, arterial stiffness, and LV-morphology; whereas, it was associated with RV systolic function in MICT.

TRAF6: A player in CVB3-induced myocarditis?

Coxsackievirus B3 (CVB3) is an important inducer of myocarditis, which, in susceptible individuals, can chronify and eventually lead to the development of dilated cardiomyopathy and heart failure. The respective mechanisms are not completely understood. Here, we analyzed expression of the TRAF6 gene, encoding TNF receptor-associated factor 6 (TRAF6), a signal transduction scaffold protein that acts downstream of cytokine receptors, in heart tissue of susceptible and non-susceptible mouse strains. We found that after infection, TRAF6 expression was upregulated in both non-susceptible C57BL/6 wildtype and susceptible A.BY/SnJ and C57BL/6-TLR3 (-/-) mice, however, to different degrees. In infected HeLa cells, we also found moderately elevated TRAF6 levels after infection, in addition, activity of the transcription factor nuclear factor kappa B (NFκB), which can be activated downstream of TRAF6, was strongly enhanced in infected cells. To functionally analyze the role of TRAF6 with regard to infection progression, TRAF6 expression was knocked down in cultured HeLa cells using specific siRNAs. We found that reduction of TRAF6 expression had no effect on NFκB activation in response to infection. Taken together, our data suggest that CVB3 infection enhances TRAF6 levels, however, this induction might not be necessary for infection-induced NFκB activation.

Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells.

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

The E3 SUMO ligase Nse2 regulates sumoylation and nuclear-to-cytoplasmic translocation of skNAC-Smyd1 in myogenesis.

Skeletal and heart muscle-specific variant of the α subunit of nascent polypeptide associated complex (skNAC; encoded by NACA) is exclusively found in striated muscle cells. Its function, however, is largely unknown. Previous reports have demonstrated that skNAC binds to m-Bop/Smyd1, a multi-functional protein that regulates myogenesis both through the control of transcription and the modulation of sarcomerogenesis, and that both proteins undergo nuclear-to-cytoplasmic translocation at the later stages of myogenic differentiation. Here, we show that skNAC binds to the E3 SUMO ligase mammalian Mms21/Nse2 and that knockdown of Nse2 expression inhibits specific aspects of myogenic differentiation, accompanied by a partial blockade of the nuclear-to-cytoplasmic translocation of the skNAC-Smyd1 complex, retention of the complex in promyelocytic leukemia (PML)-like nuclear bodies and disturbed sarcomerogenesis. In addition, we show that the skNAC interaction partner Smyd1 contains a putative sumoylation motif and is sumoylated in muscle cells, with depletion of Mms21/Nse2 leading to reduced concentrations of sumoylated Smyd1. Taken together, our data suggest that the function, specifically the balance between the nuclear and cytosolic roles, of the skNAC-Smyd1 complex might be regulated by sumoylation.

skNAC and Smyd1 in transcriptional control.

Skeletal and heart muscle-specific variant of the alpha subunit of nascent polypeptide associated complex (skNAC) is exclusively found in striated muscle cells. Its function, however, is largely unknown. Previous reports could demonstrate that skNAC binds to Smyd1 (SET and MYND domain containing protein 1). The facts that (a) SET domains have histone methyltransferase activity, and (b) MYND domains are known recruiters of histone deacetylases (HDACs), implicate the skNAC-Smyd1 complex in transcriptional control. To study potential target genes, we carried out cDNA microarray analysis on differentiating C2C12 myoblasts in which expression of the skNAC gene had been knocked down. We found and confirmed a series of targets, specifically genes encoding regulators of inflammation, cellular metabolism, and cell migration. Mechanistically, as shown by Western blot, ELISA, and ChIP analysis at selected promoter regions, transcriptional control by skNAC-Smyd1 appears to be exerted at least in part by affecting a series of histone modifications, specifically H3K4 di- and trimethylation and potentially also histone acetylation. Taken together, our data suggest that the skNAC-Smyd1 complex is involved in transcriptional regulation both via the control of histone methylation and histone (de)acetylation.

Exercise, skeletal muscle and inflammation: ARE-binding proteins as key regulators in inflammatory and adaptive networks.

