Here, we show AMPK heterotrimers containing the α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosine-binding (PTB) domains of TBC1D1 and is increased by two different pharmacological activators of AMPK (AICAR and A769962). The interaction enhances the efficiency by which AMPK phosphorylates TBC1D1 on its key regulatory site, Ser237. Furthermore, the interaction is reduced by a naturally occurring R125W mutation in the PTB1 domain of TBC1D1, previously found to be associated with severe familial obesity in females, with a concomitant reduction in Ser237 phosphorylation. Our observations provide evidence for a functional difference between AMPK α-subunits and extend the repertoire of protein kinases that interact with substrates via stabilisation mechanisms that modify the efficacy of substrate phosphorylation.AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis which achieves this through the phosphorylation of a myriad of downstream targets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucose uptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser Activation of the enzyme can be brought about by energy stress that is signalled by increases in intracellular AMP:ATP or ADP:ATP ratios, or by increases in intracellular Catrations ,2.PRKAA1 and PRKAA2 which encode the α1 and α2 subunits, respectively [Mammalian AMPK occurs as heterotrimeric complexes comprising α, β and γ subunits, each with distinct functions. Catalytic activity is conferred by two distinct genes, ectively . These cectively . T172 phectively –9 and trectively  in skele237, within the second PTB domain (PTB2), a covalent modification that promotes 14-3-3 protein binding [AMPK-mediated glucose uptake in skeletal muscle is thought to involve TBC1D1 ,12, a Ra binding ,19,24,25TBC1D1 is associated with growth- and obesity-related traits in pigs, chickens and rabbits [Sequence variation in  rabbits –29 as we rabbits ,31. A co rabbits ,32. Our  rabbits . However237. We also demonstrate that this interaction is disrupted by the naturally occurring R125W mutation.The initial aim of the present study was to identify proteins that bind to the PTB domains of TBC1D1 using Stable Isotope Labelling by Amino acids in Cell culture (SILAC). We found a stable association of AMPK heterotrimers containing α1 but not α2 subunits and demonstrate that this has functional consequences for the kinetics of phosphorylation of the critical AMPK-directed phosphorylation site on TBC1D1, Ser172 (#2531), AKT (#4685), ACC (#3876), pACC Ser79 (#3661), IRAP (#3808), rabbit FLAG (#2368), Raptor (#2280), pRaptor Ser792 (#2083) and TBC1D1 (#5929) were from Cell Signaling Technology. Other antibodies were pan-14-3-3 (sc-629) and GST (sc-138) from Santa Cruz Biotechnology Inc., GFP , AMPK-γ1 , pTBC1D1 Ser237 , β-actin (A1978) and mouse FLAG (F1804). Isoform-specific AMPK-α antibodies were described previously [Unless otherwise stated all reagents were from Sigma–Aldrich. Primary antibodies against pan-AMPK-α (#2532), AMPK-β1/2 (#4150), pAMPK Threviously . A769662Tbc1d1 mice with muscle-specific overexpression of Tbc1d1 were generated by cloning of PCR-amplified 3xFLAG-Tbc1d1 from skeletal muscle cDNA into pBSK-MCK containing exon, one of the muscle creatine kinase promoters. The construct was injected into the fertilised oocytes of FVB/N inbred mice . A founder line with high (5-fold) Tbc1d1 overexpression in heart and skeletal muscle tissue was backcrossed to the N7 generation with C57BL/6J mice using marker-assisted genotyping.Transgenic MCK-3xFLAG-ad libitum access to food and water. After weaning, animals received a standard chow diet . Male mice between 10 and 14 weeks were killed by cervical dislocation, quadricep muscle was dissected and directly snap-frozen in liquid nitrogen.Mice were kept in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals, and all experiments were approved by the Ethics Committee of the State Ministry of Agriculture, Nutrition and Forestry . Three to six mice per cage (Macrolon type III) were housed at a temperature of 22°C and a 12-h light–dark cycle (lights on at 6 a.m.) with TBC1D1 constructs and AS160 were cloned from pCMV5.HA-1 TBC1D1 and p3xFLAG-CMV-10 AS160 , Flp-In HEK293 (Invitrogen) and HEK293T cell lines were cultured in DMEM supplemented with 10% (v/v) FBS (Invitrogen), 50 IU/ml penicillin, 50 µg/ml streptomycin and 2 mM glutamine. C2C12 cells were differentiated into myotubes as previously described . HEK293TPRKAA1 and PRKAA2) in HEK293 Flp-In cells was performed via the CRISPR-Cas9 method [Knockout of AMPK-α1 and -α2 (9 method  using th9 method . As someAfter transfection with FuGENE 6 (Promega), α1/α2 DKO cells expressing either FLAG-AMPK-α1 or FLAG-AMPK-α1 K47R were selected and single foci expanded in complete medium supplemented with blasticidin (15 µg/ml) and hygromycin B (200 µg/ml).4P2O7, 1 mM Na3VO4, 0.5 mM PMSF, Calbiochem protease inhibitor cocktail), lysates incubated  with GFP-trap beads (ChromoTek), precipitates washed four times with precipitation buffer and samples pooled prior to eluting, SDS–PAGE and Nano LC–MS/MS on an Orbitrap Velos (Thermo Fisher Scientific). Mass spectrometric detection and quantification was performed as previously published [Transduced C2C12 myoblasts were expanded in R0K0 or R6K4 isotopically labelled DMEM (Dundee Cell Products) for at least six passages prior to differentiation. Myotubes were harvested in precipitation buffer  or precipitation buffer and subsequently washed. For endogenous reciprocal immunoprecipitations, AMPK-α1 antibody or sheep IgG was preconjugated to protein-G-coated sepharose beads which were then incubated  with lysates from C2C12 myotubes harvested in precipitation buffer and subsequently washed. Standard western blot procedures were performed. Signal was detected by either a LI-COR Odyssey infrared imaging system of fluorescently labelled secondary antibodies or ECL of HRP-conjugated secondary antibodies.2. Additional A769662 (25 µM) or vehicle was spiked into activation experiment pull-downs during the 4°C incubation step. After extensive washing bound proteins were detected via western blotting. Recombinant AMPK trimer complexes were expressed in E. coli and purified as previously published [Glutathione S-transferase (GST)-tagged or dual GST/His-tagged proteins were expressed upon IPTG induction in Escherichia coli BL21 (DE3) overnight at 15°C, purified using talon affinity resin (Clontech), eluted with 200 mM imidazole, 50 mM Tris–HCl (pH 8), 300 mM NaCl, 5 mM 2-mercaptoethanol, 5% glycerol and subsequently immobilised and purified on glutathione sepharose . For pull-down experiments, immobilised purified proteins (2 or 4 µg) were incubated  with Flp-In HEK293 cell lysate (5 mg) in precipitation buffer supplemented with 2 mM ATP and 5 mM MgClublished . For dir3VO4, 30 mM NaF, 10 mM Na4P2O7, 10 mM EDTA, 0.5 mM PMSF, Calbiochem protease inhibitor cocktail). To reduce basal AMPK, cells were pre-treated with STO-609  as previously described [Cells transfected for 48 h were treated as specified in figure legends and harvested in ice-cold lysis buffer  bound robustly, whereas those containing α2 subunits (α2β1γ1 and α2β2γ1) associated only weakly  inhibited AMPK binding to GFP-PTB1 + 2 . We additionally found that the kinase activity of α1 was not required for TBC1D1 binding as, surprisingly, a kinase-dead mutant expressed in a CRISPR/Cas9 HEK293 α1/α2 double knockout (HEK293-DKO) cell line  showed enhanced binding to GST-PTB1 + 2 .To explore the contribution of the enzyme/peptide substrate interaction interface  in facilitating the AMPK–TBC1D1 interaction, we mutated the AMPK-mediated phosphorylation site Sernstructs . MutatioPTB1 + 2 . Very sig Ser237  . A GFP-TBC1D1 R125W mutant transiently expressed in these cells did indeed exhibit reduced phosphorylation of Ser237 compared with the wild-type protein .The reduction in the AMPK–TBC1D1 interaction brought about by the R125W mutation would be predicted, based on our previous results , to redu protein . This wa protein . Moreove237, a key regulatory site. To the best of our knowledge, our results provide the first evidence for a substantive difference in substrate selection between the α1 and α2 subunits of AMPK in vitro or in intact cells; previous studies have revealed only very subtle differences [Our findings demonstrate that AMPK heterotrimers containing α1 but not α2 subunits form a direct stable association with TBC1D1, but not with its paralogue AS160, which enhances the efficacy of phosphorylation of a TBC1D1 on Serferences . The difTypically, protein kinases do not bind substrates with sufficiently high affinity for their interaction to survive cell lysis, immunoprecipitation and extensive washing of the complexes. Based on our results, therefore, we propose a dual interaction mechanism for the stable interaction of AMPK α1-containing heterotrimers with TBC1D1.237 phosphorylation site within the PTB2 domain. Evidence for this derives from the fact that the interaction: (i) is reduced by mutation of Ser237 and its neighbouring residues, Met232, Arg233 and Leu241, that together confer selective substrate binding by AMPK  that the presence of PTB1 substantially enhances the binding of the PTB2 domain to AMPK ; (ii) th domains . AlternaDocking sites, external to protein kinase catalytic interfaces, are well established to facilitate alignment of the substrate phospho-acceptor site with its cognate protein kinase substrate-binding groove to enhance phosphorylation efficiency (reviewed in –51). The237 phosphorylation and that this is associated with an increase in muscle glucose uptake [237 phosphorylation. This is substantiated by our observations that TBC1D1 Ser237 phosphorylation is: (i) enhanced in cells expressing α1 subunit-containing AMPK heterotrimers versus those expressing α2-containing heterotrimers  knock-in mutation promotes increased IGF1 secretion from the liver, leading to lipogenic gene expression in adipose tissue and consequent obesity [237 phosphorylation of the R125W mutant demonstrated in our experiments and (ii) IGF1 hypersecretion-mediated obesity in the knock-in mouse where Ser231 can no longer be phosphorylated; an attractive hypothesis is that the observed severe obesity phenotype in a subset of women with the R125W mutation may occur via a mechanism involving elevated circulating levels of IGF1. In summary, we have shown that α1-containing AMPK heterotrimers bind stably to TBC1D1 via its PTB domains. This interaction plays an important role in controlling the degree of Ser237 phosphorylation on TBC1D1 and is thus likely to be important in controlling energy homeostasis in tissues co-expressing α1-containing AMPK heterotrimers and TBC1D1.The binding of α1-containing complexes to TBC1D1 may have important roles in non-muscle cell types where α1 subunit expression and activity predominates over α2. In human hepatocytes, for example, expression of α1β2γ1 heterotrimers predominates , and in tivation . Interes obesity . Taken t


