OTUD5 limits replication fork instability by organizing chromatin remodelers.

Proper regulation of replication fork progression is important for genomic maintenance. Subverting the transcription-induced conflicts is crucial in preserving the integrity of replication forks. Various chromatin remodelers, such as histone chaperone and histone deacetylases are known to modulate replication stress, but how these factors are organized or collaborate are not well understood. Here we found a new role of the OTUD5 deubiquitinase in limiting replication stress. We found that OTUD5 is recruited to replication forks, and its depletion causes replication fork stress. Through its C-terminal disordered tail, OTUD5 assembles a complex containing FACT, HDAC1 and HDAC2 at replication forks. A cell line engineered to specifically uncouple FACT interaction with OTUD5 exhibits increases in FACT loading onto chromatin, R-loop formation, and replication fork stress. OTUD5 mediates these processes by recruiting and stabilizing HDAC1 and HDAC2, which decreases H4K16 acetylation and FACT recruitment. Finally, proteomic analysis revealed that the cells with deficient OTUD5-FACT interaction activates the Fanconi Anemia pathway for survival. Altogether, this study identified a new interaction network among OTUD5-FACT-HDAC1/2 that limits transcription-induced replication stress.

Design, synthesis and validation of a new Crimped Head-Piece for DNA-Encoded libraries generation.

Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.

Joining the PARty: PARP Regulation of KDM5A during DNA Repair (and Transcription?).

The lysine demethylase KDM5A collaborates with PARP1 and the histone variant macroH2A1.2 to modulate chromatin to promote DNA repair. Indeed, KDM5A engages poly(ADP-ribose) (PAR) chains at damage sites through a previously uncharacterized coiled-coil domain, a novel binding mode for PAR interactions. While KDM5A is a well-known transcriptional regulator, its function in DNA repair is only now emerging. Here we review the molecular mechanisms that regulate this PARP1-macroH2A1.2-KDM5A axis in DNA damage and consider the potential involvement of this pathway in transcription regulation and cancer. Using KDM5A as an example, we discuss how multifunctional chromatin proteins transition between several DNA-based processes, which must be coordinated to protect the integrity of the genome and epigenome. The dysregulation of chromatin and loss of genome integrity that is prevalent in human diseases including cancer may be related and could provide opportunities to target multitasking proteins with these pathways as therapeutic strategies.

Transcription-replication conflicts as a source of common fragile site instability caused by BMI1-RNF2 deficiency.

Common fragile sites (CFSs) are breakage-prone genomic loci, and are considered to be hotspots for genomic rearrangements frequently observed in cancers. Understanding the underlying mechanisms for CFS instability will lead to better insight on cancer etiology. Here we show that Polycomb group proteins BMI1 and RNF2 are suppressors of transcription-replication conflicts (TRCs) and CFS instability. Cells depleted of BMI1 or RNF2 showed slower replication forks and elevated fork stalling. These phenotypes are associated with increase occupancy of RNA Pol II (RNAPII) at CFSs, suggesting that the BMI1-RNF2 complex regulate RNAPII elongation at these fragile regions. Using proximity ligase assays, we showed that depleting BMI1 or RNF2 causes increased associations between RNAPII with EdU-labeled nascent forks and replisomes, suggesting increased TRC incidences. Increased occupancy of a fork protective factor FANCD2 and R-loop resolvase RNH1 at CFSs are observed in RNF2 CRISPR-KO cells, which are consistent with increased transcription-associated replication stress in RNF2-deficient cells. Depleting FANCD2 or FANCI proteins further increased genomic instability and cell death of the RNF2-deficient cells, suggesting that in the absence of RNF2, cells depend on these fork-protective factors for survival. These data suggest that the Polycomb proteins have non-canonical roles in suppressing TRC and preserving genomic integrity.

Active recovery vs hot- or cold-water immersion for repeated sprint ability after a strenuous exercise training session in elite skaters.

