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Physiologic and molecular bases of muscle hypertrophy and atrophy: impact of resistance exercise on human skeletal muscle (protein and exercise dose effects)1
Stuart M. Phillips

Abstract: Normally, skeletal muscle mass is unchanged, beyond periods of growth, but it begins to decline in the fourth or fifth decade of life. The mass of skeletal muscle is maintained by ingestion of protein-containing meals. With feeding, muscle protein synthesis (MPS) is stimulated and a small suppression of muscle protein breakdown (MPB) occurs, such that protein balance becomes positive (MPS > MPB). As the postprandial period subsides and a transition toward fasting occurs, the balance of muscle protein turnover becomes negative again (MPB > MPS). Thus, during maintenance of skeletal muscle mass, the long-term net result is that MPS is balanced by MPB. Acutely, however, it is of interest to determine what regulates feeding-induced increases in MPS, since it appears that, in a number of scenarios (for example aging, disuse, and wasting diseases), a suppression of MPS in response to feeding is a common finding. In fact, recent findings point to the fact that loss of skeletal muscle mass with disuse and aging is due not chronic changes in MPS or MPB, but to a blunted feeding-induced rise in MPS. Resistance exercise is a potent stimulator of MPS and appears to synergistically enhance the gains stimulated by feeding. As such, resistance exercise is an important countermeasure to disuse atrophy and to age-related declines in skeletal muscle mass. What is less well understood is how the intensity and volume of the resistance exercise stimulus is sufficient to result in rises in MPS. Recent advances in this area are discussed here, with a focus on human in vivo data. Key words: protein synthesis, protein breakdown, resistance exercise, protein. ` ` Resume : Generalement, la masse musculaire squelettique ne varie pas apres les annees de croissance, mais commence a diminuer autour de la 4e ou de la 5e decennie de vie. La masse des muscles squelettiques se maintient par lapport de pro ` teines dans les repas. Au moment de lalimentation, la synthese des proteines musculaires (MPS) est stimulee et la degra ` dation des proteines musculaires (MPB) est legerement inhibee ce qui donne un bilan proteique positif (MPS > MPB). Au ` fur et a mesure que la periode postprandiale secoule et que lorganisme est prive de nourriture, le bilan proteique dans les ` muscles passe de nouveau au negatif (MPB > MPS). Par consequent, au cours du processus de maintien a long terme de ` la masse musculaire squelettique, la MPS est equivalente a la MPB. Dentree de jeu, il serait interessant de connatre ce ` qui controle la MPS associee a lalimentation, car dans certains episodes de vie comme le vieillissement, latrophie par ` inactivite et les maladies cachectisantes, linhibition de la MPS constitue une reponse normale a lalimentation. De fait, ` des etudes recentes revelent que la perte de masse musculaire observee en periode dinactivite et au cours du vieillisse` ` ment nest pas due a des variations de MPS ou de MPB, mais bien a un emoussement de laugmentation de la MPS stimu lee par lapport alimentaire. Les exercices de force sont de puissants stimulateurs de la MPS et ameliorent probablement de facon synergique les gains stimules par lapport alimentaire. En tant que tel, les exercices de force constituent une ex cellente strategie de lutte contre latrophie par inactivite et la perte de masse musculaire squelettique associee au vieillisse ment. Neanmoins, on ne connat ni le degre dintensite ni le nombre de repetitions des exercices de force convenant pour ` accrotre la MPS. Cet article analyse les dernieres etudes dans ce champ dinteret et met laccent sur les observations in vivo chez lhumain. ` Mots-cles : synthese des proteines, degradation des proteines, exercice de force, proteines. [Traduit par la Redaction] Received 10 March 2009. Accepted 10 March 2009. Published on the NRC Research Press Web site at apnm.nrc.ca on 29 April 2009. S.M. Phillips. Exercise Metabolism Research Group, Department of Kinesiology, McMaster University, 1280 Main St. West, Hamilton, ON L8S 4K1, Canada (e-mail: phillis@mcmaster.ca).
1This

paper is one of a selection of papers published in this Special Issue, entitled 14th International Biochemistry of Exercise Conference Muscles as Molecular and Metabolic Machines, and has undergone the Journals usual peer review process.
doi:10.1139/H09-042 Published by NRC Research Press