Approximately five million United States (U.S.) adults are diagnosed with heart failure (HF), with eight million U.S. adults projected to suffer from HF by 2030. With five-year mortality rates following HF diagnosis approximating 50%, novel therapeutic treatments are needed for HF patients. Pre-clinical animal models of HF have highlighted histone deacetylase (HDAC) inhibitors as efficacious therapeutics that can stop and potentially reverse cardiac remodeling and dysfunction linked with HF development. HDACs remove acetyl groups from nucleosomal histones, altering DNA-histone protein electrostatic interactions in the regulation of gene expression. However, HDACs also remove acetyl groups from non-histone proteins in various tissues. Changes in histone and non-histone protein acetylation plays a key role in protein structure and function that can alter other post translational modifications (PTMs), including protein phosphorylation. Protein phosphorylation is a well described PTM that is important for cardiac signal transduction, protein activity and gene expression, yet the functional role for acetylation-phosphorylation cross-talk in the myocardium remains less clear. This review will focus on the regulation and function for acetylation-phosphorylation cross-talk in the heart, with a focus on the role for HDACs and HDAC inhibitors as regulators of acetyl-phosphorylation cross-talk in the control of cardiac function. Heart failure (HF) is a common condition in the United States that impacts over five million Americans. Moreover, eight hundred thousand individuals are diagnosed with HF annually ,2. HF isSubstantial evidence highlights histone deacetylases (HDACs) as an intracellular therapeutic target for the treatment of HF ,10,11,12Nucleosomes, the structural units of chromatin, are composed of DNA and histone proteins, which are essential for DNA packaging in eukaryotic cells ,19,20. TThe role of HDACs in the development and progression of disease, specifically cardiac disease, has often been observed since histone acetylation is a key component in the regulation of gene expression ,25,26,27The connection between HDACs and the heart was initially observed by the discovery that class IIa HDACs interact with the transcription factor, myocyte enhancer factor-2 (MEF2) ,36,37,38N-lysine binding channel and a “chelator” that binds a zinc ion present in the HDAC enzyme active site. This is “linked” to the cap structure by a five or six hydrocarbon chain [As mentioned above, HDACs catalyze the removal of acetyl groups from lysine residues on histone proteins and they were historically studied as epigenetic regulators of gene transcription . HDACs aon chain ,13,44. Ton chain .HDAC inhibitors are efficacious in animal models of cardiac remodeling and heart failure ,47,48,49HDAC inhibition has also been shown to inhibit pathological cardiac hypertrophy and improve muscle contractility, as well as attenuate systolic and diastolic dysfunction ,14,16,27In addition to systolic failure, chronic heart failure is also characterized by left ventricular diastolic dysfunction or heart failure with preserved ejection fraction (HFpEF) . In thisPosttranslational modification (PTM) of proteins is crucial for the modulation and regulation of many protein-protein interactions and functions ,57. PTMsProtein phosphorylation is a reversible PTM that is essential for protein synthesis, cellular proliferation, apoptosis, signal transduction, growth, development, and aging . The humSimilar to phosphorylation, acetylation occurs on lysine residues of histone and non-histone proteins ,71,72,73Given the multitude of modification sites, p53 activity and stability are likely redundantly regulated by many PTMs. Moreover, multiple modification sites on p53 suggests cross-talk between PTMs in the regulation of p53 function. This is evident from studies that report that p53 phosphorylation of Ser15 and Ser46 are necessary for p53 acetylation ,66. MoreAs discussed above, HDAC inhibitors are efficacious in pre-clinical animal models of HF ,23,43,49As phosphorylation has been shown to impact protein acetylation ,66,88, iPhosphatases can be regulated by gene expression or PTMs that control phosphatase protein stability and activity ,94. IndeThe findings above support a role for acetylation in the regulation of protein phosphatases as feedback inhibitors of protein phosphorylation. However, direct actions have been attributed to the acetylation of protein kinases in regulating protein kinase activity ,54,101. Findings for these direct actions for acetylation in the regulation of protein phosphorylation are not surprising, given recent proteomic observations that other protein kinases are regulated by acetylation. In these studies, HDAC inhibition with trichostatin A (TSA) led to increased phosphorylation of protein tyrosine kinase 2 (PTK2), mitogen-activated protein kinase 3 , and muscle pyruvate kinase, among others . It shouThis review covered the crosstalk between protein phosphorylation and lysine acetylation and highlighted an emerging role for HDAC inhibitors in the regulation of acetylation-phosphorylation cross-talk for the treatment of cardiac dysfunction and failure. In this review, we discussed the roles for HDACs as well as their inhibition in the regulation of phosphorylation that was mediated through the induction of phosphatase gene expression A or acti+-dependent HDACs, and as such, they are sensitive to changes in nutrient utilization and ATP needs, as NAD+/NADH ratios impact sirtuin activity. Ample studies have examine sirtuins in the heart [Likewise, this review specifically examined a role for zinc-dependent HDACs in the regulation of acetylation-phosphorylation crosstalk. However, class III HDACs, the sirtuins also impart acetylation changes and are important in the heart ,111,112.he heart ,115,116,In summary, current research demonstrates pre-clinical efficacy for HDAC inhibitors in small and large animal models of HF. That, paired with the FDA approval of four HDAC inhibitors  for the treatment of cancer, suggests feasibility in repurposing HDAC inhibitors for the treatment of human HF; there are currently no ongoing clinical trials using HDAC inhibitors in HF patients. However, as this review points-out, HDAC inhibitors likely target more than just histone proteins in the myocardium. As such, we would argue the importance for considering class/isoform selective HDAC inhibitors to limit negative off-target actions that may occur with treatment from pan-HDAC inhibitors that are currently FDA approved. Consistent with this, a recent systematic review of pan-HDAC inhibitors in cancer patients demonstrated mild cardiac side effects that included ST-segment and T wave (ST/T) abnormalities along with QT prolongation; this is a concern, as these measures demonstrate the potential for lethal ventricular arrhythmias . Overall


P < 0.05), but no difference was found between fiber types with respect to the level of mTOR protein. Resistance exercise led to non-significant increases (2–3-fold) in mTOR and S6K1 phosphorylation as well as a 50% decrease (P < 0.05) in eEF2 phosphorylation in both fiber types. Intake of EAA caused a 2 and 6-fold higher (P < 0.05) elevation of mTOR and S6K1 phosphorylation, respectively, in both type I and type II fibers compared to placebo, with no effect on phosphorylation of eEF2. In conclusion, protein levels of S6K1 and eEF2 were significantly higher in type II than type I fibers suggesting higher capacity of the mTOR pathway in type II fibers. Ingestion of EAA enhanced the effect of resistance exercise on phosphorylation of mTOR and S6K1 in both fiber types, but with considerable variation between single fibers of both types.Human muscles contain a mixture of type I and type II fibers with different contractile and metabolic properties. Little is presently known about the effect of anabolic stimuli, in particular nutrition, on the molecular responses of these different fiber types. Here, we examine the effect of resistance exercise in combination with intake of essential amino acids (EAA) on mTORC1 signaling in individual type I and type II human muscle fibers. Five strength-trained men performed two sessions of heavy leg press exercise. During exercise and recovery, the subjects ingested an aqueous solution of EAA (290 mg/kg) or flavored water (placebo). Muscle biopsies were taken from the vastus lateralis before and 90 min after exercise. The biopsies were freeze-dried and single fibers dissected out and weighed (range 0.95–8.1 μg). The fibers were homogenized individually and identified as type I or II by incubation with antibodies against the different isoforms of myosin. They were also analyzed for both the levels of protein as well as phosphorylation of proteins in the mTORC1 pathway using Western blotting. The levels of the S6K1 and eEF2 proteins were ~50% higher in type II than in type I fibers ( Human skeletal