This study compared the acute effects of three recovery methods: active recovery (AR), hot- and cold-water immersion (HWI and CWI, respectively), used between two training sessions in elite athletes. Twelve national-team skaters (7 males, 5 females) completed three trials according to a randomized cross-over study. Fifteen minutes after an exhaustive ice-skating training session, participants underwent 20 min of HWI (41.1 ± 0.5°C), 15 min of CWI (12.1 ± 0.7°C) or 15 min of active recovery (AR). After 1 h 30 min of the first exercise, they performed a repeated-sprint cycling session. Average power output was slightly but significantly higher for AR (767 ± 179 W) and HWI (766 ± 170 W) compared to CWI (738 ± 156 W) (p = 0.026, d = 0.18). No statistical difference was observed between the conditions for both lactatemia and rating of perceived exertion. Furthermore, no significant effect of recovery was observed on the fatigue index calculated from the repeated sprint cycling exercises (p > 0.05). Finally, a positive correlation was found between the average muscle temperature measured during the recoveries and the maximal power output obtained during cycling exercises. In conclusion, the use of CWI in between high-intensity training sessions could slightly impair the performance outcomes compared to AR and HWI. However, studies with larger samples are needed to confirm these results, especially in less trained athletes.

Unveiling the Chemical Diversity of the Deep-Sea Sponge Characella pachastrelloides.

Sponges are at the forefront of marine natural product research. In the deep sea, extreme conditions have driven secondary metabolite pathway evolution such that we might expect deep-sea sponges to yield a broad range of unique natural products. Here, we investigate the chemodiversity of a deep-sea tetractinellid sponge, Characella pachastrelloides, collected from ~800 m depth in Irish waters. First, we analyzed the MS/MS data obtained from fractions of this sponge on the GNPS public online platform to guide our exploration of its chemodiversity. Novel glycolipopeptides named characellides were previously isolated from the sponge and herein cyanocobalamin, a manufactured form of vitamin B12, not previously found in nature, was isolated in a large amount. We also identified several poecillastrins from the molecular network, a class of polyketide known to exhibit cytotoxicity. Light sensitivity prevented the isolation and characterization of these polyketides, but their presence was confirmed by characteristic NMR and MS signals. Finally, we isolated the new betaine 6-methylhercynine, which contains a unique methylation at C-2 of the imidazole ring. This compound showed potent cytotoxicity towards against HeLa (cervical cancer) cells.

The OTUD5-UBR5 complex regulates FACT-mediated transcription at damaged chromatin.

Timely stalling and resumption of RNA polymerases at damaged chromatin are actively regulated processes. Prior work showed an importance of FACT histone chaperone in such process. Here we provide a new role of OTUD5 deubiquitinase in the FACT-dependent process. Through a DUB RNAi screen, we found OTUD5 as a specific stabilizer of the UBR5 E3 ligase. OTUD5 localizes to DNA double strand breaks (DSBs), interacts with UBR5 and represses the RNA Pol II elongation and RNA synthesis. OTUD5 co-localizes and interacts with the FACT component SPT16 and antagonizes the histone H2A deposition at DSB lesions. OTUD5 interacts with UBR5 and SPT16 independently through two distinct regions, and both interactions are necessary for arresting the Pol II elongation at lesions. These analyses suggested that the catalytic (through UBR5 stabilization) as well as scaffolding (through FACT binding) activities of OTUD5 are involved in the FACT-dependent transcription. We found that a cancer-associated missense mutation within the OTUD5 Ubiquitin Interacting Motif (UIM) abrogates the FACT association and the Pol II arrest, providing a possible link between the transcriptional regulation and tumor suppression. Our work establishes OTUD5 as a new regulator of the DNA damage response, and provides an insight into the FACT-dependent transcription at damaged chromatin.

Modelling performance with exponential functions in elite short-track speed skaters.