Appl. Physiol. Nutr. Metab. 34: 403410 (2009)

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Introduction
In the years beyond those in which humans are growing, there is normally no net new accretion of skeletal muscle mass. In fact, beginning in the fourth or fifth decade of life, the mass of skeletal muscle begins to slowly decline, a condition referred to as sarcopenia (Evans 1995; Frontera et al. 1991; Hughes et al. 2001). Regardless of our age, however, muscle proteins are constantly and simultaneously being synthesized and degraded. Thus, maintenance of the mass of skeletal muscle is through the net balance between the processes of muscle protein synthesis (MPS) and muscle protein breakdown (MPB). This balance is maintained by ingestion of protein-containing meals, which results in a systemic hyperaminoacidemia that is stimulatory for the synthesis of new muscle proteins (Bennet et al. 1989; Bohe et al. 2001, 2003; Fujita et al. 2007b). It appears that this stimulation of MPS is driven almost exclusively by the hyperaminoacidemia and not by systemic hyperinsulinemia, which is merely permissive but not overtly stimulatory for MPS (Greenhaff et al. 2008; Bell et al. 2005; Fujita et al. 2006; Chow et al. 2006). Comparisons of feeding-induced changes in MPS and MPB highlight the fact that changes in MPS are severalfold higher than changes in MPB, pointing to regulation of this arm of protein turnover as the prime locus of acute regulation (Biolo et al. 1997; Fujita et al. 2007b; Paddon-Jones et al. 2004; Volpi et al. 1998, 2003; Greenhaff et al. 2008). In fact, even transcriptional changes would be dependent on well-timed and appropriately scaled increases in protein translation to manifest a change in phenotype. Thus, from a number of standpoints, the regulation of MPS is important. This is not to underestimate the importance of MPB in determining muscle protein mass or quality but, in the nondiseased state, MPB is not elevated and so does not play quantitatively as important a role as changes in MPS. For example, in states such as aging, there is an anabolic resistance to feeding, which is characterized by a blunted rise of MPS to amino acids (Cuthbertson et al. 2005; Guillet et al. 2004). A parallel result occurs in uncomplicated disuse atrophy (Glover et al. 2008b), with the major difference between the 2 states being that, in atrophy, at least in young persons, there is a decline in basal MPS (Glover et al. 2008b; Ferrando et al. 1996; Paddon-Jones et al. 2006a) and no increase in MPB (Paddon-Jones et al. 2006a; Ferrando et al. 1996). Skeletal muscle is a plastic tissue with the ability to respond to a variety of external stimuli, such as exercise. Regular performance of dynamic endurance-type exercise results in a muscle phenotype that is more fatigue resistant, highly oxidative, and has a high capacity for lipid oxidation (Holloszy and Booth 1976; Holloszy and Coyle 1984; Holloszy 2001). By contrast, regular performance of resistance exercise induces an increase in muscle fibre cross-sectional area and greater force generating capacity, and is less stimulatory for changes in oxidative capacity (Tang et al. 2006; Staron et al. 1984, 1991, 1994; Hakkinen et al. 1998). The potency of resistance exercise as a stimulator of MPS is evident in the fact that the acute increase in MPS is of greater magnitude and, especially, of far longer duration than the change after feeding (Phillips et al. 1997; Miller et al. 2005; Tang et al. 2008).

