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Type 2 deiodinase polymorphism causes ER stress and hypothyroidism in the brain

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Type 2 deiodinase polymorphism causes ER stress and hypothyroidism in the brain
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  Type 2 deiodinase polymorphism causes ERstress and hypothyroidism in the brain Sungro Jo, … , Miriam O. Ribeiro, Antonio C. Bianco J Clin Invest.  2018. https://doi.org/10.1172/JCI123176. Levothyroxine (LT4) is a form of thyroid hormone used to treat hypothyroidism. In the brain,T4 is converted to the active form T3 by type 2 deiodinase (D2). Thus, it is intriguing thatcarriers of the Thr92Ala polymorphism in the D2 gene ( DIO2  ) exhibit clinical improvementwhen liothyronine (LT3) is added to LT4 therapy. Here, we report that D2 is a cargo proteinin ER Golgi intermediary compartment (ERGIC) vesicles, recycling between ER and Golgi.The Thr92-to-Ala substitution (Ala92-D2) caused ER stress and activated the unfoldedprotein response (UPR). Ala92-D2 accumulated in the trans-Golgi and generated less T3,which was restored by eliminating ER stress with the chemical chaperone 4-phenyl butyricacid (4-PBA). An Ala92-Dio2 polymorphism–carrying mouse exhibited UPR andhypothyroidism in distinct brain areas. The mouse refrained from physical activity, sleptmore, and required additional time to memorize objects. Enhancing T3 signaling in the brainwith LT3 improved cognition, whereas restoring proteostasis with 4-PBA eliminated theAla92-Dio2 phenotype. In contrast, primary hypothyroidism intensified the Ala92-Dio2phenotype, with only partial response to LT4 therapy. Disruption of cellular proteostasis andreduced Ala92-D2 activity may explain the failure of LT4 therapy in carriers of Thr92Ala-DIO2. Research ArticleEndocrinologyMetabolism Find the latest version: http://jci.me/123176/pdf  The Journal of Clinical Investigation RESEARCH ARTICLE 1 jci.org Introduction Hypothyroidism results from autoimmune destruction or surgical removal of the thyroid gland. Hence, symptoms are due to insuf󰁦icient levels of thyroid hormones, which affects tens of millions worldwide 󰀨󰀱󰀩. T󰀴 is the main hormone secreted by the normal thyroid gland, but it needs to be converted to T󰀳 in order to gain full biological activi-ty 󰀨󰀲, 󰀳󰀩. In healthy individuals, the thyroid gland contributes only a small fraction of daily T󰀳 production 󰀨󰀼󰀲󰀰󰀥󰀩; the bulk of T󰀳 produc-tion happens outside the thyroid parenchyma via conversion from T󰀴 by 󰀲 deiodinases, D󰀱 and D󰀲. Thus, therapy consisting of daily tablets of levothyroxine 󰀨LT󰀴󰀩 to treat hypothyroidism is commonsensical, having evolved to be the standard of care for this disease 󰀨󰀱, 󰀴󰀩.About 󰀱󰀵󰀥 of LT󰀴-treated hypothyroid patients remain symp-tomatic, with impaired cognition and reduced physical activity despite appropriate treatment 󰀨󰀵󰀩. The srcin of these symptoms is not clear, with some speculating that LT󰀴 therapy is not suf󰁦icient for all patients; thus, symptoms may be due to residual