Mathematical models are used to describe and predict the effects of training on performance. The initial models are structured by impulse-type transfer functions, however, cellular adaptations induced by exercise may exhibit exponential kinetics for their growth and subsequent dissipation. Accumulation of exercise bouts counteracts dissipation and progressively induces structural and functional changes leading to performance improvement. This study examined the suitability of a model with exponential terms (Exp-Model) in elite short-track speed (ST) skaters. Training loads and performance evolution from fifteen athletes (10 males, 5 females) were previously collected over a 3-month training period. Here, we computed the relationship between training loads and performance with Exp-Model and compared with previous results obtained with a variable dose-response model (Imp-Model). Exp-Model showed a higher correlation between actual and modelled performances (R2 = 0.83 ± 0.08 and 0.76 ± 0.07 for Exp-Model and Imp-Model, respectively). Concerning model parameters, a higher τA1 (time constant for growth) value was found (p = 0.0047; d = 1.4; 95% CI [0.4;1.9]) in males compared to females with Exp-model, suggesting that females have a faster adaptative response to training loads. Thus, according to this study, Exp-model may better describe training adaptations in elite ST athletes than Imp-Model.

Molecular Regulation of Skeletal Muscle Growth and Organelle Biosynthesis: Practical Recommendations for Exercise Training.

The regulation of skeletal muscle mass and organelle homeostasis is dependent on the capacity of cells to produce proteins and to recycle cytosolic portions. In this investigation, the mechanisms involved in skeletal muscle mass regulation-especially those associated with proteosynthesis and with the production of new organelles-are presented. Thus, the critical roles of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway and its regulators are reviewed. In addition, the importance of ribosome biogenesis, satellite cells involvement, myonuclear accretion, and some major epigenetic modifications related to protein synthesis are discussed. Furthermore, several studies conducted on the topic of exercise training have recognized the central role of both endurance and resistance exercise to reorganize sarcomeric proteins and to improve the capacity of cells to build efficient organelles. The molecular mechanisms underlying these adaptations to exercise training are presented throughout this review and practical recommendations for exercise prescription are provided. A better understanding of the aforementioned cellular pathways is essential for both healthy and sick people to avoid inefficient prescriptions and to improve muscle function with emergent strategies (e.g., hypoxic training). Finally, current limitations in the literature and further perspectives, notably on epigenetic mechanisms, are provided to encourage additional investigations on this topic.

Influence of post-exercise hot-water therapy on adaptations to training over 4 weeks in elite short-track speed skaters.

This study aimed to investigate the effects of regular hot water bathing (HWB), undertaken 10 min after the last training session of the day, on chronic adaptations to training in elite athletes. Six short-track (ST) speed skaters completed four weeks of post-training HWB and four weeks of post-training passive recovery (PR) according to a randomized cross-over study. During HWB, participants sat in a jacuzzi (40 °C; 20 min). According to linear mixed models, maximal isometric strength of knee extensor muscles was significantly increased for training with HWB (p < 0.0001; d = 0.41) and a tendency (p = 0.0529) was observed concerning V ˙ O 2 m a x . No significant effect of training with PR or HWB was observed for several variables (p > 0.05), including aerobic peak power output, the decline rate of jump height during 1 min-continuous maximal countermovement jumps (i.e. anaerobic capacity index), and the force-velocity relationship. Regarding specific tasks on ice, a small effect of training was found on both half-lap time and total time during a 1.5-lap all-out exercise (p = 0.0487; d = 0.23 and p = 0.0332; d = 0.21, respectively) but no additional effect of HWB was observed. In summary, the regular HWB protocol used in this study can induce additional effects on maximal isometric strength without compromising aerobic and anaerobic adaptations or field performance in these athletes.

Glenoid Track Instability Management Score: Radiographic Modification of the Instability Severity Index Score.