muscle contains a mixture of fibers with different contractile and metabolic properties, these fibers also differ with respect to adaptability to training. Slow twitch type I fibers have, in general, more mitochondria and a higher oxidative capacity , 2. In cResistance exercise enhances the size of individual muscle fibers, in particular fast-twitch fibers , 7. AnotIn an earlier study, we demonstrated that maximal eccentric contractions evoked dissimilar responses in type I and type II fibers: phosphorylation of S6K1 and of ribosomal protein (rp) S6 was elevated 3–4-fold and 6–9-fold, respectively, in type II fibers, with no change in the type I fibers . In thisAccordingly, in the present study, the subjects performed a session of heavy resistance exercise in combination with intake of essential amino acids (EAA) or placebo. Muscle biopsies were taken before and after exercise for analysis of individual type I and type II fibers with respect to both the levels of protein and the degrees of phosphorylation of proteins of the mTORC1 pathway. For this purpose, we employed a novel application of the Western blotting technique, an approach similar to that described by Murphy . This prFive healthy men who had been performing resistance training for at least 1 year gave both their oral and written consent to participate after being fully informed about the procedure and possible risks involved. Their mean age was 25 ± 3 years, height 179 ± 4 cm, weight 85 ± 4 kg, and maximal one repetition leg press (1RM) 442 ± 18 kg. These subjects were a sub-group from our earlier study . The preFollowing a 9 h fast, subjects reported to the laboratory in the morning. A baseline biopsy was taken from the vastus lateralis muscle with a Weil-Bakesley conchotome   under loAt nine time points during the experiment , the participants consumed 150 ml of either an aqueous solution of EAA or placebo (flavored water) in a double-blind, counterbalanced order  and each fiber weighed using a quartz-fiber fish pole balance, spectrophotometrically calibrated with p-nitrophenol . Fibers 2, 1% Triton X-100, 50 mM β-glycero-P, 1 mM sodium ortovanadate, 1% Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich), 1% Halt™ Protease Inhibitor Cocktail (Thermo Scientific), and 2 mM 4,4′-DDT at pH 7.4. The samples were stored on ice for approximately 1 h throughout homogenization followed by addition of 5 μl 2X Laemmli sample buffer (Bio Rad). The samples were then heated at 95°C for 5 min and stored at −20°C. The procedure is modified from the method originally described by Murphy . A supernatant from a whole muscle homogenate of a post-exercise muscle biopsy was also loaded onto each gel to control for between membrane differences in protein phosphorylation and expression. The Western blotting protocol employed has been described in detail previously (2O−10% methanol) for 30 min at 4°C and the proteins transferred to polyvinylidine fluoride membranes (Biorad) at 300 mA for 180 min at 4°C. The membranes were subsequently stained using Pierce® Reversible Stain Kit (Thermo Scientific).In brief, electrophoresis was run for 30 min at 300 V in Tris buffer  on an ice bed in a cold room at 4°C. The gels were subsequently incubated in transfer buffer  (20 mM Tris base and 137 mM NaCl). Incubation with primary antibodies targeting the phosphorylated proteins was carried out over-night at 4°C, the membranes were then washed prior to incubation with secondary antibodies for 1 h at room temperature, and then washed once again prior to visualization with SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo Scientific). The Molecular Imager ChemiDoc™ XRS system was used to detect protein bands and the Quantity One® software, version 4.6.3 (Bio-Rad Laboratories) for quantification.2O (5 ×1 min) and TBS (3 ×3 min) and thereafter stripped for 30 min at 50°C utilizing Restore™ PLUS Western Blot Stripping Buffer (Thermo Scientific). After being washed once again with dH2O (5 ×1 min) and TBS (3 ×3 min), the membranes were incubated with primary antibodies for detection of total protein as described above. The levels of phosphorylated and total protein are expressed in relation to the weight of the fiber.The membranes were subsequently washed thoroughly with dHThe membranes were again stripped twice prior to exposure of antibodies targeting myosin heavy chain I (MHC I) and then myosin heavy chain II (MHC II). On the basis of visual inspection of the MHC I and MHC II staining, fibers were categorized either as type I, type II, or hybrid fibers (staining for both MHC I and II). The hybrid fibers were discarded; only 22 fibers (about 3% of the fibers) were classified as hybrid fibers, which is too small to give a valuable result.2448 , total S6K1 , phosphorylated S6K1 Thr389 , total eEF2 , and phosphorylated eEF2 Thr56 . The secondary antibodies, anti-rabbit HRP-linked (#7074S) and anti-mouse HRP-linked (#7076S), were both purchased from Cell Signaling and diluted 1:10,000.Both primary and secondary antibodies were diluted with TBS containing 2.5% milk. The primary antibodies used were Mouse Anti-Slow Skeletal Myosin Heavy Chain , Rabbit Anti-Fast Myosin Skeletal Heavy Chain , total mTOR , phosphorylated mTOR SerP < 0.05. Statistical analysis was carried out with Statistica software .A repeated measures three-way ANOVA  was applied to compare changes in mean levels of protein expression and phosphorylation in the type I and type II fibers in the two conditions. When a main or interaction effect was detected, planned comparisons was performed to identify where the differences occurred. The level of statistical significance was set at P < 0.05), whereas no difference was observed with respect to the mTOR protein  and S6K1, in the placebo condition  type II fibers express higher levels of the S6K1 and eEF2 proteins than type I fibers, (2) type I and type II fibers respond similarly to intake of EAA in combination with resistance exercise, and (3) there is a large variation in this response between individual fibers within each subject as well as between subjects.Type II muscle fibers had approximately 50% higher levels of both S6K1 and eEF2, signal transduction proteins downstream of mTOR. These findings are in agreement with a previous report on rat muscle where the levels of S6K1 and protein kinase B (Akt/PKB) are higher in the fast EDL than in the slow soleus muscle . These pResistance exercise alone did not increase the phosphorylation of mTOR and S6K1 significantly, although the levels were elevated by 2–3-fold 90 min after exercise. This differs from our previous observation that maximal eccentric contractions markedly elevated phosphorylation of S6K1 and reduced phosphorylation of eEF2 in type II fibers only . The faiWhen the subjects ingested a mixture of EAA, mTOR, and S6K1 were stimulated considerably in both type I and type II fibers following resistance exercise . InteresHowever, data on resting muscle do not support a larger responsiveness in type I fibers , 35. InfOur observations of elevated phosphorylation of eEF2 in type II fibers in resting muscle are in accordance with previous findings . Here, tWe observed here considerable variation in the levels of both protein and phosphorylation between individual fibers of both type I and type II. The large variation in signaling response indicates that some fibers are not recruited, despite the heavy work load. Similar large variations in substrate levels (glycogen) and ATP within fibers of the same type have been reported following endurance exercise or electrical stimulation of the muscle , 40, furIn the present study, the number of type II fibers dissected out and analyzed was more than double the number of type I fibers . SimilarIn summary, the levels of both S6K1 and eEF2 proteins were higher in type II than type I muscle fibers, suggesting that in the former the mTORC1 pathway has a greater capacity. Heavy resistance exercise in combination with consumption of EAA elevates phosphorylation of both mTOR and S6K1 in both fiber types above the levels seen at rest and following resistance exercise alone. The only minor differences in the response to intake of EAA was that when normalized to protein content, S6K1 was slightly more phosphorylated in type I fibers. Large variations in the responses of individual fibers to the different stimuli were observed, with some fibers demonstrating high levels of phosphorylation following exercise and others no stimulation at all. Future studies exploring possible relationship between signaling response, on one hand, and substrate levels or metabolic changes, on the other, within individual type I and type II fibers may help to understand the mechanism involved in training adaptation.All datasets generated for this study are included in the manuscript and/or the supplementary files.Five healthy men who had been performing resistance training for at least 1 year gave both their oral and written consent to participate after being fully informed about the procedure and possible risks involved. Their mean age was 25 ± 3 years, height 179 ± 4 cm, weight 85 ± 4 kg, and maximal one repetition leg press (1RM) 442 ± 18 kg. These subjects were a sub-group from our earlier study . The preSE, KS, and EB designed the study. MM and WA carried out the experiment. SE dissected and analyzed muscle fibers and performed statistical analyses. SE and EB wrote the manuscript. KS, MM, and WA edited the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rat and human subcellular fractions, relatively specific substrates, corresponding coenzymes and HPLC, spectrophotometric or spectrofluorometric analysis of product formation were used. The results showed significant inhibition of cytochromes P450  activities by all tested sesquiterpenes in rat as well as in human hepatic microsomes. On the other hand, all tested sesquiterpenes did not significantly affect the activities of carbonyl-reducing enzymes and conjugation enzymes. The results