An important consideration is the substantive differences between signaling events that are thought to trigger a rise in MPS as a result of feeding and resistance exercise in humans and those observed in rodent models (reviewed in Kimball et al. 2002; Bolster et al. 2004). In addition, when it comes to resistance exercise, the magnitude of change and the temporal pattern of activation of MPS in rodents are markedly different from that seen in humans. In rodents, there are also marked fibre-type-dependent differences in rates of protein turnover. For example, Mittendorfer et al. (2005) reported that biopsies taken from the soleus, vastus, and triceps muscles of men exhibited virtually identical rates of MPS at rest and in response to feeding, a finding also reported for vastus and soleus by Carroll et al. (2005), despite observing differing levels of phosphorylated and total 4EBP-1 between muscles. These findings are in marked contrast to data from rats (Goldspink et al. 1986, 1983; Goldspink 1978; Loughna et al. 1986; Lewis et al. 1984; Kelly et al. 1984), in which differences as high as 2-fold in rates of protein turnover have been reported between muscles comprised of slow- and fast-fibre types. Therefore, when designing studies that aim at characterizing the regulation of protein metabolism, in an effort to develop interventions aimed at combating muscle wasting, there is a substantive advantage to studying a human model. Despite understanding of the way feeding and resistance exercise affect MPS, only now are researchers beginning to study how the system responds, in terms of signaling pathways that are activated with either stimuli in humans (Dreyer et al. 2006, 2008; Fujita et al. 2007a, 2007b; Koopman et al. 2006, 2007; Witard et al. 2009; Glover et al. 2008a; Eliasson et al. 2006; Karlsson et al. 2004; Kumar et al. 2009). While the elucidation of these pathways has ostensibly meant progress in the understanding of the regulation of MPS, there are reports of substantial mismatches between signaling and actual measured MPS in rats (Greenhaff et al. 2008). Thus, there is still much to learn about how MPS is regulated from a signaling standpoint. It is known, at this point, that proteins of the Akt-mammalian target of rapamycin (mTOR)-S6 kinase (K) pathway (Fig. 1) are intimately involved in turning on MPS, but the extent of signal to response is far from clear. Moreover, redundancies in signaling are highly likely and may not be unraveled quickly without the use of some broad-based protein and phosphorprotein screening approaches.

Changes in human MPS as a result of feeding

Feeding protein or amino acids stimulates MPS, an effect that appears to be due, almost exclusively, to the amino acids themselves (Biolo et al. 1997; Fujita et al. 2007b; Paddon-Jones et al. 2004; Volpi et al. 1998, 2003; Greenhaff et al. 2008). It appears that only the indispensable amino acids are required to manifest this effect (Tipton et al. 1999b; Volpi et al. 2003). In particular, the amino acid leucine occupies a position of prominence, in that it alone can act as a stimulatory signal for MPS (Anthony et al. 2000, 2002). In humans, the ability of leucine alone to act as a signal for activating MPS has been tested only once (Smith et al. 1992); however, many lines of evidence point to the abilPublished by NRC Research Press

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Fig. 1. Schematic representation of signal pathways for activation of mammalian target of rapamycin (mTOR), leading to ribosomal assembly, biogenesis, and global protein synthesis by both amino acids (leucine) and insulin. The cell-surface receptors could be the insulin and (or) insulin-like growth-factor receptor, integrins (Int), or other growth-factor and (or) mitogenic receptors. Adapted from Proud (2007) and Bolster et al. (2004). IR, insulin receptor; IRS, insulin receptor substrate; TSC, tuberous sclerosis complex; Rheb, rat homologue enriched in brain.

ity of leucine to act in a stimulatory manner for feeding-induced increases in MPS (Katsanos et al. 2006; Karlsson et al. 2004; Greiwe et al. 2001). It should be noted, however, that even if leucine alone were to stimulate a rise in MPS by activating proteins in the mTOR pathway, in the absence of substrate (i.e., a full complement of indispensable amino acids), MPS would ultimately slow and eventually revert to basal levels. Thus, complete mixtures of amino acids, both infused (Biolo et al. 1997; Bohe et al. 2001, 2003) and ingested (Tipton et al. 1999a), or ingestion of intact proteins (Tipton et al. 2004; Wilkinson et al. 2007; Symons et al. 2007; Katsanos et al. 2008; Paddon-Jones et al. 2006b) have led to increases in MPS. Interestingly, the process of MPS is saturable and appears to be a function of the extracellular rather than the intracellular amino acid concentrations (Bohe et al. 2003). The response of MPS in both young and older persons was found to be curvilinear, with an approximate plateau at 10 g of indispensable amino acids (Cuthbertson et al. 2005). A notable difference existed in the response to exogenous amino acids between young and old, in that the gain for each amino acid dose and the overall