PURPOSE: The purpose of this study is (1) to test the proposed treatment algorithm, the Glenoid Track Instability Management Score (GTIMS), which incorporates the glenoid track concept into the instability severity index score (ISIS), and (2) to compare treatment decision-making using either GTIMS versus ISIS in 2 cohorts of patients with operatively treated anterior instability. METHODS: A multicenter, retrospective review of two consecutive groups consisting of 72 and 189 patients treated according to ISIS and GTIMS, respectively, was conducted. Inclusion criteria for all patients were ≥2 confirmed traumatic anterior shoulder instability events and a physical examination demonstrating a positive anterior apprehension and relocation test. The GTIMS was graded for all 189 patients in the cohort, which uses 3-dimensional computed tomography as the sole radiographic parameter to assess on-track (0 points) versus off-track (4 points) Hill-Sachs lesions. This method differs from ISIS, which uses multiple plain radiographs for the 4-point imaging portion of the score. Outcomes scores were compared within the GTIMS and ISIS groups, as well as between them for overall comparisons based on the Western Ontario Shoulder Instability Index (WOSI), the Single Assessment Numerical Evaluation (SANE) score, and the mean rates of recurrent instability. RESULTS: A total of 261 consecutive patients from 2009 to 2014 who presented with recurrent anterior shoulder instability were treated according to either ISIS (n = 72/261, 27.6%) or GTIMS (n = 189/261, 72.4%). At a mean follow-up time of 33.2 months (range 24-49 months), the overall cohort mean ISIS of 2.9 ± 2.2 (range 0-9) was significantly higher than the mean GTIMS of 1.9 ± 1.9 (range = 0-9, P < .001). Of the 72 ISIS treated patients, 50 (69.4%) had an ISIS score of ≥ 4 and underwent a Latarjet, and the 22 patients (30.6%) with an ISIS score of < 4 underwent an arthroscopic Bankart repair. Based on GTIMS in the 189-patient cohort, using the same cutoff of 4 to indicate the need for a Latarjet, 162 patients were treated with arthroscopic Bankart repair (85.7%) and 27 with Latarjet (14.3%). The overall outcomes improved for patients treated with a Latarjet in both groups (GTIMS WOSI from 1099 [47.7% normal] to 395 [81.3% normal]; GTIMS SANE from 48 to 81; ISIS WOSI from 1050 [50% normal] to 345 [83.4% normal]; ISIS SANE from 50 to 84; P < .01). Similar positive outcomes were seen in patients treated with arthroscopic Bankart repair (GTIMS WOSI from 1062 [49.2% normal] to 402 [80.6% normal]; GTIMS SANE from 49 to 82; ISIS WOSI from 1080 [51.8% normal] to 490 [76.7% normal]; ISIS SANE from 48 to 77; P < .01). Of note, the patients with arthroscopically indicated ISIS had significantly worse outcomes scores than those treated arthroscopically according to GTIMS (P < .01). Of the 189 patients graded with GTIMS, there would have been 33 more Latarjet procedures recommended based on ISIS score. Thus the distribution of procedures based on ISIS versus GTIMS was significantly different (χ2 = 45.950; P < .001), indicating a higher rate of recommending Latarjets when using ISIS versus GTIMS. CONCLUSIONS: When ISIS scoring and plain radiograph parameters only are used, this predicted a 2-fold increase in recommending a Latarjet versus GTIMS scoring criteria, which uses advanced imaging and the on- and off-track principle to more conservatively delineate anterior instability treatment with promising postoperative patient outcomes. Overall, there were minimal differences in outcomes between GTIMS and ISIS Latarjet patients; however, better outcomes were seen in patients indicated for arthroscopic Bankart repair according to GTIMS and on-off track computed tomography scanning indications. LEVEL OF EVIDENCE: II, Prospective Cohort Study.

Increase in muscle power is associated with myofibrillar ATPase adaptations during resistance training.

