Devaraj Ezhilarasan

Department of Pharmacology, the Blue Lab, Molecular Medicine and Toxicology Division, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Chennai 60 0 077, Tamil Nadu, India

Keywords:Hepatic stellate cells Glutaminolysis Glycolysis Hedgehog Myofibroblasts

ABSTRACT Background: Upon liver injury,quiescent hepatic stellate cells (qHSCs),reside in the perisinusoidal space,phenotypically transdifferentiate into myofibroblast-like cells (MFBs).The qHSCs in the normal liver are less fibrogenic,migratory,and also have less proliferative potential.However,activated HSCs (aHSCs) are more fibrogenic and have a high migratory and proliferative MFBs phenotype.HSCs activation is a highly energetic process that needs abundant intracellular energy in the form of adenosine triphosphate (ATP)for the synthesis of extracellular matrix (ECM) in the injured liver to substantiate the injury.Data sources: The articles were collected through PubMed and EMBASE using search terms“mitochondria and hepatic stellate cells”,“mitochondria and HSCs”,“mitochondria and hepatic fibrosis”,“mitochondria and liver diseases”,and“mitochondria and chronic liver disease”,and relevant publications published before September 31,2020 were included in this review.Results: Mitochondria homeostasis is affected during HSCs activation.Mitochondria in aHSCs are highly energetic and are in a high metabolically active state exhibiting increased activity such as glycolysis and respiration.aHSCs have high glycolytic enzymes expression and glycolytic activity induced by Hedgehog(Hh) signaling from injured hepatocytes.Increased glycolysis and aerobic glycolysis (Warburg effect) endproducts in aHSCs consequently activate the ECM-related gene expressions.Increased Hh signaling from injured hepatocytes downregulates peroxisome proliferator-activated receptor-γ expression and decreases lipogenesis in aHSCs.Glutaminolysis and tricarboxylic acid cycle liberate ATPs that fuel HSCs to proliferate and produce ECM during their activation.Conclusions: Available studies suggest that mitochondria functions can increase in parallel with HSCs activation.Therefore,mitochondrial modulators should be tested in an elaborate manner to control or prevent the HSCs activation during liver injury to subsequently regress hepatic fibrosis.

Introduction

Chronic liver disease (CLD) causes significant morbidity and mortality worldwide [1] .Chronic alcohol consumption,hepatitis B and C virus infections,aflatoxin B1 contaminated food,and nonalcoholic steatohepatitis are considered primary risk factors for the progression of various manifestations of CLD [2] .Advanced liver fibrosis results in impaired metabolic function,liver contraction,cirrhosis,portal hypertension,and liver failure and often needs liver transplantation [3] .Advanced liver fibrosis and cirrhosis are also major risk factors for hepatocellular carcinoma (HCC) with up to 90% of HCC cases occurring on the background of a cirrhotic liver [3-5] .Among the various manifestations of CLD,hepatic fibrosis is considered a converging point for the deterioration of liver architecture.Hepatic fibrosis is now considered a reversible process [6] .However,pesistant fibrosis may progress into cirrhosis,HCC,and subsequently liver failure [7] .

Hepatic stellate cells (HSCs)

Fig.1.Progression of hepatic fibrosis.CLD:chronic liver disease;qHSCs:quiescent hepatic stellate cells;aHSCs:activated hepatic stellate cells;PTD:portal triad;KC:Kupffer cell;ECM:extracellular matrix;SEC:sinusoidal endothelial cells;CLD:chronic liver disease;HVPG:hepatic venous pressure gradient;PT:portal hypertension.