indicate that acyclic sesquiterpenes might affect CYP1A, CYP2B and CYP3A mediated metabolism of concurrently administered drugs and other xenobiotics. The possible drug–sesquiterpene interactions should be verified in in vivo experiments.Sesquiterpenes, 15-carbon compounds formed from three isoprenoid units, are the main components of plant essential oils. Sesquiterpenes occur in human food, but they are principally taken as components of many folk medicines and dietary supplements. The aim of our study was to test and compare the potential inhibitory effect of acyclic sesquiterpenes,  Sesquiterpenes, defined as 15-carbon compounds formed from three isoprenoid units, are an extremely diverse, heterogeneous and large group of natural compounds. Sesquiterpenes, together with monoterpenes, represent the major components of plant essential oils, widely used in folk medicines, health-supporting preparations and cosmetics [Farnesol occurs in, among others, cabreuva, ambrette, jasmine, ylang, and rose essential oils. FAR has a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-allergic, chemo-preventive and anticancer effects. For example, FAR ameliorates serum allergic antibody titres and lipid profiles in ovalbumin-challenged mice , and migtrans and a cis form. They can be found in cabreuva, niaouli and neroli oils, among others. NER is a common ingredient in many food and cosmetic products. In organisms, it exerts many biological effects e.g., antioxidant, antibacterial, anti-parasite and anticancer [Nerolidol (NER) is the allylic isomer of farnesol and exists in two geometric isomers, a ticancer . For exaticancer  and schiticancer . Moreoveticancer . Neuroprticancer . Using Hticancer .Taken together, both acyclic sesquiterpenes mentioned above (structures are presented in Cytochrome P450 (CYP)-catalyzed oxidative reactions are the most common phase I reactions in drug metabolism. However, CYPs are not the only enzymes involved in the phase I metabolism of drugs and other xenobiotics—carbonyl reduction is the major phase I reaction for an array of xenobiotics . Carbonytrans-nerolidol (TNER) and cis-nerolidol (CNER) on the activities of CYPs, CBR1, NQO1, AKRs, GSTs, UGTs and SULTs. With respect to possible inter-species differences in an action of inhibitors on drug-metabolizing enzymes, both rat and human hepatic subcellular fractions were used as consequent in vivo experiments in rat are being planned.Therefore, the present study was designed to evaluate the inhibitory effect of three abundant acyclic and biologically active sesquiterpenes farnesol, cis-nerolidol (CNER), trans-nerolidol (TNER), and farnesol (FAR), which are commonly found in the human diet, on cytosolic and microsomal biotransformation enzymes, were tested in human and rat liver subcellular fractions.In the present study, inhibitory effects of acyclic sesquiterpenes First, the inhibitory effect of sesquiterpenes at final concentrations of 100 µM on the main hepatic biotransformation enzymes was assessed by measuring enzyme activities towards specific substrates. This high concentration was tested first to find out whether there is any inhibition at all. Specific activities of selected cytochromes P450 , with and without sesquiterpenes in rat and human microsomes, are given in From all the tested enzymes, only CYP1A2 (EROD activity) and CYP2B/3A (BROD activity), both in human and rat microsomal fractions, were inhibited by CNER, TNER and FAR. Non-enzymatic interference of sesquiterpenes, with some compounds in the reaction mixtures (fluorescence quenching), was considered and excluded.50) in human and rat microsomes. Obtained IC50 values and their 95% confidence intervals are given in CYP1A2 (EROD activity) and CYP2B/3A (BROD activity) were strongly inhibited by testing sesquiterpenes in screening experiment. These enzymes were selected for further testing including determination of 50% inhibition concentration  and CYP3A4 inhibitor ketoconazole (KET) were used for comparison of the inhibitory effect with sesquiterpenes. The 5 µM ANF inhibited EROD activity by 92% but 5 µM FAR, CNER and TNER, only by 77%, 50%, and 38%, respectively. On the other hand, the 5 µM KET inhibited BROD activity by 66%, while the inhibitory effect of 5 µM sesquiterpenes was stronger: FAR inhibited BROD activity by 85%, CNER by 71%, and TNER by 68%.50 values were determined, as high inhibitory effect of acyclic sesquiterpenes toward EROD and BROD activities was found in screening experiment. However, no such a high inhibition was observed in other CYP activities. Data are presented in The inhibitory effect of CNER, TNER and FAR on other human CYP activities catalysed by CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 and corresponding ICThe type of inhibition for CYP1A2 (EROD activity) and CYP2B/3A (BROD activity) by TNER was determined in human and rat microsomal fractions. When the inhibition of CYPs was tested, microsomes were incubated with different concentrations of ethoxyresorufin or benzyloxyresorufin (0.5–5.0 µM) in the presence or absence of TNER (5 µM).M and apparent K’M values and lower apparent V´MAX than VMAX values were found in both species. Kinetic parameters are given in The results of kinetic study are presented in Inhibition of drug-metabolizing enzymes is important from several points of view. Regarding drugs and toxic xenobiotics, the inhibition of the enzymes catalyzing their deactivation leads to augmented plasma concentration of biologically active/toxic substance. By this way, drug efficacy can be prolonged and, together with increased risk of undesired effects and the toxic effect of xenobiotics, can get worse. On the other hand, the inhibition of enzymes that activate xenobiotics into more reactive metabolites can decrease cellular toxicity of these xenobiotics and could contribute to chemo-prevention. In every way, it is necessary to know if all components of human food, as well as all potential drugs, are able to inhibit the main drug-metabolizing enzymes. For that reason, the inhibitory effects of many natural compounds, such as flavonoids, towards drug-metabolizing enzymes have been intensively studied e.g., ,20,21,22Curcuma aromatica, were evaluated for their ability to inhibit CYPs, the sesquiterpene -(+)-germacrone-4,5-epoxide inhibited certain CYPs very potently with IC50 = 1.0 µM [50 value of 3.6 µM in human liver microsomes [Although sesquiterpenes as highly lipophilic compounds can be considered as probable inhibitors of drug-metabolizing enzymes, the information about their potential inhibitory effects is limited. Moreover, all studies have been focused only on cytochromes P450 (CYPs). When ten sesquiterpenes, isolated from the rhizomes of = 1.0 µM . Sesquit= 1.0 µM . Anothercrosomes . Cedrol,crosomes .In our study, the potential inhibitory effect of three acyclic sesquiterpenes FAR, CNER and TNER on the main drug-metabolizing enzymes was studied in rat and human hepatic subcellular fractions. In addition to CYPs, the activities of carbonyl-reducing enzymes and the main conjugation enzymes were also included in the study, with the aim to obtain more complex information. With respect to the necessity of further verifying of the results obtained in vitro in subsequent in vivo experiments, both rat and human samples were used in the present study. We aim to know if the sensitivity of drug-metabolizing enzymes to sesquiterpenes in rats is similar to those in humans, and thus rats could be a suitable experimental animal for planned evaluation of drug–sesquiterpene interactions in vivo.Firstly, the inhibitory potency of acyclic sesquiterpenes at fixed concentration (100 µM) toward the main drug-metabolizing enzymes was screened in rat and human hepatic cytosols and microsomes. While marked inhibition of two CYP-mediated activities (EROD and BROD) by all three sesquiterpenes was observed, the activities of other enzymes were not affected by any sesquiterpene. Although the specific activities of individual enzymes mostly differed between rat and human, the effect of sesquiterpenes seemed to be similar: acyclic sesquiterpenes inhibited only EROD and BROD activities in both species.50 values, and to find out the type of inhibition. EROD activity is ascribed mainly to CYP1A2 in liver microsomes, while BROD activity mainly to CYP2B1 in rat, and to CYP3A4/2B6 activities in human [Based on these results, the inhibitory effects of acyclic sesquiterpenes toward EROD and BROD activities were tested in details to obtain ICin human .50 values of three sesquiterpenes tested were similar  and they were slightly lower in humans (1.3–2.4 µM) than in rats (4.4–6.4 µM) microsomes. Interestingly, the inhibitory effect of acyclic sesquiterpenes was stronger than the effect of KET, a well-known CYP3A4 inhibitor. As CYP3A4 metabolizes about 50% of all drugs, its inhibition by sesquiterpenes might result in drug–sesquiterpene interactions. In the kinetic study, TNER (as a selected representative) acted as non-competitive inhibitor in both species.Concerning BROD activities, ICIn the case of EROD activity, the inhibitory potency differed more among sesquiterpenes, as well as between rat and human microsomes. Model CYP1A inhibitor ANF had a stronger effect than acyclic sesquiterpenes. TNER inhibited EROD competitively in humans but non-competitively in rats. The sesquiterpene mediated inhibition of CYP1A could represent a protective effect of sesquiterpenes, as CYP1A often catalyzes formation of toxic metabolites from environmental pollutants.50 values were determined. However, none or weak inhibition by acyclic sesquiterpenes have been observed. Surprisingly, hydroxylation of midazolam and testosterone, both ascribed to CYP3A enzymes, were inhibited only mildly, although BROD activity  was strongly inhibited by all sesquiterpenes tested. TNER was the most potent inhibitor of testosterone hydroxylation with IC50 51 µM, while CNER was the most effective toward midazolam hydroxylation with