The role of inflammation in skeletal muscle adaptation to exercise is complex and has hardly been elucidated so far. While the acute inflammatory response to exercise seems to promote skeletal muscle training adaptation and regeneration, persistent, low-grade inflammation, as seen in a multitude of chronic diseases, is obviously detrimental. The regulation of cytokine production in skeletal muscle cells has been relatively well studied, yet little is known about the compensatory and anti-inflammatory mechanisms that resolve inflammation and restore tissue homeostasis. One important strategy to ensure sequential, timely and controlled resolution of inflammation relies on the regulated stability of mRNAs encoding pro-inflammatory mediators. Many key transcripts in early immune responses are characterized by the presence of AU-rich elements (AREs) in the 3'-untranslated regions of their mRNAs, allowing efficient fine-tuning of gene expression patterns at the post-transcriptional level. AREs exert their function by recruiting particular RNA-binding proteins, resulting, in most cases, in de-stabilization of the target transcripts. The best-characterized ARE-binding proteins are HuR, CUGBP1, KSRP, AUF1, and the three ZFP36 proteins, especially TTP/ZFP36. Here, we give a general introduction into the role of inflammation in the adaptation of skeletal muscle to exercise. Subsequently, we focus on potential roles of ARE-binding proteins in skeletal muscle tissue in general and specifically exercise-induced skeletal muscle remodeling. Finally, we present novel data suggesting a specific function of TTP/ZFP36 in exercise-induced skeletal muscle plasticity.

skNAC depletion stimulates myoblast migration and perturbs sarcomerogenesis by enhancing calpain 1 and 3 activity.

skNAC (skeletal and heart muscle specific variant of nascent polypeptide-associated complex α) is a skeletal and heart muscle-specific protein known to be involved in the regulation of sarcomerogenesis. The respective mechanism, however, is largely unknown. In the present paper, we demonstrate that skNAC regulates calpain activity. Specifically, we show that inhibition of skNAC gene expression leads to enhanced, and overexpression of the skNAC gene to repressed, activity of calpain 1 and, to a lesser extent, calpain 3 in myoblasts. In skNAC siRNA-treated cells, enhanced calpain activity is associated with increased migration rates, as well as with perturbed sarcomere architecture. Treatment of skNAC-knockdown cells with the calpain inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal) reverts both the positive effect on myoblast migration and the negative effect on sarcomere architecture. Taken together, our data suggest that skNAC controls myoblast migration and sarcomere architecture in a calpain-dependent manner.

Nicotinic acid adenine dinucleotide phosphate regulates skeletal muscle differentiation via action at two-pore channels.

Calcium signaling is essential for the differentiation of many cell types, including skeletal muscle cells, but its mechanisms remain elusive. Here we demonstrate a crucial role for nicotinic acid adenine dinucleotide phosphate (NAADP) signaling in skeletal muscle differentiation. Although the inositol trisphosphate pathway may have a partial role to play in this process, the ryanodine signaling cascade is not involved. In both skeletal muscle precursors and C2C12, cells interfering with NAADP signaling prevented differentiation, whereas promoting NAADP signaling potentiated differentiation. Moreover, siRNA knockdown of two-pore channels, the target of NAADP, attenuated differentiation. The data presented here strongly suggest that in myoblasts, NAADP acts at acidic organelles on the recently discovered two-pore channels to promote differentiation.

The effect of the NF-kappa B inhibitors curcumin and lactacystin on myogenic differentiation of rhabdomyosarcoma cells.

Rhabdomyosarcoma is a soft tissue sarcoma mainly seen in children. Despite considerable progress within the last few years, therapeutic approaches for this type of tumor are still limited. The respective tumor cells originate from myogenic precursor cells and are characterized by a blockade in their differentiation program. Interestingly, there is a direct inverse correlation between the differentiation status of a specific rhabdomyosarcoma cell and its metastatic potential. Thus, here, we tested whether the ubiquitous transcription factor NF-κB, which regulates myogenic differentiation and is also a promising therapeutic target in the treatment of other types of tumors, might be an interesting candidate for the development of novel rhabdomyosarcoma treatment strategies. For this purpose, we analyzed NF-κB activity (classical pathway) in myoblasts with different differentiation potential, specifically in three different rhabdomyosarcoma cell lines. In addition, we inhibited NF-κB activity in these cells and analyzed the effects on myogenic differentiation. We show that after the induction of differentiation, NF-κB activity declines rapidly in normal myoblasts, but only slightly in rhabdomyosarcoma cells. However, after treatment of the cells with two different small-molecule NF-κB-inhibiting compounds, the IKK inhibitor curcumin and the proteasome inhibitor lactacystin, we found that neither curcumin nor lactacystin promoted myogenic differentiation in either normal myoblasts or rhabdomyosarcoma cells. Taken together, our data suggest that treatment with curcumin or lactacystin might not be a suitable approach in the treatment of rhabdomyosarcoma.

Adipokines as Predictive Biomarkers for Training Adaptation in Subjects with Multimorbidity-A Hypothesis-Generating Study.