NEW FINDINGS: What is the central question of this study? The aim of this study was to examine the effects of resistance training on gains in the external mechanical power output developed during climbing and myofibrillar ATPase activity in rats. What is the main finding and its importance? Using rapid flow quench experiments, we show that resistance training increases both the power output and the myofibrillar ATPase activity in the flexor digitorum profundus, biceps and deltoid muscles. Data fitting reveals that these functional ameliorations are explained by an increase in the rate constant of liberation of ATP hydrolysis products and contribute to performance gains. ABSTRACT: Skeletal muscle shows a remarkable plasticity that permits functional adaptations in response to different stimulations. To date, modifications of the proportions of myosin heavy chain (MHC) isoforms and increases in fibre size are considered to be the main factors providing sarcomeric plasticity in response to exercise training. In this study, we investigated the effects of a resistance training protocol on the myofibrillar ATPase (m-ATPase) cycle, muscle performance (power output) and MHC gene expression. For this purpose, 8-week-old Wistar Han rats were subjected to 4 weeks of resistance training, with five sessions per week. Muscle samples of flexor digitorum profundus (FDP), biceps and deltoid were collected and subjected to RT-qPCR analyses and assessment of m-ATPase activity with rapid flow quench apparatus. Training led to a significant increase in muscle mass, except for the biceps, and in total mechanical power output (+135.7%, P < 0.001). A shift towards an intermediate fibre type (i.e. MHC2x-to-MHC2a isoform transition) was also observed in biceps and FDP but not in the deltoid muscle. Importantly, rapid flow quench experiments revealed an enhancement of the m-ATPase activity during contraction at maximal velocity (kF ) in the three muscles, with a more marked effect in FDP (+242%, P < 0.001). Data fitting revealed that the rate constant of liberation of ATP hydrolysis products (k3 ) appears to be the main factor influencing the increase in m-ATPase activity. In conclusion, the data showed that, in addition to classically observed changes in MHC isoform content and fibre hypertrophy, m-ATPase activity is enhanced during resistance training and might contribute significantly to performance gains.

Bathyptilones: Terpenoids from an Antarctic Sea Pen, Anthoptilum grandiflorum (Verrill, 1879).

: An Antarctic coral belonging to the order Pennatulacea, collected during the 2013 austral autumn by trawl from 662 to 944 m depth, has yielded three new briarane diterpenes, bathyptilone A-C (1-3) along with a trinorditerpene, enbepeanone A (4), which bears a new carbon skeleton. Structure elucidation was facilitated by one- and two-dimensional NMR spectroscopy, mass spectrometry and confirmed by X-ray crystallography. The three compounds were screened in four cancer cell lines. Bathyptilone A displayed selective nanomolar cytotoxicity against the neurogenic mammalian cell line Ntera-2.

Anverenes B⁻E, New Polyhalogenated Monoterpenes from the Antarctic Red Alga Plocamium cartilagineum.

The subtidal red alga Plocamium cartilagineum was collected from the Western Antarctic Peninsula during the 2011 and 2017 austral summers. Bulk collections from specific sites corresponded to chemogroups identified by Young et al. in 2013. One of the chemogroups yielded several known acyclic halogenated monoterpenes (2-5) as well as undescribed compounds of the same class, anverenes B-D (6-8). Examination of another chemogroup yielded an undescribed cyclic halogenated monoterpene anverene E (9) as its major secondary metabolite. Elucidation of structures was achieved through one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy and negative chemical ionization mass spectrometry. Compounds 1-9 show moderate cytotoxicity against cervical cancer (HeLa) cells.

BMI1-UBR5 axis regulates transcriptional repression at damaged chromatin.

BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1), which plays a key role in maintaining epigenetic silencing during development. BMI1 also participates in gene silencing during DNA damage response, but the precise downstream function of BMI1 in gene silencing is unclear. Here we identified the UBR5 E3 ligase as a downstream factor of BMI1. We found that UBR5 forms damage-inducible nuclear foci in a manner dependent on the PRC1 components BMI1, RNF1 (RING1a), and RNF2 (RING1b). Whereas transcription is repressed at UV-induced lesions on chromatin, depletion of the PRC1 members or UBR5 alone derepressed transcription elongation at these sites, suggesting that UBR5 functions in a linear pathway with PRC1 in inducing gene silencing at lesions. Mass spectrometry (MS) analysis revealed that UBR5 associates with BMI1 as well as FACT components SPT16 and SSRP1. We found that UBR5 localizes to the UV-induced lesions along with SPT16. We show that UBR5 ubiquitinates SPT16, and depletion of UBR5 or BMI1 leads to an enlargement of SPT16 foci size at UV lesions, suggesting that UBR5 and BMI1 repress SPT16 enrichment at the damaged sites. Consistently, depletion of the FACT components effectively reversed the transcriptional derepression incurred in the UBR5 and BMI1 KO cells. Finally, UBR5 and BMI1 KO cells are hypersensitive to UV, which supports the notion that faulty RNA synthesis at damaged sites is harmful to the cell fitness. Altogether, these results suggest that BMI1 and UBR5 repress the polymerase II (Pol II)-mediated transcription at damaged sites, by negatively regulating the FACT-dependent Pol II elongation.