IC50 66 µM. The discrepancy of these results can be explained by different substrate concentrations used in the enzyme assays , and by different binding sites of these substrates [Consequently, the inhibitory effects of sesquiterpenes were followed up in other CYP-mediated activities in human liver microsomes. Nine substrates relatively specific for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 were used, and corresponding ICbstrates .Until now, the inhibitory effect of FAR on CYPs was tested only in rabbit liver microsomes. The obtained results showed that ethoxycoumarin deethylase and diclofenac-4-hydroxylase activities were most sensitive to FAR, while caffeine-8-hydroxylation and taxol-6-hydroxylation were not inhibited at all. The observed inhibition appeared to be reversible, and was not strictly competitive, but rather mixed in nature . Concerncis-nerolidol (CNER), trans-nerolidol (TNER), farnesol (FAR), and other chemicals benzyloxyresorufin, ethoxyresorufin, resorufin, coumarin, 7-ethoxy-4-(trifluoromethyl)coumarin, S-mephenytoin, diclofenac, bufuralol, testosterone, midazolam, chlorzoxazone, menadione, 1-chloro-2,4-dinitrobenzene, p-nitrophenol, p-nitrophenylsulphate, 2-napthol, 3’-phosphoadenosine-5’-phosphate, acenaphthenol, reduced glutathione, UDP-glucuronic acid, cytochrome c, NADH, NADP and NADPH, were purchased from Sigma-Aldrich . All other chemicals used were of HPLC or analytical grade.Sesquiterpenes Male Wistar rats (10–12 weeks old) were obtained from Velaz . They were housed in air-conditioned animal quarters with a 12 h light/dark cycle. Food (a standard rat chow diet) and water were provided ad libitum. The animal protocols used in this work were evaluated and approved by the Ethic Committee of the Ministry of Education, Youth and Sports (Protocol MSMT-24185/2015-11). The protocols were in accordance with the Guide for the Care and Use of Laboratory Animals .Human liver samples were obtained from livers excluded from transplantation for medical reasons  and were used in compliance with National Ethic Laws.v/v), using a Potter-Elvehjem homogenizer and sonication with Sonopolus . The subcellular fractions were isolated by differential centrifugation of the tissue homogenate [Frozen liver (both rat and human) was thawed at room temperature for up to 15 min and processed to microsomal and cytosolic fractions. Briefly, livers were homogenized in a 0.1 M sodium phosphate buffer, pH 7.4, at the ratio of 1:6 (mogenate  and storProtein concentrations were assayed using the bicinchoninic acid (BCA) assay according to manufacturer’s instructions (Sigma-Aldrich). Human liver microsomal fraction contains 8.1 mg of protein in 1 mL, and cytosolic fraction 12.1 mg of protein in 1 mL. Rat liver microsomal fraction contains 8.5 mg of protein in 1 ml, and cytosolic fraction 13.1 mg of protein in 1 mL.v/v).Enzyme activities were assayed in human and rat liver cytosolic and microsomal fractions. The enzyme assays (each performed in 4–8 replicates) were repeated three times  and UV or fluorescence detection, according to the cited literature. Further incubation details are described either in the corresponding references, as well as in the papers from this laboratory e.g., [Enzyme activities of individual CYP forms were measured according to established protocols . The folxylation , CYP2D6 xylation , CYP2E1,xylation  and CYP3xylation , and midxylation ,41. Actiry e.g., ,43,44. CS-transferase , UDP-glase (GST ).50) value and the mechanism of enzyme inhibition was revealed. Sesquiterpenes CNER, TNER and FAR, non-volatile liquids, were pipetted, and proper concentration was calculated according to density. All compounds were dissolved in DMSO. The final solvent concentration in the inhibition assays was 1%, which had no effect on the enzyme activity (data not shown). Non-enzymatic interference of sesquiterpenes with some compounds in the reaction mixtures  was considered and excluded. All experiments were performed in three replicates, if not stated otherwise.Sesquiterpenes were first screened for inhibition of biotransformation enzymes in subcellular fractions in vitro, then the half-maximal inhibitory concentration . The IC50 values for each sesquiterpene were determined by plotting sigmoidal dose response curves of specific enzyme activity vs. logarithm of sesquiterpene concentration using GraphPad Prism version 6.07 for Windows .The concentration of sesquiterpenes required for 50% inhibition of enzyme activity, the ICThe kinetic inactivation study of the CYP1A2 (EROD activity) and CYP2B/3A (BROD activity), both in rat and human, with TNER, was performed to find out the mechanism of enzyme inhibition by this sesquiterpene. Mechanism of EROD and BROD inhibition was tested at the final ethoxyresorufin or benzyloxyresorufin concentrations 0.5–5.0 µM. The concentration of TNER was 5.0 µM.Experimental data were fitted to the Michaelis-Menten equation, basic kinetic parameters were determined using the program GraphPad Prism version 6.07 for Windows. The type of inhibition was estimated using a Lineweaver-Burk  plot.p < 0.05. Data are presented as the mean ± standard deviation.All experiments (each sample performed in four replicates) were repeated three times (HPLC measurement of CYP activities only twice). All calculations were done using Microsoft Excel and GraphPad Prism 6.07. Statistical significance was tested by one-way Anova, and differences were considered statistically significant when Taken together, acyclic sesquiterpenes FAR, CNER, and TNER showed certain potency to inhibit CYP1A, CYP2B and CYP3A activities in liver. On the other hand, these sesquiterpenes did not seriously affect other CYPs, carbonyl-reducing enzymes and conjugation enzymes. As CYP3A4 metabolizes about 50% of all drugs, its inhibition by sesquiterpenes might result in drug—sesquiterpene interactions. Nevertheless, the results obtained in vitro should be verified in vivo. Rat seems to be suitable model for these in vivo studies, as comparable sensitivity of drug-metabolizing enzymes to these sesquiterpenes have been observed in rat and human samples.


The influence of the Madden-Julian oscillation (MJO) on Antarctic sea ice extent has not been extensively explored. This study investigates intraseasonal variability of the sea ice extent induced by the MJO and its physical mechanism. During austral winter, the sea ice extent anomaly exhibits considerable melting and freezing as the MJO evolves. Numerical experiments and the Rossby wave theory show that the high-latitude circulation anomalies in response to the MJO are responsible for the sea ice change. The MJO-induced Rossby waves propagate into the Southern Hemisphere through the northerly ducts over the western Indian Ocean–central Africa and the Maritime Continent. The MJO-induced circulation anomalies reach high latitudes and lead to anomalous meridional temperature advection, causing changes in the sea ice extent. The time difference between the meridional wind and sea ice anomalies is ~5 days. As the MJO moves, the sea ice extent anomaly also exhibits eastward-migrating behavior. Strong sea ice melting in the total anomaly is synchronous to the evolution of the MJO, suggesting the practical usefulness of the location of the MJO for the prediction of the sea ice decrease. Antarctic sea ice is found to be affected by atmospheric and oceanic internal modes on the seasonal to interannual and interdecadal time scales, such as the El Niño–Southern Oscillation (ENSO)9, Southern Annular Mode11, Antarctic Circumpolar Wave12, and Atlantic Multidecadal Oscillation13. These variabilities induce high-latitude circulation anomalies, leading to warm or cold air advection, anomalous sea ice motion, and ocean current change, and ultimately changes in the sea ice. On the intraseasonal time scale, there may be atmospheric or oceanic processes responsible for the changes in Antarctic sea ice. In particular, remote forcing from the tropics to the polar region needs to be examined.Sea ice in the polar region plays a crucial role in modulating the stratospheric polar vortex14. The MJO is known as a source of the subseasonal-to-seasonal predictability of weather and climate15. Many previous studies have explored the effects of the MJO on global circulation17, regional precipitation19, and extratropical temperature23. Recently, the MJO is proposed to influence the polar region28. For example, a study showed that enhanced diabatic heating over the far western Pacific induces warming over the Arctic through the zonal mean fluid dynamics26, and therefore, it is anticipated that the sea ice over this region can be changed29. There were also earlier studies to examine the MJO effect on the change in Antarctic sea ice31. However, these were on a short intraseasonal time scale , and its impact was not found. The latter arises because an MJO index that is not separated by season was used, thus, the genuine seasonal effect was not captured well. While the MJO moves eastward along the equator during boreal winter, northward or northwestward propagation occurs along with the typical eastward movement during austral winter32. Additionally, it is likely that the synoptic-scale internal variability in the extratropics is larger than the atmospheric response forced from the tropics on this rather short intraseasonal time scale31. In this study, we particularly investigate the effects of the tropical lower frequency variation with time scale ranging from 20 to 90 days. The time scale has a significant impact on the extratropical circulation and teleconnection, and, in fact, it is commonly used in studies associated with the MJO-related teleconnection to higher latitudes.This study demonstrates the impact of the Madden–Julian oscillation (MJO) on the intraseasonal variability of Antarctic sea ice. The MJO is the dominant physical mode on the intraseasonal time scale with planetary-scale circulation coupled with a large-scale convective complex in