BACKGROUND: Physical exercise exerts a positive effect on many chronic conditions, specifically lifestyle-related diseases such as overweight and obesity, type 2 diabetes mellitus (T2DM), cardiovascular conditions and osteoarthritis (OA). As a result of common risk factors, most of these patients present with multiple conditions. Exercise- and disease-related biomarkers, such as adipokines, are emerging tools in training supervision and regulation; however, their significance in subjects with multimorbidities is unknown. SUBJECTS AND METHODS: To address this issue, adipokines leptin, adiponectin and resistin were assessed in a cohort of subjects with multimorbidities (n = 39) presenting with at least two of the abovementioned conditions or relevant risk factors before and after a six-month exercise and lifestyle intervention program ('MultiPill-Exercise'), and correlated with training adaptation, namely changes in relative maximum oxygen uptake (V·O2max). RESULTS: There was a significant negative correlation between baseline leptin concentrations and training effect for relative V·O2max (after three months: rho = -0.54, p = 0.020 *; after six months: rho = -0.45, p = 0.013 *), with baseline leptin explaining 35% of the variance in delta relative V·O2max after three months and 23% after six months. CONCLUSIONS: Leptin might be a suitable surrogate biomarker in the context of exercise-based lifestyle intervention programs in subjects with multimorbidity.

Skeletal Muscle MicroRNA Patterns in Response to a Single Bout of Exercise in Females: Biomarkers for Subsequent Training Adaptation?

microRNAs (miRs) have been proposed as a promising new class of biomarkers in the context of training adaptation. Using microarray analysis, we studied skeletal muscle miR patterns in sedentary young healthy females (n = 6) before and after a single submaximal bout of endurance exercise ('reference training'). Subsequently, participants were subjected to a structured training program, consisting of six weeks of moderate-intensity continuous endurance training (MICT) and six weeks of high-intensity interval training (HIIT) in randomized order. In vastus lateralis muscle, we found significant downregulation of myomiRs, specifically miR-1, 133a-3p, and -5p, -133b, and -499a-5p. Similarly, exercise-associated miRs-23a-3p, -378a-5p, -128-3p, -21-5p, -107, -27a-3p, -126-3p, and -152-3p were significantly downregulated, whereas miR-23a-5p was upregulated. Furthermore, in an untargeted approach for differential expression in response to acute exercise, we identified n = 35 miRs that were downregulated and n = 20 miRs that were upregulated by factor 4.5 or more. Remarkably, KEGG pathway analysis indicated central involvement of this set of miRs in fatty acid metabolism. To reproduce these data in a larger cohort of all-female subjects (n = 29), qPCR analysis was carried out on n = 15 miRs selected from the microarray, which confirmed their differential expression. Furthermore, the acute response, i.e., the difference between miR concentrations before and after the reference training, was correlated with changes in maximum oxygen uptake (V̇O2max) in response to the training program. Here, we found that miRs-199a-3p and -19b-3p might be suitable acute-response candidates that correlate with individual degrees of training adaptation in females.

c-Met is essential for wound healing in the skin.

Wound healing of the skin is a crucial regenerative process in adult mammals. We examined wound healing in conditional mutant mice, in which the c-Met gene that encodes the receptor of hepatocyte growth factor/scatter factor was mutated in the epidermis by cre recombinase. c-Met-deficient keratinocytes were unable to contribute to the reepithelialization of skin wounds. In conditional c-Met mutant mice, wound closure was slightly attenuated, but occurred exclusively by a few (5%) keratinocytes that had escaped recombination. This demonstrates that the wound process selected and amplified residual cells that express a functional c-Met receptor. We also cultured primary keratinocytes from the skin of conditional c-Met mutant mice and examined them in scratch wound assays. Again, closure of scratch wounds occurred by the few remaining c-Met-positive cells. Our data show that c-Met signaling not only controls cell growth and migration during embryogenesis but is also essential for the generation of the hyperproliferative epithelium in skin wounds, and thus for a fundamental regenerative process in the adult.

TRAF6 regulates proliferation and differentiation of skeletal myoblasts.

We could recently demonstrate an important role of receptor interacting protein-2 (RIP2), an activator of nuclear factor kappa B (NF-κB) and a target of activated receptors of the tumor necrosis factor receptor (TNFR) type, in myogenic differentiation and regeneration. Here, we analyze a potential role of TNFR associated factor 6 (TRAF6), which also associates with the cytoplasmic domain of TNFR type, but also IL-1-R and TLR type receptors, and activates NF-κB, in these processes. Specifically, we show that during myogenic differentiation in vitro, traf6 gene expression is downregulated in normal myoblasts, but not in rhabdomyosarcoma cells, suggesting a role of the TRAF6 protein in this process. Inhibition of traf6 expression using specific siRNAs led to an inhibition of both myoblast proliferation and differentiation, whereas inhibition of the TRAF6 effector NF-κB alone in our system only blocked proliferation. Finally, we demonstrate that the traf6 gene is downregulated in skeletal muscle tissue of the dystrophic mdx mouse. Taken together, these data argue for a role of TRAF6 in the regulation of skeletal muscle differentiation and regeneration.