hypothy-roidism. In fact, LT󰀴-treated hypothyroid patients lack thyroidal T󰀳 secretion; all T󰀳 is produced in tissues other than the thyroid gland, mostly via D󰀲. While it has been assumed that deiodinases restore T󰀳 homeostasis in LT󰀴-treated patients 󰀨󰀶󰀩, preclinical and clinical studies indicate that this pathway alone is not suf󰁦icient to fully restore daily T󰀳 production during LT󰀴 therapy 󰀨󰀷󲀓󰀱󰀰󰀩.Thyroidectomized rats on LT󰀴 exhibit hypothyroidism in the liver, skeletal muscle, and brain, despite normal serum thy-roid-stimulating hormone 󰀨TSH󰀩 levels 󰀨󰀸󰀩. Individuals on LT󰀴 with normal serum TSH exhibit higher BMI and tend to experi-ence greater use of beta blockers, statins, or antidepressant med-ication 󰀨󰀹󰀩 and lower energy expenditure 󰀨󰀱󰀱󰀩; they also exhibit dif󰁦iculty in weight management, fatigue, or low energy levels and problems with mood and memory 󰀨󰀱󰀲󰀩. The brain, in particu-lar, depends on T󰀳 produced via the D󰀲 pathway, which is located in glial cells. Indeed, most T󰀳 bound to nuclear thyroid hormone receptors 󰀨TR󰀩 in the brain is produced locally via the D󰀲 path-way 󰀨󰀱󰀳󰀩. This occurs via a paracrine-signaling mechanism in which glial cell–derived T󰀳 activates neuronal gene expression 󰀨󰀱󰀴󰀩. Thus, adequate D󰀲 functionality is critical in LT󰀴 treatment for hypothyroid patients, producing most circulating T󰀳 and also directly affecting intracellular T󰀳 levels. Levothyroxine (LT4) is a form of thyroid hormone used to treat hypothyroidism. In the brain, T4 is converted to the active form T3 by type 2 deiodinase (D2). Thus, it is intriguing that carriers of the Thr92Ala polymorphism in the D2 gene ( DIO2 ) exhibit clinical improvement when liothyronine (LT3) is added to LT4 therapy. Here, we report that D2 is a cargo protein in ER Golgi intermediary compartment (ERGIC) vesicles, recycling between ER and Golgi. The Thr92-to-Ala substitution (Ala92-D2) caused ER stress and activated the unfolded protein response (UPR). Ala92-D2 accumulated in the trans-Golgi and generated less T3, which was restored by eliminating ER stress with the chemical chaperone 4-phenyl butyric acid (4-PBA). An Ala92-Dio2 polymorphism–carrying mouse exhibited UPR and hypothyroidism in distinct brain areas. The mouse refrained from physical activity, slept more, and required additional time to memorize objects. Enhancing T3 signaling in the brain with LT3 improved cognition, whereas restoring proteostasis with 4-PBA eliminated the Ala92-Dio2 phenotype. In contrast, primary hypothyroidism intensified the Ala92-Dio2 phenotype, with only partial response to LT4 therapy. Disruption of cellular proteostasis and reduced Ala92-D2 activity may explain the failure of LT4 therapy in carriers of Thr92Ala-DIO2. Type 2 deiodinase polymorphism causes ER stress and hypothyroidism in the brain Sungro Jo, 1  Tatiana L. Fonseca, 2  Barbara M. L. C. Bocco, 2  Gustavo W. Fernandes, 2  Elizabeth A. McAninch, 1  Anaysa P. Bolin, 1,3  Rodrigo R. Da Conceição, 1,4  Joao Pedro Werneck-de-Castro, 1  Daniele L. Ignacio, 1  Péter Egri, 5  Dorottya Németh, 5  Csaba Fekete, 