Characterization of Posterior Glenoid Bone Loss Morphology in Patients With Posterior Shoulder Instability.

PURPOSE: To systemically describe posterior bone defects in the setting of posterior shoulder instability based on several parameters, including surface area, slope and version, defect height from the base of the glenoid, and extent of bone loss at equal intervals along the long axis of the fossa. METHODS: A total of 40 young, active individuals with recurrent posterior shoulder instability and a bony injury confirmed on either computed tomography (n = 18; mean age, 26.3 ± 4.0 years) or magnetic resonance imaging (n = 22; mean age, 20.0 ± 4.9 years) were identified. The posterior glenoid bone defect was characterized using the following measures: (1) percentage of bone loss, (2) glenoid vault version, (3) slope of the posterior defect relative to the glenoid surface, (4) superior-inferior length of the defect, and (5) anterior-posterior width of the defect at 5 intervals along the glenoid fossa. RESULTS: The mean age of the 40 patients was 22.9 ± 5.5 years (range, 14.9-35.5 years). The mean surface area of glenoid bone loss was 9.7% ± 4.7%. Glenoid version measured at 5 equal intervals along the inferior two-thirds of the glenoid was 12.8° ± 4.9°, 11.9° ± 5.0°, 10.1° ± 6.3°, 10.5° ± 6.5°, and 8.7° ± 7.2° from superior to inferior. The mean slope of the posterior defect relative to the glenoid fossa was 26.8° ± 11.5°. The mean superior-inferior height of the bony defect was 21.9 ± 0.4 mm. The anterior-posterior sloped width of the defect at 5 equal intervals along the glenoid fossa was 0.9 ± 1.5 mm, 2.8 ± 2.4 mm, 4.0 ± 1.7 mm, 4.0 ± 2.1 mm, and 2.9 ± 2.6 mm from superior to inferior. Low-grade (<10%) bone loss was diagnosed in most shoulders (23 of 40 evaluated), whereas 15 had moderate bone loss (10% to <20%) and 2 had high-grade bone loss (≥20%). CONCLUSIONS: Posterior glenoid bone loss is characterized by a loss of posterior bony concavity, increased slope from anterior to posterior, and increased posterior version. The most anterior-posterior sloped width was quantified at the third and fourth intervals of 5 equal intervals from superior to inferior. This study highlights that patients with posterior instability have bone loss that is sloped relative to the glenoid fossa and suggests that management must be appropriately tailored given the distinctiveness of posterior bone loss. LEVEL OF EVIDENCE: Level IV, case series.

Systems model and individual simulations of training strategies in elite short-track speed skaters.

This study aimed to evaluate the effect of simulated training strategies on performance potential in elite short-track speed skaters. Training load and field-based criterion performances from fifteen athletes (10 males, 5 females) were collected over a 3-month training period and the relationship between training loads and performance was computed with a variable dose-response model using a genetic algorithm. Individual simulations of tapers preceded or not preceded by an overload training (OT) were assessed. We obtained a significant correlation between actual and modelled performances (R2 = 0.76 ± 0.07). Regarding model parameters, no significant difference was found between males and females but the time to recover performance tended to be lower in females. Simulations in which the taper parameters were free highlighted that an exponential or a step taper were the most effective for increasing performance compared to a linear taper (p < 0.05). Optimal exponential taper duration after OT was 10.7 ± 2.4d and the optimal load reduction was 75.9 ± 3.7%. OT intensity had the greatest influence on the predicted performance, followed by OT duration, taper decay, and to a lesser extent load reduction during taper and taper duration. Thus, a variable dose-response systems model allows the evaluation of different taper strategies and their potential effect on performance changes.