Proof of Concept of a 6-Month Person-Oriented Exercise Intervention 'MultiPill-Exercise' among Patients at Risk of or with Multiple Chronic Diseases: Results of a One-Group Pilot Trial.

Physical exercise has been shown to be effective in the treatment of non-communicable chronic diseases. However, patients with multiple chronic diseases (multimorbidity) have received little attention in health policy. This pilot trial served as a proof of concept of a 6-months person-oriented exercise intervention for people at risk of or with diagnosed cardiovascular diseases, diabetes mellitus type 2, overweight and/or hip/knee osteoarthritis, regarding effects on health outcomes as well as adherence and safety. The intervention ('MultiPill-Exercise') was designed to promote physical exercise participation, considering an individual perspective by addressing personal and environmental factors. Outcomes were assessed at baseline (t0) and after three- (t3) and six-months (t6). The primary outcome was self-reported physical exercise participation in minutes/week comparing t3 and t6 vs. t0. Secondary outcomes included cardio-respiratory fitness (maximum oxygen uptake VO2peak during incremental cycling ergometry), isometric peak torque of knee extensors and flexors, health-related quality of life (Veterans Rand 12 with its subscales of perceived general health (GH), mental health (MCS), and physical health (PCS)) and blood levels. Adherence to exercise (% of attended sessions during the first 12-weeks of the intervention) and adverse events were monitored as well. Data were analyzed using a non-parametric procedure for longitudinal data, estimating rank means (MRank) and relative treatment effects (RTE) as well as linear-mixed effect models for parametric data. The primary endpoint of physical exercise participation was significantly higher at t3 and t6 compared to baseline (t3 vs. t0: MRank = 77.1, p < 0.001, RTE: 0.66; t6 vs. t0: MRank = 70.6, p < 0.001, RTE = 0.60). Improvements at both follow-up time points compared to t0 were also found for relative VO2peak (t3 vs. t0 = 2.6 mL/kg/min, p < 0.001; t6 vs. t0 = 2.0 mL/kg/min, p = 0.001), strength of knee extensors (t3 vs. t0 = 11.7 Nm, p = 0.007; t6 vs. t0= 18.1 Nm, p < 0.001) and GH (t3 vs. t0 = 16.2, p = 0.003; t6 vs. t0 = 13.4, p = 0.008). No changes were found for MCS, PCS and for blood levels. Overall exercise adherence was 77%. No serious adverse events were recorded. Results of this pilot trial represent a first proof of concept for the intervention 'MultiPill-Exercise' that will now be implemented and evaluated in a real-world health care setting.

miRNAs as markers for the development of individualized training regimens: A pilot study.

Small, non-coding RNAs (microRNAs) have been shown to regulate gene expression in response to exercise in various tissues and organs, thus possibly coordinating their adaptive response. Thus, it is likely that differential microRNA expression might be one of the factors that are responsible for different training responses of different individuals. Consequently, determining microRNA patterns might be a promising approach toward the development of individualized training strategies. However, little is known on (1) microRNA patterns and their regulation by different exercise regimens and (2) possible correlations between these patterns and individual training adaptation. Here, we present microarray data on skeletal muscle microRNA patterns in six young, female subjects before and after six weeks of either moderate-intensity continuous or high-intensity interval training on a bicycle ergometer. Our data show that n = 36 different microRNA species were regulated more than twofold in this cohort (n = 28 upregulated and n = 8 downregulated). In addition, we correlated baseline microRNA patterns with individual changes in VO2 max and identified some specific microRNAs that might be promising candidates for further testing and evaluation in the future, which might eventually lead to the establishment of microRNA marker panels that will allow individual recommendations for specific exercise regimens.

RIP4 is a target of multiple signal transduction pathways in keratinocytes: implications for epidermal differentiation and cutaneous wound repair.

Receptor interacting protein 4 (RIP4) is an important regulator of epidermal morphogenesis during embryonic development. We could previously show that expression of the rip4 gene is strongly downregulated in cutaneous wound repair, which might be initiated by a broad variety of growth factors and cytokines. Here, we demonstrate that in keratinocytes, rip4 expression is controlled by a multitude of different signal transduction pathways, such as the p38 mitogen-activated protein kinase (MAPK) and the nuclear factor kappa B (NF-kappaB) cascade, in a unique and specific manner. Furthermore, we show that the steroid dexamethasone abolishes the physiological rip4 downregulation after injury and might thus contribute to the phenotype of reduced and delayed wound reepithelialization seen in glucocorticoid-treated patients. As a whole, our data indicate that rip4 expression is regulated in a complex manner, which might have therapeutic implications.