5  Maria Martha Bernardi, 6  Victoria D. Leitch, 7  Naila S. Mannan, 7  Katharine F. Curry, 7  Natalie C. Butterfield, 7  J.H. Duncan Bassett, 7  Graham R. Williams, 7  Balázs Gereben, 5  Miriam O. Ribeiro, 8  and Antonio C. Bianco 2 1 Division of Endocrinology and Metabolism, Rush University Medical Center, Chicago, Illinois, USA. 2 Section of Adult and  Pediatric Endocrinology, Diabetes &  Metabolism, Department of Medicine, University of Chicago, Chicago, Illinois, USA. 3 Department of Pharmacology, Biomedical Science Institute, University of São Paulo, and 4 Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Federal University of São Paulo, São Paulo, SP, Brazil. 5 Department of Endocrine Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary. 6 Graduate Program of Environmental and Experimental Pathology, Graduate Program of Dentistry, Universidade Paulista, São Paulo, SP, Brazil. 7 Molecular Endocrinology Laboratory, Department of Medicine, Imperial College London, London, United Kingdom. 8 Developmental Disorders Program, Center of Biological Science and Health, Mackenzie Presbyterian University, São Paulo, SP, Brazil.   Related Commentary: https://doi.org/10.1172/JCI125203 Authorship note:  SJ, TLF, and BMLCB contributed equally to this work. Conflict of interest:  ACB is a consultant for Sentier LLC and Synthonics Inc. License:  Copyright 2018, American Society for Clinical Investigation. Submitted:  June 25, 2018; Accepted:  October 11, 2018. Reference information:    J Clin Invest . https://doi.org/10.1172/JCI123176.  The Journal of Clinical Investigation RESEARCH ARTICLE 2 jci.org Figure 1. D2 recycles between Golgi apparatus and ER.  ( A – F ) Immunofluorescence of Thr92-D2 HY  stably expressing cells using the indicated antibodies. On the far right is the Pearson’s plot for each immu-nofluorescence image. The right top number is the Pearson’s coefficient for that specific cell. Pearson’s coefficient was calculated as follows: Thr92-DIO2 × α -GM130 (0.32 ± 0.06); Ala92-DIO2 × α -GM130 (0.36 ± 0.07). ( G – L ) Same as A – F , except that cells stably express Ala92-D2 HY . Arrows point to Golgi AlaD2 staining. Pearson’s coefficient: Thr92-DIO2 × α -p230 (0.33 ± 0.05); Ala92-DIO2 × α -p230 (0.65 ± 0.12; P   < 0.01 versus Thr92-DIO2 × α -p230). ( M  and N ) Thr92-D2 HY  (Thr) or Ala92-D2 HY  (Ala) pulldown, followed by Western blot analysis with the indicated antibodies. ( O ) Same as M  and N , except that cells transiently express Δ 18-D2 HY . ( P – R ) Same as A – C  except that cells transiently express Δ 18-D2 HY . ( S ) Pearson’s coefficient between the indicated D2 proteins and cis- Golgi marker GM130; Δ 18-D2 is Δ 18-D2 HY  ( P – R ). D2T is Thr92-D2 HY  ( A – C ). Δ C-D2T is Δ 10C-Thr92-D2 HY  ( T – V ). Δ C-D2A is Δ 10C-Ala92-D2 HY  ( W – Y ). ( T – Y ) Same as A – C , except that cells transiently express Δ 10C-Thr92-D2 HY  or Δ 10C-Ala92-D2 HY . Original magnification, APO ×60/1.40 oil objective. Values are shown in box-and-whiskers plot indicating median and quartiles. n  = 21/group. Statistical analysis used was Mann- Whitney U  test in comparison with D2T. *** P   ≤ 0.0001.  