the tropics10 immediately or with some time lag through the thermal forcing and the mechanical forcing. The former is related to temperature advection by wind and the latter is related to the effect of wind stress on the sea ice velocity and Ekman drift in the ocean. Between the two, thermal forcing contributes dominantly to the intraseasonal variability of Antarctic sea ice33. On the intraseasonal time scale, atmospheric circulation variation correlates coherently with sea ice extent variation30. Since the circulation plays an important role, it is essential to investigate the dynamical processes of the MJO-induced circulation response in the Southern Hemisphere.Atmospheric circulation modulates the sea ice concentration and coverThis study aims to explore the influence of the MJO on Antarctic sea ice extent during austral winter and to investigate the dynamical mechanism generating the large-scale circulation response to the MJO. Since teleconnection pattern due to tropical forcing is prominent in the winter hemisphere due to the existence of strong potential vorticity in the background dynamic field, it is expected that the MJO influences the climate over the Antarctic region. For this, a series of simulations using a linear baroclinic model is performed, and ray tracing for poleward propagating waves is applied based on Rossby wave theory.34. Enhanced (suppressed) convection arises over the Indian Ocean in phase 1 (5) and disappears over the western North Pacific  in phase 8 (4). When the dipole convection anomalies are located over the Indian Ocean and western North Pacific , the Pacific-South America (PSA)-like teleconnection pattern develops in the mid-to-high latitudes of the Southern Hemisphere  and 250-hPa geopotential height anomalies is performed with respect to the MJO index (see the Methods section for details about the composite and the definition of the index). During austral winter , MJO convection moves northward or northeastward with a northwest-to-southeast-tilted structure. The MJO lifecycle is divided into 8 phases according to their convection location Fig. 34. EnhanOverall, as the MJO advances eastward, the MJO-induced circulation anomaly in the high latitudes (approximately from 80° S to 50° S) also propagates eastward. For example, a cyclonic circulation anomaly . Because it takes about two weeks for the circulation response to tropical convection to reach the high latitudesThe result of the model experiment shows that the MJO phase-2 forcing  generates a teleconnection pattern, with the anticyclonic circulation anomaly over the MD and the cyclonic circulation anomaly over the RS and Amundsen-Bellingshausen Seas Fig. . This paOn the other hand, the MJO phase-4 forcing  produces anticyclonic circulation over the MD and cyclonic circulation over the Amundsen-Bellingshausen Seas and AP Fig. , similar39. This is confirmed by calculating the stationary total wavenumber (Ks), which implies the preferred region for wave propagation40. The inset plot in Fig. The results of the linear model simulations show that the high-latitude circulation anomaly propagates eastward as does the diabatic forcing , produces a cyclonic circulation over the RS and Amundsen-Bellingshausen Seas at phases 3–4. The anomalous circulation is responsible for the sea ice extent anomaly, with an increase in the MD and a decrease in the RS. Meanwhile, the enhanced convection over the Indian subcontinent and Maritime Continent, representing MJO phase 4, induces an anticyclonic circulation anomaly over the MD and a cyclonic one over the Amundsen-Bellingshausen Seas and AP at phases 5–6, causing an increase in the sea ice extent in the RS and EW and a decrease in the AP. The physical processes are schematically summarized in Fig. The anomalous circulation generates the temperature advection by the meridional wind anomaly that modulates the sea ice variation. That is, the meridional wind anomaly leads to a temperature change within a day, while the latter tends to produce sea ice melting or freezing during the next 4 days. As the MJO propagates eastward from the Indian Ocean to the western North Pacific, the teleconnection also shifts to the east . Following the high-latitude circulation anomaly, the sea ice extent anomaly exhibits eastward-migrating behavior as well. It is suggested that the induced circulation pattern over the sea ice region near Antarctic is nearly antisymmetric for the opposite MJO convective forcing.Intraseasonal variability of the sea ice extent accounts for up to 29% of the total variation in the sea ice extent. Although the amount seems to be rather small, this may have an implication on the real prediction of the sea ice extent using the MJO phase because the change in the total sea ice extent anomaly is also synchronous to the evolution of the MJO.36, but the major causes of this movement have not been elucidated. This study shows that the variation in Antarctic sea ice extent is driven by the austral winter MJO.The eastward-migrating property of Antarctic sea ice concentration anomaly has been reported in earlier studies44. All obtained variables has 2.5° horizontal resolution. Intraseasonal variability for each variable is obtained by removing the annual cycle, which is calculated as the first three harmonics of the calendar mean cycle, and then by applying a 20–90-day Lanczos bandpass filter. Only austral winter, from June to August [JJA], is considered for the period of 1979 to 2015.The 25 × 25 km gridded ice concentration data provided in the National Snow and Ice Data Center (NSIDC) are used. The data are derived by the brightness temperature field obtained from Nimbus-7 SMMR and DMSP SSM/I-SSMIS. Missing values in the data are replaced by linear temporal interpolation. Using the sea ice concentration, the sea ice extent is calculated by summing the areas of the grid cells where the sea ice concentration is greater than 15%. The local sea ice extent is reinterpolated into a regular 2.5° × 2.5° grid and meridionally summed over the Southern Hemisphere. The local sea ice extent is linearly detrended before analysis to exclude the global warming effect. The daily outgoing longwave radiation (OLR) data from the National Oceanic and Atmospheric Administration (NOAA) are utilized as a proxy for the tropical deep convection. For other atmospheric variables such as circulation and near surface temperature, the ERA-Interim reanalysis data provided by the European Centre for Medium-range Weather Forecasts (ECMWF) are used34. The phase rotates from phase 1 to 8, and phases 1–4 represent an antisymmetric structure with respect to phases 5–8. The MJO index is calculated with the amplitude of the normalized first two PCs  analysis is performed with the filtered OLR anomaly in the tropics from 30°N to 30°S. The obtained first two EOF modes are used to divide the MJO propagation into 8 phases, according to the location of MJO convectione-holding decay time of 6 h for the largest wavenumber, and the vertical diffusion is set with a damping time scale of 1000 days to suppress the vertical computational mode. The time scale of linear drag, which mimics Rayleigh friction and Newtonian damping, is set to 0.5 days for the lowest three levels, 1 day for the topmost two levels, and 20 days for the mid-levels. The experiment is initialized with the JJA-climatological background state, which contains the 3-dimensional wind, temperature, geopotential height, specific humidity, and mean sea level pressure. To imitate the MJO convection in the experiment, the areas where the OLR is greater (lesser) than 10 (−10) W m−2 in the composite map are chosen as diabatic cooling (heating). Since the MJO convection is not spatially stationary, the diabatic forcing is given for the first 7 days and then turned off. Note that the simulated circulation fields have weaker intensity than that observed due to the switching-off of the forcing. If the forcing is persistently applied, the intensity is similar to that of the observation, although the circulation pattern in the subtropics is somewhat different than that indicated from the observation.To investigate the circulation response to the MJO, a numerical experiment is performed using an atmospheric linear baroclinic model (LBM) with T42 horizontal resolution and 20 vertical levels in sigma coordinates. The LBM is based on the primitive equations and its code is available from http://ccsr.aori.u-tokyo.ac.jp/~lbm/sub/lbm.html. The horizontal diffusion is set such that the 46. The dispersion relationship, which includes the effect of the basic meridional flow on a beta plane, is written as47:ω, k, and l are the angular frequency and the zonal and meridional wavenumber, respectively, and f is the planetary vorticity. The case of ω = 0 produces the stationary Rossby wave. The zonal and meridional group velocities, calculated by the partial derivatives of equation  in the Mercator coordinate system is written as Ksvalue indicates the preferred region for wave propagation40.The stationary total wavenumber (Supplementary Figures