amplitude of the curve was greater for the young vs. the old, indicating a greater sensitivity and overall net anabolism in response to amino acids. This anabolic resistance of MPS in aging was accompanied by a reduced signaling response in the elderly, indicating a potential dampening of signaling with aging. The underlying mechanism for this adaptation remains unclear. Recently, Moore et al. (2009) reported on the dose-response of MPS following resistance exercise, using intact isolated egg protein as a dietary source. Similar to what was reported previously (Cuthbertson et al. 2005), at 20 g of ingested protein, a plateau in MPS was observed. Figure 2 shows a plot of the MPS data, as well as a corresponding plot of leucine oxidation as an index of amino acid catabolism, as percentage of the basal (i.e., 0 g) protein dose. Surprisingly, no notable changes in signaling protein phosphorylation were observed that would explain the changes in MPS. It is possible that this was due to the fact that exercise alone had already stimulated (estimated via phosphorylation of activating sites) the signaling machinery maximally and that protein ingestion could not increase
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406 Fig. 2. Percentage increases (from basal level, or 0 g) in muscle protein synthesis (MPS) and leucine oxidation after resistance exercise in young men as a function of ingested protein dose. The ingested protein was isolated egg protein. Data from Moore et al. (2009).

Appl. Physiol. Nutr. Metab. Vol. 34, 2009

in athletes trained in either dynamic endurance events or in high-intensity resistance exercise are remarkably different when exposed to the opposite training stimulus (Coffey et al. 2006). By contrast, when exposed to their regular training stimulus, there was very little change in signaling (Coffey et al. 2006). This dampened training-induced, but contraction mode-specific, response is not surprising, and is something Wilkinson et al. (2008) have recently shown in a longitudinal study from both the signaling and MPS responses. Indeed, it stands to reason that the phenotype induced by each contraction mode (i.e., mitochondrial proliferation by dynamic endurance work and myofibrillar proteins by high-intensity resistive work) is manifest as an increased synthetic rate of the proteins that are increased by chronic performance of that exercise mode. It has long been held by those who study the phenomenon of resistance-training-induced hypertrophy that a contraction-intensity threshold exists to induce hypertrophy. This belief extends back to the classic work of Delorme (1945), who, in 1945, made the following conclusions:
Low-repetition, high-resistance exercises produce power. High-repetition, low-resistance exercises produce endurance. Each of these two types of exercise is incapable of producing results obtained by the other. In order to obtain rapid hypertrophy in weakened, atrophied muscle, the muscle should be subjected to strenuous exercise and, at regular intervals, to the point of maximum exertion.

the level of phosphorylation any further (Moore et al. 2009). Such a conclusion may seem at odds with the idea that exercise and feeding are synergistic in their ability to stimulate rises in protein synthesis (Tang et al. 2007; Moore et al. 2005; Biolo et al. 1997); however, Glover et al. (2008a) observed this synergism in S6K1 and ribosomal protein S6, critical proteins of the Akt-mTOR pathway (Fig. 1), but at 6 h postexercise. Nonetheless, in the absence of a difference in signaling protein phosphorylation, it was concluded that the increase in MPS would simply have been a substrate (i.e., amino acid) -driven response. At this point, regrettably, it is still largely unknown exactly how amino acids act to affect changes in MPS, either alone or in combination with resistance exercise. Bohe et al. (2003) proposed the existence of a leucine sensor, which they hypothesized would be a membrane-bound protein responsive to extracellular amino acid concentrations. This is an intriguing possibility and one that deserves further investigation.

Changes in MPS as a result of resistance exercise

There are a number of reports that have shown resistance, both alone (Chesley et al. 1992; Welle et al. 1993, 1995; Yarasheski et al. 1993, 1999; Phillips et al. 1997, 1999) and in combination with feeding (Rasmussen et al. 2000; Tipton et al. 1999a, 2003; Wilkinson et al. 2007), a marked stimulator of human MPS and, in the absence of nutrition, also MPB. It is only when feeding is superimposed on the postexercise stimulus, however, that a net positive protein balance and protein accretion occur (Rasmussen et al. 2000; Tipton et al. 1999a, 2003; Wilkinson et al. 2007; Biolo et al. 1997). Until recently, the acute events of varying intensities of resistance exercise on both signaling and the response of MPS were largely unknown; however, that is beginning to change. For example, responses of muscle signaling proteins