Modelling performance and skeletal muscle adaptations with exponential growth functions during resistance training.

System theory is classically applied to describe and to predict the effects of training load on performance. The classic models are structured by impulse-type transfer functions, nevertheless, most biological adaptations display exponential growth kinetics. The aim of this study was to propose an extension of the model structure taking into account the exponential nature of skeletal muscle adaptations by using a genetic algorithm. Thus, the conventional impulse-type model was applied in 15 resistance trained rodents and compared with exponential growth-type models. Even if we obtained a significant correlation between actual and modelled performances for all the models, our data indicated that an exponential model is associated with more suitable parameters values, especially the time constants that correspond to the positive response to training. Moreover, positive adaptations predicted with an exponential component showed a strong correlation with the main structural adaptations examined in skeletal muscles, i.e. hypertrophy (R2 = 0.87, 0.96 and 0.99, for type 1, 2A and 2X cross-sectional area fibers, respectively) and changes in fiber-type composition (R2 = 0.81 and 0.79, for type 1 and 2A fibers, respectively). Thus, an exponential model succeeds to describe both performance variations with relevant time constants and physiological adaptations that take place during resistance training.

Regulation of ULK1 Expression and Autophagy by STAT1.

Autophagy involves the lysosomal degradation of cytoplasmic contents for regeneration of anabolic substrates during nutritional or inflammatory stress. Its initiation occurs rapidly after inactivation of the protein kinase mammalian target of rapamycin (mTOR) (or mechanistic target of rapamycin), leading to dephosphorylation of Unc-51-like kinase 1 (ULK1) and autophagosome formation. Recent studies indicate that mTOR can, in parallel, regulate the activity of stress transcription factors, including signal transducer and activator of transcription-1 (STAT1). The current study addresses the role of STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. We show that STAT1-deficient human fibrosarcoma cells exhibited enhanced autophagic flux as well as its induction by pharmacological inhibition of mTOR. Consistent with enhanced autophagy initiation, ULK1 mRNA and protein levels were increased in STAT1-deficient cells. By chromatin immunoprecipitation, STAT1 bound a putative regulatory sequence in the ULK1 5'-flanking region, the mutation of which increased ULK1 promoter activity, and rendered it unresponsive to mTOR inhibition. Consistent with an anti-apoptotic effect of autophagy, rapamycin-induced apoptosis and cytotoxicity were blocked in STAT1-deficient cells but restored in cells simultaneously exposed to the autophagy inhibitor ammonium chloride. In vivo, skeletal muscle ULK1 mRNA and protein levels as well as autophagic flux were significantly enhanced in STAT1-deficient mice. These results demonstrate a novel mechanism by which STAT1 negatively regulates ULK1 expression and autophagy.

AMP-activated protein kinase stabilizes FOXO3 in primary myotubes.

AMP-activated protein kinase (AMPK) is a critical enzyme in conditions of cellular energy deficit such as exercise, hypoxia or nutritional stress. AMPK is well known to regulate protein degradation pathways notably through FOXO-related axis. In this study, we investigated the implication of AMPK activation in FOXO3 expression and stability in skeletal muscle primary myotubes. First, time course and dose response studies revealed optimal AICAR treatment duration and dose in skeletal muscle cells. Then, experiments with cycloheximide treatment of primary myotubes highlighted that AICAR infusion extends FOXO3 protein half-life. Our results also showed that AICAR treatment or nutrient depletion increases FOXO3 expression in primary myotubes and the expression of the mitochondrial E3 ligase Mul1 involved in mitochondrial turnover (mitophagy). In AMPK KO cells, nutrient depletion failed to alter the level of some FOXO3-dependent atrophic genes, including LC3B, BNIP3, and the mitochondrial E3 ligase Mul1, but not the expression of other genes (i.e. FOXO1, Gabarapl1, MAFbx, MuRF1). In summary, our data highlight that AMPK stabilizes FOXO3 and suggest a role in the first initiation step of mitochondrial segregation in muscle cells.