Drosophila, a paradigm for gradient formation taught to biology students for more than two decades. “Seeing is believing” also summarizes the erroneous steps that were needed to elucidate the mechanisms of gradient formation and the path of movement of Bcd. Initially proclaimed as a dogma in 1988 and later incorporated into the SDD model where the broad diffusion of Bcd throughout the embryo was the predominant step leading to gradient formation, the SDD model was irrefutable for more than two decades until first doubts were raised in 2007 regarding the diffusion properties of Bcd associated with the SDD model. This led to re-thinking of the issue and the definition of a new model, termed the ARTS model which could explain most of the physical constraints that were inherently associated with the SDD model. In the ARTS model, gradient formation is mediated by the mRNA which is redistributed along cortical microtubules to form a mRNA gradient which is translated to form the protein gradient. Contrary to the SDD model, there is no Bcd diffusion from the tip. The ARTS model is also compatible with the observed cortical movement of Bcd. I will critically compare the SDD and the ARTS models as well as other models, analyze the major differences, and highlight the path where Bcd is localized during early nuclear cycles.In this commentary, I will review the latest findings on the Bicoid (Bcd) morphogen in  Drosophila during the late 80’s [bcd resides at the top of the hierarchy of segmentation genes [The Bicoid (Bcd) protein and its gradient Fig.  is one oate 80’s , 2, it wate 80’s . bcd reson genes  to patteon genes  via its on genes . These, on genes  that fursynthesis”, D for “diffusion” and D for uniform “degradation”) was initially used to explain the establishment of the gradient [bcd mRNA stays strictly at the tip during all developmental stages (Fig. The SDD model (S standing for “gradient , 9. The ges Fig.  serving ges Fig. , followeAttempts to validate the model were inconclusive for a long time and it took almost two decades to realize that the diffusion constant of Bcd was far too low to move to the posterior  and thatactive RNA transport and synthesis”. The ARTS model incorporates the formation of a bcd mRNA gradient and involves active transport of the mRNA by means of microtubules forming a mRNA gradient first (Fig. In 2009, a new model for Bcd gradient formation was proposed solving most of the physical constraints associated with the apparent slow diffusion constant of Bcd: the ARTS model –21, stanrst Fig. , followerst Fig. . One reprst Fig. , f, it iIn 2007, a model was proposed to involve Bcd diffusion combined with nucleocytoplasmic shuttling, but no Bcd degradation . The nucTo precisely monitor the path of Bcd movement during early development, a sensitive approach was developed that allowed for the study of the spatial Bcd movement during the early nuclear cycles using single confocal sections . This stDrosophila, e. g. that of the blow fly Lucilia sericata [The drug treatment data of  revealedsericata , 23 solvbcd mRNA along the cortex [Data on the content and structure of the inner yolk is limited, primarily due to microscopic constraints, because the laser of a confocal microscope cannot penetrate deeply into the optically dense yolk layer. Actin microfilaments in the inner yolk were described , 25, bute cortex . It is pe cortex  describebcd gene resulting in 5 different isoforms, some of which have been characterized [D. melanogaster, D. pseudoobscura [bcd cDNA in the past were done based on the original c53.46.6 bcd cDNA clone [bcd-null phenotype [bcd+ 5 + 8 embryos that allowed the rescue of embryos with severely altered Even-skipped stripe patterns and thus substantially changed segmental anlagen [Currently, little data is known regarding the alternative splicing of the cterized , 30. Of cterized  represencterized . The foucterized . Interesoobscura , that gaNA clone  encodinghenotype , however anlagen , 34. A l anlagen . Consist anlagen , despite anlagen . Unfortu anlagen  did not bcd mRNA gradient as the template for the protein gradient [This question was discussed by  in an efgradient , 17. MorWhy does it make sense to observe Bcd residing and moving along the cortex and not moving towards the interior? Arguably, the most important finding is the cortical location of the mRNA Fig.  and the


Ice containing essential oils (EOs) nanoencapsulated in β-cyclodextrins (β-CD) (named as EOs+β-CD ice) was used for stunning/slaughtering by hypothermia in ice slurry, and for ice storage of gilthead seabream. Clove essential oil (CEO) was used at fish stunning/slaughtering, while ice storage of whole fish was performed using a combination of carvacrol, bergamot and grapefruit EOs (CBG). Inclusion complexes CBG+β-CD were characterized, and antimicrobial effect was also evaluated. The kneading method used to form inclusion complexes with CBG showed a good complexation efficiency. Microbial, physical-chemical and sensory analyses were carried out to assess the quality changes of fresh whole seabream during ice storage at 2 °C for 17 days. Results  indicated that seabream stunning/slaughtering and storage using EOs+β-CD ice  improved the quality of fresh fish and extended the shelf-life up to 4 days. For example, In these studies, the EOs doses used are relatively high. For example, CDs are cyclic oligomers of α-D-glucopyranose, of which β-cyclodextrins (β-CD) are the most used in food technology. The β-CD molecule exhibits the shape of a truncated hollow cone, which interior cavity is hydrophobic, and the exterior is hydrophilic. These characteristics allow β-CD be a good tool for increasing water solubility and protecting EOs against oxidation and evaporation. Hence, β-CD have been used in food technology for molecular encapsulation of EOs –forming an inclusion complex named as EOs+β-CD in this work–, which have been recognised as GRAS . KneadinCitrus bergamia Risso) EOs, which have a fresh and elegant aroma, are limonene, linalyl acetate, linalool, γ-terpinene, and β-pinene. Grapefruit EOs are a mixture of volatile compounds like terpenes and oxygenated derivatives such as aldehydes , alcohols and esters, their main component being D-limonene. Bergamot and grapefruit extracts have been studied to inhibit the oxidation of fish lipid with successful results, and were the most preferred citrus in terms of sensory analyses  for storage of fresh whole fish in ice has not been studied. Thus, this work characterizes the quality consistency of the newly prepared EOs+β-CD and also studies the use of EOs+β-CD ice for ice-storage of fresh seabream to improve its freshness quality and shelf-life, and in combination or not with the use of clove EOs (CEO) nanoencapsulated in β-CD, and embedded in ice, for stunning and slaughtering of farmed seabream and for decreasing its stress, as described in 22.1Bergamot, carvacrol and grapefruit essential oils were obtained from Lluch Essence (Spain). β-CDs was provided by Roquette (France). The EOs+β-CD inclusion complex powder was prepared using 1:1 molar ratio (1g of CBG and 7.6 g of β-CD) following the kneading method , and theIn previous studies, the most effective dose of EOs  for the conservation of fish on ice was selected. Doses were evaluated from 25 to 100 mg EOs/kg of ice, and the dose of 50 mg EOs/kg of ice was finally selected because it was adequately effective in keeping the freshness of the fish on ice.2.22.2.1−1 and under a nitrogen atmosphere with a flow rate of 50 mL min−1. Thermograms were obtained for pure carvacrol essential oil, pure mix of carvacrol, bergamot and grapefruit , pure β-cyclodextrine and the EOs+β-CD complexes.The Differential Scanning Calorimetry (DSC) technique was utilized to analyze the thermal behavior of the EOs+β-CD inclusion complexes. The analyses were performed using a differential scanning calorimeter DSC 822e . Ten mg of each sample were placed in 100 μL sealed aluminum crucibles whose lips were perforated with a 50 μm diameter pin. Thermograms were obtained at temperatures of 25 °C–400 °C, with a heating rate of 10 °C min2.2.2The amount of EOs entrapped in the inclusion complex was determined by UV-Vis spectroscopy  at 254 nm. 5 mg of sample was dissolved in 5 mL of acetonitrile and left for 48 h after being well mixed to allow enough time for all entrapped active compound to be in solution. After this procedure, the solutions were centrifuged at 3200 rpm for 15 min to remove any β-CD from the solution, leaving only the active compound . A standard curve of carvacrol was prepared with concentration ranging of 2.5–30 μg/mL, under the same conditions. The EE% was calculated as follow: complex .2.2.3FITR used 10 mg of each samples, pure mix of carvacrol, bergamot and grapefruit  and the EOs+β-CD complexes. Attenuated total reflectance–Fourier transform infrared spectra were obtained by means of a Nicolet 5700 spectrometer  using a single-bounce diamond attenuated total reflection accessory  at room temperature (25 °C).−1 with OMNIC 8.0 software (Thermo Fisher Scientific Inc.), although the complete spectrum was collected in the 4000–400 cm−1 range.Spectral data were treated in the range of 3600–2400 cm2.2.4The morphology of β-cyclodextrins and the EOs+β-CD complexes was analyzed using a Scanning Electron Microscope (Model S-3500N Hitachi). The samples were coated with a thin film of platinum and analyzed at different magnifications: 1000x and 2000x.2.3The seabream fish  were obtained from a fish farm of the company Servicios Atuneros del Mediterráneo SL  located in South-eastern Spain. The stunning/slaughtering of farmed fish was carried out by hypothermia in crushed ice slurry . It was used normal crushed ice (without EOs+β-CD), and crushed ice including CEO inclusion complex in β-CD (CEO+β-CD) in two concentrations: 10 and 15 mg CEO/kg ice. The stunning/slaughtering of seabream was conducted as described in -Current slaughtering and ice storage using normal crushed ice (control).-Current slaughtering with normal ice and ice storage using CBG+β-CD ice.-Stunning/slaughtering with 10 mg CEO+β-CD ice slurry, and storage with normal crushed ice.-Stunning/slaughtering with 10 mg CEO+β-CD ice slurry, and storage with CBG+β-CD crushed ice.-Stunning/slaughtering with 15 mg CEO+β-CD ice slurry, and storage with normal ice.