In fact, the propensity to advocate the practice of lifting heavier loads to induce hypertrophy and strength is an idea still inherent in even the most up-to date reviews on this topic (ACSM Position Stand 2009). Acute studies appear to support, at least in part, the lift heavier paradigm (Kumar et al. 2009), inasmuch as a rise in MPS is only seen when intensities of load lifted exceed 60% of the single repetition maximum (1RM). Of note, however, is that, at intensities beyond 60% and up to 90% of 1RM, there was a similar stimulation of MPS. This finding may indicate that chronic performance of resistance exercise at higher intensities (>60% of 1RM) has little additional value, as far as stimulating MPS, and possibly hypertrophy, is concerned. This supposition is predicated on the assumption that acute changes are meaningful in terms of predicting long-term hypertrophic changes, which is a concept that does have support (Hartman et al. 2007; Wilkinson et al. 2007). Other studies have leant credence to the concept that it may not be the intensity of the lift that is the active variable in determining the response of MPS. Fujita et al. (2007a) reported that even low-intensity exercise (20% of 1RM) resulted in stimulation of MPS, with an accompanying rise in S6K1 phosphorylation, when blood flow was occluded. This acute finding is perhaps not surprising when one considers that a number of training studies have shown that this practice of blood flow occlusion can result, when practiced chronically and even when lifting at low intensities (30% 40% of 1RM), in substantial hypertrophy and strength gains equivalent to those seen at 80% of 1RM (Abe et al. 2006). It is not known exactly why occlusion has this effect, but the most likely explanation is that the venous occlusion induces a local fatigue that forces recruitment of type II muscle fibres that would not normally otherwise be recruited at such low intensities. An alternative explanation put forward has
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407 ACSM Position Stand. 2009. Progression models in resistance training for healthy adults. Med. Sci. Sports Exerc. 41: 687 708. doi:10.1249/MSS.0b013e3181915670. PMID:19204579. Anthony, J.C., Yoshizawa, F., Anthony, T.G., Vary, T.C., Jefferson, L.S., and Kimball, S.R. 2000. Leucine stimulates translation initiation in skeletal muscle of postabsorptive rats via a rapamycin-sensitive pathway. J. Nutr. 130: 24132419. PMID: 11015466. Anthony, J.C., Reiter, A.K., Anthony, T.G., Crozier, S.J., Lang, C.H., MacLean, D.A., et al. 2002. Orally administered leucine enhances protein synthesis in skeletal muscle of diabetic rats in the absence of increases in 4EBP1 or S6K1 phosphorylation. Diabetes, 51: 928936. doi:10.2337/diabetes.51.4.928. PMID: 11916909. Baar, K., and Esser, K. 1999. Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise. Am. J. Physiol. 276: C120C127. PMID:9886927. Bell, J.A., Fujita, S., Volpi, E., Cadenas, J.G., and Rasmussen, B.B. 2005. Short-term insulin and nutritional energy provision do not stimulate muscle protein synthesis if blood amino acid availability decreases. Am. J. Physiol. Endocrinol. Metab. 289: E999 E1006. doi:10.1152/ajpendo.00170.2005. PMID:16030064. Bennet, W.M., Connacher, A.A., Scrimgeour, C.M., Smith, K., and Rennie, M.J. 1989. Increase in anterior tibialis muscle protein synthesis in healthy man during mixed amino acid infusion: studies of incorporation of [113C]leucine. Clin. Sci. (Lond.), 76: 447454. PMID:2714054. Biolo, G., Tipton, K.D., Klein, S., and Wolfe, R.R. 1997. An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein. Am. J. Physiol. 273: E122E129. PMID:9252488. Bohe, J., Low, J.F., Wolfe, R.R., and Rennie, M.J. 2001. Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids. J. Physiol. 532: 575 579. doi:10.1111/j.1469-7793.2001.0575f.x. PMID:11306673. Bohe, J., Low, A., Wolfe, R.R., and Rennie, M.J. 2003. Human muscle protein synthesis is modulated by extracellular, not intramuscular amino acid availability: a dose-response study. J. Physiol. 