The Journal of Clinical Investigation RESEARCH ARTICLE 3 jci.org tor Atx-󰀳 bind to and direct D󰀲 to the proteasomes 󰀨󰀳󰀲󰀩. Here, we saw that both Thr󰀹󰀲󰀭D󰀲 HY  and Ala󰀹󰀲󰀭D󰀲 HY  pulldowns contained the p󰀹󰀷 adaptor UBXD󰀭󰀱, known to bind ERGIC󰀵󰀳 󰀨󰀳󰀳󰀩 󰀨Figure 󰀱, M and N󰀩. Thus, both forms of D󰀲 can be directed to the ERGIC via interaction with the p󰀹󰀷/UBXD󰀭󰀱/ERGIC󰀵󰀳 complex. The 󰀱󰀸-resi-due loop in D󰀲 󰀨󰀱󰀵󰀩 is also critical for interaction with p󰀹󰀷/UBXD󰀭󰀱: its truncation 󰀨 Δ 󰀱󰀸󰀭D󰀲 HY 󰀩 prevents ERGIC󰀵󰀳 pulldown 󰀨Figure 󰀱O󰀩 and reduces by 󰀳󰀰󰀥 colocalization with GM󰀱󰀳󰀰 󰀨Figure 󰀱, P󲀓S󰀩.Scanning the D󰀲 sequence led us to 󰀲 carboxyl target dibasic peptide ER-retrieval motifs, i.e., SKRUKKTR, where underlines indi-cate dibasic peptides 󰀨󰀳󰀴, 󰀳󰀵󰀩. Indeed, truncation of this sequence 󰀨 Δ 󰀱󰀰C󰀭ThrD󰀲 HY  or Δ 󰀱󰀰C󰀭AlaD󰀲 HY 󰀩 increased colocalization with GM󰀱󰀳󰀰 by approximately 󰀵󰀰󰀥 󰀨Figure 󰀱, S󲀓Y󰀩. Together, these stud-ies indicate that a bidirectional traf󰁦ic of D󰀲 between ER and Golgi exists, with both Thr󰀹󰀲󰀭D󰀲 HY  and Ala󰀹󰀲󰀭D󰀲 HY  recycling between ER and Golgi. These observations, however, do not explain why or how only Ala󰀹󰀲󰀭D󰀲 HY  accumulates in the trans-Golgi network.  ER stress pushes Ala󰀹󰀲󰀭D󰀲  HY   to the trans-Golgi . Ala󰀹󰀲󰀭D󰀲 HY  exhibits a longer half-life 󰀨󰀱󰀹󰀩, an indication that the Ala󰀹󰀲󰀭D󰀲 slows D󰀲 targeting to the proteasome and could lead to its det-rimental accumulation in the ER. Indeed, we detected UPR in Ala󰀹󰀲󰀭D󰀲 HY  cells 󰀨Figure 󰀲, A󲀓N󰀩. In these cells, there is mostly activation of the IRE󰀱 α  and ATF󰀶 pathways, which increase pro-tein-folding ability via chaperones and degradation of misfolded proteins. There was an approximately 󰀲-fold increase in IRE󰀱 α  phosphorylation 󰀨Figure 󰀲, A and B󰀩 and an approximately 󰀳-fold increase in spliced  XBP󰀱  󰀨 sXBP󰀱󰀩 mRNA levels 󰀨Figure 󰀲C󰀩 in cells expressing Ala󰀹󰀲󰀭D󰀲 HY . While levels of uncleaved ATF󰀶 protein remained stable 󰀨Figure 󰀲D󰀩, there was an approximately 󰀲-fold increase in cleaved ATF󰀶 protein 󰀨Figure 󰀲, D and E󰀩 and the mRNA levels of its downstream targets CHOP   󰀨~󰀳-fold; Figure 󰀲F󰀩 as well as the ER chaperone/folding protein BIP 󰀨~󰀴-fold; Figure 󰀲, G󲀓I󰀩. PERK phosphorylation 󰀨Figure 󰀲, J and K󰀩, which attenuates protein synthesis, as well as its downstream targets, such as EIF󰀲 α  phosphorylation 󰀨Figure 󰀲, L and M󰀩 and  ATF󰀴  mRNA levels, was not affected 󰀨Figure 󰀲N󰀩.In some settings, the Golgi quality control contributes to UPR by capturing misfolded proteins that emanate from the ER, divert-ing them for lysosomal degradation 󰀨󰀳󰀶󰀩. In fact, it is known that cleaved ATF󰀶 induces ERGIC󰀵󰀳 expression 󰀨󰀳󰀷󰀩 and its redistri-bution so that it is closer to the cis-Golgi 󰀨󰀳󰀸󰀩, both of which are features present in cells expressing Ala󰀹󰀲󰀭D󰀲 HY . Here, we found a 󰀲.