HSCs are liver-specific mesenchymal cells that reside in the perisinusoidal space or space of Disse between hepatocytes and sinusoidal endothelial cells [8,9].In the normal liver,HSCs account for 15% of total resident cells [10] .HSCs are quiescent,contain lipid droplets and store vitamin A in the normal liver and also they have diverse roles in the regulation of matrix degradation and synthesis,epithelial cell fate,immune modulation,and tissue health [11,12].After a chronic liver injury,quiescent HSCs (qHSCs)undergo phenotypical transdifferentiation to maintain the pathophysiological status of the injured liver [13] .Firstly,in response to chronic liver injury,qHSCs undergo phenotypic transdifferentiation into activated HSCs (aHSCs) and acquire proliferative,contractile,and myofibroblast (MFBs)-like phenotype known as initiation.Secondly,the aHSCs or MFBs-like cells secrete several cytokines and chemokines to maintain the pathophysiological status of the injured liver is termed as perpetuation (Fig.1) [7,14].For instance,in the injured liver,α-smooth muscle actin (α-SMA)positive aHSCs secrete transforming growth factor-beta (TGF-β),platelet-derived growth factor (PDGF),krüppel-like factor 6 (KLF6),matrix metalloproteinases (MMPs),tissue inhibitor of metalloproteinases (TIMPs) and several other signaling molecules.These signaling molecules provoke HSCs for their migration,proliferation,and synthesis and accumulation of a variety of extracellular matrix(ECM) proteins including fibril forming collagens Iα1 and III in the perisinusoidal space that cause metabolic hindrance and portal hypertension (Fig.2) [3,13,15].Hepatocytes mitochondria have unique features compared to other cell’s mitochondria,as they perform metabolism of carbohydrates,lipids,and proteins [16] .Hepatocyte mitochondrial dysfunctions are associated with various liverrelated ailments including hepatic fibrogenesis [17-19] .Ample evidence suggests that increased mitochondrial activity can protect hepatocytes from various injuries [16] .Although numerous studies have described the role of mitochondria in hepatocytes during fibrogenesis,studies on the role of mitochondria in aHSCs during hepatic fibrosis are meager.This review exclusively focuses on the mitochondrial changes during the HSCs activation in the chronically injured liver.

Mitochondria in fibrogenic HSCs

Mitochondria are an important hub for biosynthetic processes and are said to regulate stress response and cell energy metabolism [20] .Mitochondria act as both source and target for intracellular reactive oxygen species (ROS) and are involved in the progression of oxidative stress mediated hepatic fibrosis [2] .In hepatic fibrosis,activation of HSCs is an important and highly dynamic process involving the synthesis of an enormous amount of ECM.Mitochondria fuel for the activation of HSCs by modulating their function.Interestingly,aHSCs have higher mitochondrial functions such as increased mitochondrial respiration (basal and maximal),adenosine triphosphate (ATP) and calcium production,spare respiratory capacity,and proton leak as compared to qHSCs [21,22].The mitochondria positively regulate the metabolic activities like glycolysis and glutaminolysis in aHSCs than in qHSCs.Mitochondrial membrane potential (Δψm) also alters during HSCs activation [23] .The requirement of increased energy for HSCs activation is also achieved by increased mitochondrial biogenesis and gene expressions associated with mitochondrial biogenesis that are significantly increased in aHSCs than in qHSCs [22] .

Fig.2.Chronic liver injury and hepatic stellate cells (HSCs) activation associated events.qHSC:quiescent HSCs;aHSC:activated HSCs;MFBs:myofibroblasts;α-SMA:alpha smooth muscle actin;PDGF:platelet derived growth factor;TGF-β:transforming growth factor-β;MMPs:matrix metalloproteinases;TIMPs:tissue inhibitors of metalloproteinases;ECM:extracellular matrix;GLS:glutaminase;GDH:glutamate dehydrogenase;GS:glutamine synthetase;AST:aspartate aminotransferase;Δψm:mitochondria membrane potential;PPAR-γ:peroxisome proliferator-activated receptor gamma.

HSCs activation associated changes in mitochondrial respiration

Mitochondrial dysfunction can lead to aberrant metabolism and cell death via apoptosis [9] .Increasing evidence suggests that mitochondrial dysfunction in hepatocytes contributes to the activation of HSCs,thereby resulting in hepatic fibrogenesis [17-19] .However,studies have also shown the mitochondrial dysfunction in HSCs during its activation in the fibrotic liver [18,24].An increased mitochondrial activity only in fibrogenic or activated human primary,LX-2 (a human fibrogenic HSCs cell line),and in rat HSCs were reported.For instance,mitochondrial respiration analysis showed an elevated oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in fibrogenic or aHSCs.Energy maps based on the OCR and ECAR analysis have confirmed that fibrogenic HSCs have the higher energy level and this is suggested to substantiate the functional demand in mitochondria [23] .In a very recent study,significant upregulation of mitochondrial respiration and glycolytic activity was seen in TGF-βinduced LX-2 cells [25] .TheΔψm was shown to characteristically increase only in aHSCs as compared to their respective less fibrogenic phenotypes.Only aHSCs exhibited an increased and distinct elevation of mitochondrial functions and increasedΔψm rendering them sensitive to mitotropics and this effect was not seen in HSCs with quiescent phenotype and healthy human primary hepatocytes [23] .For instance,a combination of doxorubicin with triphenylphosphonium,a mitochondrial homing moiety contains high lipophilicity inducing cytotoxicity only in aHSCs but not in qHSCs [23] .Activation of Hedgehog (Hh) signaling has been implicated for increased mitochondrial function in aHSCs while Hh signaling inhibitors reduced mitochondrial respiration in aHSCs and their proliferation [26] .