-Stunning/slaughtering with 15 mg CEO+β-CD ice slurry, and storage with CBG+β-CD crushed ice.The different treatments studied in this work are the followings:The polystyrene boxes were provided with holes for water drainage, and were transported to the pilot plant of the Universidad Politécnica of Cartagena to be stored in a cold room at 2 ± 0.5 °C for 17 days. Melting ice was replaced daily with a fresh quantity and excess water was drained.Fish samples (4 units) were taken on day 1, 7, 11, 15 and 17 from ice storage to perform the different analyses. One fish unit was for physicochemical and microbiological analyses, and the other three for the sensory analysis.The experiments described were approved by the General Directorate of Agriculture, Livestock, Fisheries and Aquaculture  of the Autonomous Community of the Region of Murcia . These experiments were conducted according to established animal welfare guidelines, and this study complies with all regulations.2.4For microbiological analyses, 25 g of fish muscle sample from dorsal region was diluted with 225 mL sterile buffered peptone water (Scharlau Chemie) in a sterile stomacher bag  and homogenized for 2 min using a masticator . Serial dilutions of each suspension were carried out in sterile peptone water (Scharlau Chemie), and analysed for microbial counts.Pseudomonas sp. were determined on Cetrimide Agar (Scharlau Chemie) after incubation at 37 °C for 48 h. Lactic acid bacteria were counted on Man Rogosa and sharpe Agar (Scharlau Chemie) incubated at 31 °C for 48 h. Enterobacteriaceae spp. were counted on violet red bile glucose agar (Scharlau Chemie) incubated at 37 °C for 24 h. All plates were performed in duplicate and the results were expressed as logarithms of the number of colony-forming units per gram (log CFU/g).Appropriate aliquots (0.1 or 1 mL) were spread on agar plates. Plate Count Agar (Scharlau Chemie) was used for enumeration of mesophilic and psychrotrophic aerobic bacteria, and plates were incubated for 48 h at 30 °C and 7 days at 4 °C respectively. 2.5(TMA-N) was determined using the Picric Acid Method  and measured by spectrophotometer at 410 nm. TMA-N content was expressed as mg N/100 g fish muscle. The Water-holding capacity (WHC) analysis was based on the method described by The pH was measured with a pH meter . Fish muscle (5 g) was homogenized thoroughly with 10 mL of distilled water and the homogenate was used for pH determination. Trimethylamin nitrogen 2.6Colour was measured on muscle from dorsal region surface by L*, a* and b* system CIE (1986), using a Minolta Chroma Meter CR400 . Each value was the average of three determinations per samples. In the CIE Lab system, L* denotes lightness on a 0–100 scale from black to white; a*, (+) red or (-) green; and b*, (+) yellow or (-) blue .2.7The sensory attributes of fish were evaluated by a panel of seven experienced judges and for each day of sampling. The assessment of fish was conducted using the Tasmanian Food Research unit (TRFU) system modified by Furthermore, sensory evaluation by panellists was also realised for cooked seabream muscle. Fish muscle sample (25 g) from dorsal region was cooked individually in a microwave oven at full power (850 W) for 2 min, and then presented to panellists. Sensory evaluation was conducted in individual booths under controlled conditions of light, temperature and relative humidity. Panellists were asked to score odour, taste and texture of fish using a 0–10 descriptive hedonic scale .2.8p˃0.05. Data were subjected to analysis of variance (ANOVA), followed by Tukey HSD multiple range tests to evaluate the significant difference (p˂0.05) among the different treatments. A regression analysis was used to determine the shelf-life of seabream. The data were fitted by a linear model, Y = a+bX, where Y represents mesophilic aerobic count and X represents the time of ice storage.Results are reported as mean values ± standard error. For data analysis, ANOVA and standard derivation were used. Significant differences were defined as 33.13.1.1) found similar results. The DSC curve of mix of carvacrol, bergamot and grapefruit (3:1:1) and β-CD+carvacrol, bergamot and grapefruit (β-CD+CBG) inclusion complexes are shown in Santos et al., 2015). Thermograms for β-CDs structure were relatively stable between temperatures of 100 °C and 250 °C as shown in Figs. Piletti et al., 20173.1.2EE is used to determine the amount of EOs entrapped in the inclusion complex. The percentage of complexation of EOs in the cavity of β-CD was 68.16%. Other authors found similar results. 3.1.3−1. FTIR is used to confirm the presence of specific chemical groups in the inclusion complexes . omplexes . The speomplexes . The FIT3.1.4In order to assess morphological structure and shape of inclusion complexes, SEM imaging was performed. Images obtained by SEM are shown in 3.2th day.The changes in the microbial flora of seabream from different treatments along ice storage are shown on Figs. Enterobacteriaceae counts showed a very low initial population indicating good fishing practises and handling. In addition, this low value indicates good hygiene of the marine environment where seabream was farmed and caught. In fact, the contribution of Enterobacteriaceae to the microflora of fish and its potential spoilage must be taken into consideration especially in the case of polluted water or delay in chilling after catch (Enterobacteriaceae counts (log CFU/g) ranged from below 1 in the different treatments. All these counts increased with ice storage time and from day 7 of ice storage. Counts in seabream stunned/slaughtered with CEO+β-CD crushed ice and ice stored with CBG+β-CD crushed ice were below counts of seabream control (4.5 log CFU/g). From the results shown in er catch . The iniPseudomonas has been reported to be the specific spoilage bacteria in fresh Mediterranean fish stored in ice (Pseudomonas count (day 1) was lower than the detection limit in all treatments  low count in all sample groups (between 1 and 2 log CFU/g) indicates good fish quality . MAB couThe low initial psychrophilic bacteria count indicates good fish quality . PsychroPsychrophilic bacteria are the major group of microorganisms responsible for aerobic spoilage of chilled stored fish . In the According to 3.3The pH values of the fish muscle varies during post-mortem storage according to different parameters as species, season and others factors. The initial pH of seabream was between 6.35 and 6.50 and these values increased during storage at 2 °C . The incp˃ 0.05) between control treatment group and seabream treated with EOs+β-CD ice (at stunning/slaughtering and ice-storage). Higher values were detected in seabream slaughtered and stored with control ice. These values were lower to those found for seabream when kept under storage with slurry and crushed ice at 4 °C , panellists found fresh odour and typical attributes of the raw seabream in all conditions, indicating the maximum quality and freshness of seabream. Differences in sensory characteristic were found on 15th day. On day 15 seabream control  presented worst attributes: skin was dull, eyes concave and opaque cornea, gills were brown and with spoiled fish odour. Thus, the estimate shelf-life for the seabream control could be 13 days. However, on day 17 seabream stunned/slaughtered and ice stored using EOs+β-CD crushed ice was sensory acceptable but at limit of acceptability. In this way it is shown that, in this case, the count of psychrophilic microorganisms is more useful in estimating the shelf-life of the fish than MAB counts.According to the panellists, EOs odours were not detected during ice storage. Thus, panellists did not detect differences between samples stored in control ice and samples stored with EOs+β-CD ice. Hence, EOs+β-CD ice (used with low doses in stunning/slaughtering and ice storage) had not any adverse effect on the sensory acceptability of seabream.Similar results were found in cooked seabream (data no shown). Control seabream were worst evaluated along cold storage by the panellists. Neither odour nor taste of the cooked seabream slaughtered and stored with EOs-β-CD ice was negatively affected. The panellists did not detect unpleasant odours and taste due to EOs aroma. Thus, the use of EOs-β-CD ice in stunning/slaughtering and ice storage do not affect sensorial characteristics of fresh and cooked seabream, when low doses are used, as the handled in this work.Our previous work showed that using 10 and 15 mg CEO+β-CD crushed ice during stunning/slaughtering promoted a decrease in plasmatic glucose levels, which confirm CEO+β-CD ice decreases the stress in fish at slaughtering . FurtherSeabream slaughtered or stored in ice including encapsulated EOs showed lower microbiological and chemical values due to the antimicrobial and antioxidant properties of the essential oils. In fact, the microbiological shelf-life was established in 18 days for seabream from control treatment while seabream slaughtered or stored in antimicrobial ice had an extended shelf-life (up to 6 days). Use of antimicrobial ice extended the shelf-life of seabream stored at 2 °C between 4 and 6 days if comparing with seabream from control treatment.According to the results of sensory analyses, up to 15 days all the conditions were determined as fresh fish but on day 17 control seabream was no longer acceptable for consumption. Antimicrobial CBG+β-CD crushed ice avoids unpleasant sensorial attributes and improve sensorial acceptability. Thus, the application of CEO+β-CD during stunning/slaughtering, in combination with ice storing of seabream did not show off-flavour and off-odour along storage time. Thus, antimicrobial ice including CBG+β-CD can be a good option to extend shelf-life for marine species by fishing industry due to beneficial effects and the low price of EOs+β-CD crushed ice.Laura Navarro-Segura, Amanda E López Cánovas, Isabel Cabas: Performed the experiments; Analyzed and interpreted the data.María Ros-Chumillas: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Garcia Ayala Alfonsa: Analyzed and interpreted the data; Wrote the paper.Antonio Lopez Gomez: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.PESCAMUR SL Company  and CDTI , Project Number: IDI-20150100, and co-financed by the European Regional Development Fund (ERDF) through the Pluriregional Operational Programme for Intelligent Growth.This work was funded by The authors declare no conflict of interest.No additional information is available for this paper.