󰀲-fold increase in ERGIC󰀵󰀳 protein levels 󰀨Figure 󰀲, O and P󰀩 and a 󰀵󰀰󰀥 increase in  ERGIC󰀵󰀳  mRNA levels 󰀨Figure 󰀲󰁑󰀩. The images con󰁦irm an increase in ERGIC󰀵󰀳 protein 󰀨Figure 󰀲, R and S󰀩, revealing a 󰀳󰀵󰀥 higher colocalization with GM󰀱󰀳󰀰 󰀨Figure 󰀲, T󲀓W󰀩 and indicating redistribution to cis-Golgi. Furthermore, colocalization between Ala󰀹󰀲󰀭D󰀲 HY  and GM󰀱󰀳󰀰 was approximate-ly 󰀳󰀰󰀥 lower in  ERGIC󰀵󰀳 –/–  cells 󰀨Figure 󰀳, A󲀓H󰀩, con󰁦irming that ERGIC󰀵󰀳 is involved in the distribution of Ala󰀹󰀲󰀭D󰀲 HY  to the Golgi.The 󰀱󰀸-residue loop in D󰀲 mediates binding to the WD󰀴󰀰 domain in WSB󰀱, a D󰀲 ubiquitin ligase 󰀨󰀱󰀵, 󰀱󰀶, 󰀳󰀹󰀩. Typically, WD󰀴󰀰 domains exhibit low levels of sequence conservation, making them promiscuous binding partners 󰀨󰀴󰀰󰀩. Therefore, we tested to determine whether Ala󰀹󰀲󰀭D󰀲 HY  interacts with other ER WD󰀴󰀰-containing proteins that could provide an additional exit route to the Golgi. The SREBP cleavage-activating protein 󰀨SCAP󰀩 D󰀲 is a type I ER-resident protein, which enables D󰀲-gen-erated T󰀳 to access and bind TRs 󰀨󰀳󰀩. D󰀲 is atypical among the deiodinases in that it displays a relatively short half-life as a result of ubiquitination and proteasomal degradation. This is caused by an exclusive 󰀱󰀸-residue loop that mediates binding to 󰀲 ubiquitin ligases 󰀨󰀱󰀵󲀓󰀱󰀷󰀩. This loop also harbors a Thr󰀹󰀲-to-Ala substitution 󰀨Ala󰀹󰀲󰀭D󰀲󰀩 caused by a SNP present in 󰀱󰀲󰀥–󰀳󰀶󰀥 of the population 󰀨󰀱󰀸󰀩. Not surprisingly, the single amino acid substitution slows down the rate of D󰀲 turnover; it is also asso-ciated with ectopic presence of D󰀲 in the Golgi apparatus 󰀨󰀱󰀹󰀩. Clinically, carriers of the Thr󰀹󰀲Ala-DIO󰀲 polymorphism were found more likely to have hypertension, insulin resistance, type 󰀲 diabetes, bipolar disorder, mental retardation, low I󰁑, suscep-tibility to lung injury, osteoarthritis, Alzheimer’s disease 󰀨󰀲󰀰󰀩, and increased bone turnover, but perhaps unsurprisingly, these associations have not been reproduced in all population studies 󰀨󰀶, 󰀲󰀱󰀩. It is intriguing, however, that carriers of the Thr󰀹󰀲Ala polymorphism in the D󰀲 gene 󰀨 DIO󰀲 󰀩 exhibited symptomatic improvement when liothyronine 󰀨LT󰀳󰀩 was added to LT󰀴 thera-py 󰀨󰀲󰀲, 󰀲󰀳󰀩, suggesting that Thr󰀹󰀲Ala-DIO󰀲 carriers do not pro-duce suf󰁦icient amounts of T󰀳 via D󰀲. Indeed, 󰀲 studies suggest that Thr󰀹󰀲Ala-D󰀲 is less catalytically active 󰀨󰀲󰀴, 󰀲󰀵󰀩, potential-ly explaining why only a small percentage of the LT󰀴-treated hypothyroid patients exhibit residual symptoms.Here, we studied the Thr󰀹󰀲Ala polymorphism in cell and ani-mal models, having found that D󰀲 is a cargo protein in ER Golgi intermediary compartment 󰀨ERGIC󰀩 vesicles, recycling between ER and Golgi. Ala󰀹󰀲󰀭D󰀲 causes ER stress and activates the unfold-ed protein response 󰀨UPR󰀩 in different brain areas. Our 󰁦ind-ings suggest that disruption of cellular proteostasis and reduced Ala󰀹󰀲󰀭D󰀲 catalytic activity explain the failure of LT󰀴 therapy in carriers of the Thr󰀹󰀲Ala-DIO󰀲 polymorphism; the data support a need for further studies with chemical chaperones and combina-tion therapy with LT󰀳 in humans. Results To understand how Thr󰀹󰀲Ala-DIO󰀲 affects D󰀲, we studied HEK-󰀲󰀹󰀳 cells stably expressing either form of D󰀲 double tagged with His and yellow fluorescent protein 󰀨YFP󰀩 󰀨󰀱󰀹󰀩. Thr󰀹󰀲󰀭D󰀲 HY  distrib-uted predominantly to the ER with low-level