Mitochondrial uncoupling is defined as a dissociation between mitochondrial membrane potential generation and its use for mitochondrial-dependent ATP synthesis [27] .Originally,this process was considered a mitochondrial dysfunction.However,studies also indicated that“mild”mitochondrial uncoupling and decrease in ATP levels,can produce a beneficial effect in metabolic diseases like obesity,diabetes and nonalcoholic fatty liver disease(NAFLD) [28,29].Studies have shown that mild mitochondrial uncoupling in cells can trigger the orchestration of peroxisome proliferator-activated receptor-γ(PPAR-γ) coactivator -1αsignaling cascade to compensate for the reduction in ATP levels [27,30].Taking as a lead,a study has showed that mild mitochondrial uncoupling and associated ATP loss can interfere with fibrogenic functions of aHSCs [29] .The mitochondrial chemical uncouplers such as valinomycin and carbonyl cyanide-ptrifluoromethoxyphenylhydrazone treatments inhibited proliferation and reduced ROS levels in mouse and human HSCs [29] .Further,mild mitochondrial uncoupling significantly reduced the fibrogenic markers such asα-SMA,actin alpha 2 (ACTA2),PDGF,and procollagen 1 protein expressions and interferes with profibrogenic TGF-βsignaling [22,29].The main role of aHSCs in the fibrotic liver is ECM synthesis [7],which is a highly dynamic process and HSCs activation requires a high amount of intracellular energy in the form of ATP and therefore,alterations in mitochondrial homeostasis and ATP loss can decrease HSCs activation and fibrogenic functions.

HSCs activation associated metabolic changes in mitochondria

Increased mitochondrial activity was reported during HSCs activation and studies have shown increased mitochondria number in aHSCs.In contrast to qHSCs,the aHSCs express higher level of glycolytic enzymes such as hexokinase 2 (HK2),phosphofructokinase (PFK),pyruvate kinase M2,glucose transporter (GLUT) 1,and monocarboxylate transporter 4 [31,32] suggesting an urgent demand for mitochondrial processes that promote energy production and anabolism.Inhibition of glycolysis by 2-Deoxy-D-glucose causes a phenotypic reversion of aHSCs to qHSCs suggesting increased mitochondrial activity in aHSCs.Induction of aerobic glycolysis (Warburg effect) in HSCs induces their phenotypic activation and fibrogenic function.The rate and the transformation level of glucose to lactate are accumulated intracellularly in aHSCs even when amounts of oxygen are available [33] .Therefore,the inhibition of pyruvate to lactate conversion in aHSCs by a lactate dehydrogenase A inhibitor results in an imbalance of lactate/pyruvate ratio,inhibition of their proliferation,suppression of fibrogenic gene expression,and induces lipid accumulation and upregulation of genes involved in lipogenesis [31,33].