552: 315324. doi:10.1113/jphysiol.2003.050674. PMID: 12909668. Bolster, D.R., Jefferson, L.S., and Kimball, S.R. 2004. Regulation of protein synthesis associated with skeletal muscle hypertrophy by insulin-, amino acid- and exercise-induced signalling. Proc. Nutr. Soc. 63: 351356. doi:10.1079/PNS2004355. PMID: 15294054. Carroll, C.C., Fluckey, J.D., Williams, R.H., Sullivan, D.H., and Trappe, T.A. 2005. Human soleus and vastus lateralis muscle protein metabolism with an amino acid infusion. Am. J. Physiol. Endocrinol. Metab. 288: E479E485. doi:10.1152/ajpendo. 00393.2004. PMID:15507532. Chesley, A., MacDougall, J.D., Tarnopolsky, M.A., Atkinson, S.A., and Smith, K. 1992. Changes in human muscle protein synthesis after resistance exercise. J. Appl. Physiol. 73: 13831388. PMID:1280254. Chow, L.S., Albright, R.C., Bigelow, M.L., Toffolo, G., Cobelli, C., and Nair, K.S. 2006. Mechanism of insulins anabolic effect on muscle: measurements of muscle protein synthesis and breakdown using aminoacyl-tRNA and other surrogate measures. Am. J. Physiol. Endocrinol. Metab. 291: E729E736. doi:10.1152/ ajpendo.00003.2006. PMID:16705065. Coffey, V.G., Zhong, Z., Shield, A., Canny, B.J., Chibalin, A.V., Zierath, J.R., and Hawley, J.A. 2006. Early signaling responses to divergent exercise stimuli in skeletal muscle from welltrained humans. FASEB J. 20: 190192. PMID:16267123.
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been that the acute rise in growth hormone is somehow responsible for increases in skeletal muscle growth. Evidence to support the notion that growth hormone is in any way anabolic for skeletal muscle, or even affects MPS, is lacking; however, there is a lot of evidence to support the contrary position (Yarasheski et al. 1995; Rennie 2003; Liu et al. 2008). Thus, the possibility that it is the recruitment of type II fibres per se, independent of the exercise intensity, that is the prime variable affecting the stimulation of MPS and, ultimately, training-induced hypertrophy provides an interesting future avenue of study. It has been known for some time that, in response to resistance training, the larger type II fibres display a greater degree of hypertrophy than the smaller type I fibres (McCall et al. 1996; Staron et al. 1990, 1994). Moreover, recent evidence demonstrates that following resistance exercise, it is primarily the type II fibres that display an activation of many of the critical signaling proteins (S6K1, ribosomal protein S6, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase) involved in the regulation of MPS (Tannerstedt et al. 2008). Of these proteins, the response of S6K1 requires emphasis, since it was reported to be 3- to 4-fold higher in type II than in type I fibres (Tannerstedt et al. 2008). This is notable since the phosphorylation of this protein has been reported to be predictive of hypertrophy in rats (Baar and Esser 1999) and humans (Terzis et al. 2008), and is also predictive, at least in young persons, of the intensity-dependent rise in MPS (Kumar et al. 2009). Therefore, collectively, these data suggest that, regardless of exercise intensity, a prerequisite condition to maximize the anabolic effect of resistance exercise may be the activation of these highly trainable type II muscle fibres.

Conclusion
It is clear that we are beginning to understand how feeding and exercise influence changes in MPS and, ultimately, muscle mass. The next frontier in these investigations will be to determine how specific protein fractions (mitochondrial, myofibrillar, and sarcoplasmic) and, ultimately, specific proteins themselves, respond to exercise and nutrition. Integration of these data with signaling, gene, and metabolomic approaches will no doubt accelerate our understanding of these important stimuli and the effects they have on skeletal muscle.

Acknowledgements
Thanks to the graduate students who performed the work cited by the author for much of this work. A special thanks to Dan Moore for his conscientious and informative discussion and editing of this manuscript. This work was supported by grants to the author from NSERC, CIHR, the US National Dairy Council, and the US National Cattlemans Beef Association. The author declares no conflicts of interest.

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