colocalization with the cis-Golgi marker GM󰀱󰀳󰀰 󰀨Figure 󰀱, A󲀓C󰀩 and the trans-Golgi marker p󰀲󰀳󰀰 󰀨Figure 󰀱, D󲀓F󰀩. Whereas Ala󰀹󰀲󰀭D󰀲 HY  also exhibited low-level colocalization with the cis-Golgi marker 󰀨Figure 󰀱, G󲀓I󰀩, it was clearly present in the trans-Golgi 󰀨Figure 󰀱, J󲀓L󰀩.A network of ERGIC vesicles exists that shuttles proteins back and forth between ER and cis-Golgi, namely the COPII and COPI vesicles 󰀨󰀲󰀶, 󰀲󰀷󰀩. COPII vesicles recognize, concentrate, and export ER proteins to the Golgi 󰀨󰀲󰀸󰀩 based on the presence of export signals, none of which can be found in D󰀲. Other proteins are concentrated in COPII vesicles via speci󰁦ic transport adaptors 󰀨󰀲󰀹󰀩. To 󰁦ind out whether D󰀲 traf󰁦ics through this network, we analyzed pulldowns of both Thr󰀹󰀲󰀭D󰀲 HY  and Ala󰀹󰀲󰀭D󰀲 HY . While we failed to identify COPII proteins 󰀨data not shown󰀩, these pull-downs contained the transport adaptor ERGIC󰀵󰀳 󰀨󰀳󰀰󰀩 along with p󰀹󰀷 and UBXD󰀭󰀱. The ATP-driven chaperone p󰀹󰀷 is involved in quality control and uses adaptors to process ubiquitinated proteins for recycling or degradation 󰀨󰀳󰀱󰀩. For example, p󰀹󰀷 and the adap-  The Journal of Clinical Investigation RESEARCH ARTICLE 4 jci.org carboxyl end󰀩 in cells depleted of cholesterol 󰀨Figure 󰀳I󰀩 through a mechanism that required the WD󰀴󰀰 domain in SCAP 󰀨Figure 󰀳J󰀩.To 󰁦ind out how much the presence of Ala󰀹󰀲󰀭D󰀲 HY  in the trans-Golgi depends on ER stress and/or SCAP activation, we explored whether overnight incubation of Ala󰀹󰀲󰀭D󰀲 HY  cells with is a WD󰀴󰀰-containing ER cholesterol sensor that transports SREBP󰀱 from the ER, via ERGIC 󰀨󰀴󰀱󰀩, to the Golgi 󰀨󰀴󰀲󰀩, a pathway that is activated by ER stress 󰀨󰀴󰀳, 󰀴󰀴󰀩. Fluorescence resonance energy transfer 󰀨FRET󰀩 studies in live cells indicated that Ala󰀹󰀲-D󰀲 HY , but not Thr󰀹󰀲󰀭D󰀲 HY , interacted with SCAP ECFP  󰀨ECFP in the Figure 2. Expression of Ala92-D2 causes ER stress, triggers UPR response.  UPR markers in Thr92-D2 HY –expressing (Thr) or Ala92-D2 HY –expressing (Ala) cells. ( A ) Western blot of pIRE1 α . ( B ) Quantification of pIRE1 α  shown in A . ( C ) s  XBP1  mRNA levels. RQ, relative quantification. ( D ) Western blot of uncleaved (uc) and cleaved (c) ATF6. ( E ) Quantification of cATF6 shown in D . ( F ) CHOP   mRNA levels. ( G ) Western blot of BIP. ( H ) Quantification of BIP shown in G . ( I ) BIP   mRNA levels. (  J ) Western blot of pPERK. ( K ) Quantification of pPERK shown in  J . ( L ) Western blot of pEIF2 α . ( M ) Quantification of pEIF2 α  shown in L . ( N )  ATF4  mRNA levels. ( O ) Western blot of ERGIC53. ( P ) Quantification of ERGIC53 shown in O . ( Q ) ERGIC53  mRNA levels. ( R – W ) Immuno-fluorescence of Thr92-D2 HY  or Ala92-D2 HY  stably expressing cells using the indicated antibodies. Original magnification, APO ×60/1.40 oil objective. Values are shown in a box-and-whiskers plot indicating median and quartiles. n  = 4–5/group. Statistical analysis used was the Mann-Whitney U  test or Kruskall-Wallis test followed by the Dunn’s multiple comparison test. * P   ≤ 0.05; ** P   ≤ 0.01.
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