Hh signaling pathway plays an important role in acute and chronic liver injuries by controlling the fate of HSCs,and promotes liver fibrosis [34,35].Hh signaling acts as a metabolic switch and is responsible for mitochondrial reprogramming in qHSCs to convert them into MFBsinvitroandinvivo[26] .At a molecular level,Hh signaling activates the Warburg effect during HSCs activation.The resultant glycolytic end-products i.e.lactate metabolically reprogram qHSC into highly proliferative and fibrogenic MFBs [31] .The gene expressions that regulate glycolysis,Hh signaling,and hypoxia-inducible factor 1α(HIF1α) are upregulated upon HSCs activation.Hypoxia-induced migration of MFBs involves an early mitochondrial-dependent ROS-mediated activation of extracellular signal-regulated kinases (ERKs),and the c-Jun N-terminal kinases(JNKs) [36] .Therefore,inhibitors of glycolysis,or lactate accumulation,inhibitors of Hh signaling,and HIF1αcan significantly decrease lactate accumulation,Hh signaling,and HIF1αrespectively in MFBs and cause phenotypic reversion i.e.MFBs to qHSCs [31] .Upon chronic liver injury,HSCs not only undergo phenotypic transdifferentiation towards an MFB-like phenotype but also accompanied by a loss of the intracellular lipid droplets characterizing their quiescent state [37] .In a bile duct ligated hepatic fibrosis model,injured hepatocytes release Hh ligands activates Hh signaling in qHSCs and inhibits lipogenesis and gluconeogenesis through downregulation of PPAR-γand other lipogenic genes [31] .This study states that Hh signaling is not only responsible for the glycolytic changes in MFBs but also causes lipid loss,a characteristic feature of HSCs activation.

Glutamine is a non-essential amino acid abundantly present in the circulation and has multiple metabolic functions in cells [38] .Glutamine metabolism (glutaminolysis) has emerged as a critical regulator of many cellular processes in diverse pathologies [39] .The glutaminolytic pathway enzymes such as glutaminase (GLS),glutamate dehydrogenase (GDH),and transaminases which convert glutamine to glutamate and glutamate to alphaketoglutarate (α-KG) involve in glutamine metabolism.Glutaminederivedα-KG can supplementα-KG that is regenerated during the tricarboxylic acid (TCA) cycle to enhance TCA cycle activity,increasing both ATP production and key metabolic intermediates for the biosynthesis of nucleic acids and other biological macromolecules [26,39].Interestingly,increased glutamine metabolism is identified as a key metabolic characteristic during HSCs activation [26] .Disruption of the glutaminolysis pathway decreases aHSCs proliferation and collagen production ability.HSCs proliferation is critically dependent on glutamine that is used to generateα-KG and non-essential amino acid.Glutamine synthetase (GS) has been reported to be induced during HSC activation at the protein level [40] .Glutaminolysis has been implicated as one of the main mechanisms for regulation of HSCs activation because glutaminolytic activity fuels anapleurosis to meet the elevated demands of bioenergetic and biosynthetic pathways needed for the maintenance of MFB-like phenotype in the fibrotic liver [33] .In vitro(LX-2 cells) and CCl 4 -induced liver fibrosis rats,the aHSCs utilize more glutamine and upregulate the gene expressions responsible for enzymes such as GLS,aspartate transaminase (AST),and GDH that involved in glutamine metabolism,while inhibition of these enzymes or depletion of glutamine prevents HSCs activation.These experimental studies were correlated with a clinical investigation,in which,gene expressions ofGLS,GDH1,AST1,andAST2were significantly increased in the human fibrotic liver tissue [40] .

Hh signaling not only is responsible for the increased glycolytic activity but also induces glutaminolysis to satisfy the increased demands for energy and anabolic substrates when qHSC transdifferentiate to MFBs [26] .Interestingly,conditional deletion or inhibition of Hh signaling suppresses the phenotypic transformation of qHSCs into MFBs.For instance,in CCl 4 -induced hepatic fibrotic mice,administration of cyclopamine,an Hh signaling pathway inhibitor,has remarkably decreased mitochondrial respiration and proliferation of MFBs [26] .Hippo signaling and its downstream effectors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are involved in cell proliferation,survival,development,differentiation,metabolism,and cross-talk with the immune system [41] .YAP is an important downstream effector of the Hh pathway during liver repair [26] .The high Hh signaling induces the expression of glutaminolysis associated genes during the phenotypic transition of qHSCs to MFBs.In a transfection study,when mice primary HSCs were treated with cyclopamine or verteporfin,an YAP inhibitor,the expressions of GLS,collagen 1α1,and MMP-2 were decreased [26] .This study showed that inhibition of glutaminolysis and its associated signaling with their specific inhibitors could suppress glutaminolysis,mitochondrial respiration,cell growth and migration,and fibrogenesis potentials of aHSCs in the fibrotic liver.

ATP-citrate lyase (ACL) is a key lipogenic enzyme that converts mitochondria-derived citrate in the cytoplasm to acetyl-CoA [42] .Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl-CoA for fatty acid biosynthesis,regulation,and metabolism [43] .5 ′ -AMP-activated protein kinase (AMPK) is a member of the serine/threonine kinase family,plays a pivotal role in fatty acid biosynthesis by negatively regulating ACC.Increased ACC expression and inhibition of AMPK signaling were reported during HSCs activation in the fibrotic liver [44,45].In a study,activation of AMPK expression inhibits PDGF induced HSCs proliferation,activation,and migration,and these effects were associated with the inhibition of Akt and extracellular ERK signaling pathways [46] .Invitro,ACC inhibition represses the tension and TGF-βmediateds activation of primary rat and human HSCs.In the dimethylnitrosamine-induced hepatic fibrosis model,ACC inhibition significantly reduces fibrosis in rats.Increased ACC activity is responsible for the upregulation of mitochondrial respiration and glycolytic activity seen in TGF-βinduces LX-2 cells.ACC inhibition decreases mitochondrial respiration and glycolytic activity induced by TGF-βin HSCs.ACC inhibitors treated HSCs display a quiescent phenotype with normal mitochondrial function and metabolism [25] .This study showed that ACC inhibition could act as a metabolic switch to prevent glycolysis and oxidative phosphorylation during HSC activation.The overall mitochondria-associated changes during HSCs activation is depicted in Fig.3 .

Other signaling molecules involved in mitochondria-mediated HSCs activation

PDGF signaling is important for HSCs proliferation and their activation [47] .PDGF induces cell proliferation and migration via ERK activation and Akt/mammalian target of rapamycin (mTOR) pathways [48] .Mitochondrial ROS production is also an important factor for HSCs activation [2,49,50].Several experimental studies have shown that intracellular ROS accumulation by hepatocytes in the injured liver is responsible for HSCs activation via paracrine signaling [51-56] .Studies on the status of mitochondrial ROS accumulation during HSCs activation are meager.However,a few studies reported the mitochondrial ROS role in HSCs activation.For instance,p66Shc is a redox enzyme that mediates mitochondrial ROS generation.Invivo,p66Shc expression was increased upon CCl 4 -induced fibrotic liver in mice.In primary HSCs,p66Shc expression caused mitochondrial ROS production and altered mitochondrial morphology and cellular metabolism.Invivoknockdown andinvitrop66Shc silencing inhibited ROS accumulation in mitochondria and activation of NOD-like receptor protein 3 (NLRP3) inflammasome in aHSCs [57] .In another study,lipopolysaccharides (LPS)treatment induced ROS expression in aHSCs,leading to NLRP3 inflammasome activation.LPS treatment also increased mitochondrial superoxide dismutase 2 (SOD2) and ROS in aHSCs which was negatively correlated with mitophagy [58] .These studies show that activation of inflammasome plays an important role in mitochondrial ROS mediated HSCs activation.Expressions of augmenter of liver regeneration (ALR) were decreased in hepatic tissues both in human and mice with liver fibrosis.ALR expression was decreased following HSCs activation.The knockdown of ALR deteriorates liver fibrosis and activates HSCs,thereby promoting F-actin assembly which accelerates the HSCs migration in the fibrotic liver.HSCs activation caused by ALR knockdown was associated with increased mitochondrial calcium and increased ATP synthesis [21] .A mitochondrial serine protease,high-temperature requirement protein A2 (HtrA2/Omi) was found to function as quality control in mitochondrial homeostasis.HtrA2/Omi deficiency caused accumulation of mitochondrial ROS and mitochondrial defect in mouse primary hepatocytes and in the CCl 4 -induced fibrotic liver.Therefore,the role of HtrA2/Omi should be studied studied in an eloborate manner in aHSCs and isolated HSCs from the fibrotic liver in rats and humans [24] .

Modulators of HSCs activation via mitochondrial functions

Apart from specific inhibitors of glycolysis,Yap,Hh signaling,ACC,and glutaminolysis,only a few natural and synthetic compounds have been reported to modulate the mitochondrial functions related to HSCs activation.For instance,metformin,a known AMPK activator,suppressed HSCs activation via inhibiting the Akt/mTOR and ERK pathways in LX-2 cells and CCl 4 -induced liver fibrosis in mice [59] .Curcumin was shown to inhibit aerobic glycolysis in aHSCs.Curcumin treatments decreased HSCs activation and downregulated the protein and gene expressions of important glycolytic enzymes such as HK2,PFK2,and glucose transporter 4 .Activation of AMPK was implicated in curcumin-induced glycolysis inhibition in aHSCs [32] .Costunolide,a plant-derived sesquiterpene lactone treatment has reduced fibrosis markers such asα-SMA and collagen expressions,and also reduced HSCs activation by repressing the expression and activity of HK2,an important rate-limiting enzyme that regulates glycolysis [60] .Astaxanthin,a xanthophyll carotenoid,decreased mitochondrial respiration in aHSCs (LX-2 cells).Further,primary HSCs isolated from astaxanthin treated mice showed a decrease in mitochondrial cristae junction and lumen width,and area without altering cristae length,and also these cells had lower mitochondrial respiration and glycolysis than qHSCs [22] .

Future prospective

Increased mitochondrial functions in aHSCs are directly correlated with their activation.Mitochondria are a critical hub for ROS generation.Unlike hepatocytes,the status of mitochondrial antioxidants such as SOD2 and other enzymic and non-enzymic antioxidants are not studied elaborately in aHSCs.Such studies are important to understand the mitochondrial ROS role in HSCs activation and to target potential antioxidant to control the oxidative stressinducing potential of mitochondria in aHSCs.At present,the available information suggests that mitochondrial biogenesis additionally fuels HSCs during their activation via increased ATP.However,to date,it is not clear whether enhanced ATP production in aHSC is due to emergence of increased mitochondrial numbers (biogenesis) or increased functions of existing mitochondria,or both are responsible for HSCs activation in the fibrotic liver.The mitochondrial link to the TGF-βsignaling remains elusive and it is very important to understand the profibrogenic singling from mitochondria to the nucleus during HSCs activation and should be studied in the near future.TheΔψm is increased during HSCs activation and this leads us a question whether aHSCs are resistant to intrinsic or mitochondrial apoptosis.The shape and structure of mitochondria in aHSCs should be investigated using a transmission electron microscope to check the presence of megamitochondria during HSCs activation.At present,we do not know whether induction of mitophagy in aHSCs is useful to control HSCs activation and several such questions remaining unanswered.Further,decreased AMPK and increased Hh signaling impairs mitochondrial homeostasis and induces HSCs activation.The mechanism of Hh signaling mediated lipid loss during HSCs activation is also not clear.Therefore,AMPK agonists and Hh signaling antagonists should be tested in an elaborate manner.

Conclusion

Ample evidence suggests that HSCs activation does not depends on a single entity and it is a complex process involving multiple fibrogenic singling in the injured liver.The qHSCs have less fibrogenic,migratory,and proliferative potential.Once,qHSCs are activated,it acquires a high fibrogenic,migratory and proliferative MFBs-like phenotype.This phenotypic transition is a highly energetic process and hence,it needs an uninterrupted supply of intracellular energy in the form of ATP for the synthesis of ECM in the injured liver.Available studies suggest that mitochondria functions increase in parallel with HSCs activation.Unsurprisingly,mitochondria homeostasis is affected during HSCs activation.Studies have confirmed that aHSCs contain a highly metabolically active state of mitochondria and exhibit increased respiration,glycolysis,glutaminolysis,calcium level,and so on.The Warburg effect and glutaminolysis are not only the metabolic characteristics of cancer cells but also involve in HSCs activation in the fibrotic liver.Glutaminolysis and TCA cycle derived ATP fuels HSCs to proliferate and produce ECM during their phenotypic activation.An upregulation of Hh signaling is responsible for increased mitochondrial functions such as glycolysis,glutaminolysis,and TCA cycle.Therefore,mitochondrial modulators should be tested in a more elaborate manner to control or prevent the HSCs activation during liver injury to subsequently regress hepatic fibrosis.

Acknowledgments

None.

CRediTauthorshipcontributionstatement

DevarajEzhilarasan:Conceptualization,Writing -original draft,Writing -review &editing.

Funding

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Ethicalapproval

Not needed.

Competinginterest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.