Introduction
Parkinson’s disease (PD) is one of the most frequently diagnosed neurodegenerative diseases, prevailing in the population over sixty [32]. Pathologically, PD results from loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and depletion of dopamine in a progressive fashion, leading to clinical manifestations, including bradykinesia, rigidity, resting tremor, and postural instability, and even high fall rate and loss of independence at the advanced stage [30,32]. Such neuronal damage caused by PD activates the neuroimmune system, including microglia, astrocytes, inflammatory cytokines, and chemokines, consequently triggering neuroinflammation [21,29]. Under the context of PD, microglia as the resident macrophages in the central nervous system is clearly evidenced to be hyper-activated as a response to signals stem from damaged neurons, secreting pro- and anti-inflammatory factors [11,38]. At present, the strategy for the suppression of DA neuronal death remains lacking in the clinic [30]. Therefore, the discovery of effective molecules sensitive to microglia activation may help prevent neuroinflammation, thus beneficial for the alleviation of PD symptoms.
Transcription factors, sequence-specific DNA-binding proteins that manipulate chromatin and transcription, are associated with PD symptoms, including DA neuron death, microglia activation, and neuroinflammation [3,36,39]. Heat shock transcription factor 1 (HSF1) emerges as a regulator of heat shock proteins that refold and solubilize neurodegeneration-related proteins to exert neuroprotective function in neurodegenerative diseases [19]. For instance, HSF1 enhances heat shock protein 70 to degrade a-synuclein aggregation to protect SH-SY5Y neuroblastoma cells from a-synuclein-mediated toxicity, which is a hallmark of PD [23]. However, whether HSF1 regulates microglia activation in PD remains unknown.
During PD, enhanced oxidative stress and chronic inflammation may also lead to alterations of micro-RNAs (miRNAs, small transcripts with about 20-24 nucleotides) and their target proteins, and dysfunctional miRNAs, in turn, exacerbate neurodegeneration [20,28]. HSF1 is known to positively regulate miR-214 under the context of interstitial pulmonary fibrosis [5]. Among the miR-214 family members, miR-214-3p is reported to be declined in PD animal and cell models and alleviate cytotoxicity in damaged neurons [41]. Through the database prediction of Starbase, TargetScan, and miRDB, the nuclear factor of activated T cells 2 (NFATc2) was predicted to be a downstream target of miR-214-3p. Loss of NFATc2 is demonstrated to repress microglia activation in Alzheimer’s disease [26]. In PD, NFATc2 is phosphorylated along with increased nuclear transcriptional activity and activates a cascade of neuroinflammatory responses [18,37]. Nevertheless, the crosstalk between HSF1 and miR-214-3p/NFATc2 in PD has not been discussed before and warrants profound investigation.
In lieu of the aforementioned data, we speculated that HSF1 modulates microglial activation to affect neuroinflammation in PD via regulating the miR-214-3p/NFATc2 axis. Then, we established PD mouse models using treatment of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to explore the role of HSF1 and its downstream mechanism in PD mice so as to enrich theoretical knowledge of PD treatment.
Material and methods
Experimental animals
The protocol of animal experiments was approved by the Animal Ethics of Huanggang Central Hospital and followed the Guidelines for the care and use of laboratory animals [17]. C57BL/6 mice (5-6 weeks old, 18-22 g) were procured from the Center for Animal Experiment of Wuhan University (Approval No. SCXK (Hubei) 2019-0004, Hubei, China) and raised at room temperature of 22°C under 12 h day/night cycles and with free access to standard food and water. After one week of adaptive feeding, mice underwent experimental treatments.
Animal modelling
As previously described [25], PD mouse models were established via injection of MPTP. During surgery, mice were anesthetized by a combination of sodium pentobarbital (50 mg/kg, i.p.) and sevoflurane inhalation. Mice were induced by MPTP (30 mg/kg, i.p.) for 5 consecutive days, with an injection of the same volume of normal saline (NS) in the control group.
Stereotaxic injection
AntagomiR-214-3p, lentivirus-packaged oe-HSF1 (LV-oe-HSF1), lentivirus-packaged oe-NFATc2 (LV-oe-NFATc2), and the corresponding controls were all provided by GenePharma (Shanghai, China) and stereotaxically injected into mice according to the previous method [34]. Briefly, after anaesthesia, the mouse was fixed on a stereotaxic apparatus (Render Biotech Co., Ltd, Shenzhen, China) and a hole was drilled for single unilateral injection at the following stereotactic coordinates: 3.0 mm anteroposterior from bregma;
1.2 mm mediolateral from bregma; 5.0 mm dorsoventral from bregma. Subsequently, the right side of SNpc was injected with 2 µl NS containing 0.75 nmol antagomiR-214-3p, or 5 µl oe-HSF1 (LV-oe-HSF1), or oe-NFATc2 (LV-oe-NFATc2) (20 µl per mice, 109 TU/ml) at the rate of 0.2 µl/min. Upon injection, the needle was slowly withdrawn and the mouse was placed on a heating pad for recovery. A week after stereotaxic injection, MPTP was administered for 5 consecutive days as previously mentioned.
Behavioural tests
Motor coordination and locomotor activity tests were conducted according to the previous method [25]. For motor coordination testing, prior to the formal test, mice received 10-min training on a rotating lever for 10 min every day for 3 consecutive days, making them habituated to accelerated rotation. In the formal test, the time when mice stayed in the rotating lever was recorded as the latency period of falling off the rotating lever to assess motor coordination capacity. For locomotor activity testing, mice were gently placed in an open field installation containing 9 grids with a camera above to monitor tracks of mouse motion. The number of grids covered by mouse movement and the average moving speed within 2 min were recorded as locomotor activity.
Tissue treatment
After behavioural tests, all mice were anesthetized by pentobarbital (40 mg/kg, i.p.), followed by heart perfusion [0.01 M phosphate-buffered saline (PBS), pH 7.4]. Then, the brain was removed and two cerebral hemispheres were separated on ice. SNpc (4-7 mm posterior to the bregma) was extracted from one cerebral hemisphere and fixed with 4% paraformaldehyde for 24 h for immunohistochemical analysis. The other hemisphere was rapidly frozen in liquid nitrogen for biochemical and inflammatory analyses.
Immunohistochemistry
After 24-h fixation in paraformaldehyde, SNpc tissues were sliced into 30 µm-thick sections and incubated overnight with tyrosine hydroxylase (TH) antibody (ab137869, 1 : 500, Abcam, Cambridge, MA, USA). After washing with PBS, sections were incubated with the biotin-labelled secondary antibody (ab6112, 1 : 500, Abcam) for 15 min and then with streptavidin-peroxidase for 15 min. After washing, sections were colour-developed with the diaminobenzidine kit (Abcam) and photographed under a microscope (Olympus, Tokyo, Japan).
Immunofluorescence
The 30 µm-thick sections were blocked with 5% bovine serum albumin for 0.5 h and incubated with IBA-1 antibody (ab178847, 1 : 100, Abcam) at room temperature for 1 h and then with the secondary antibody (ab150077, 1 : 200, Abcam) for 15 min. After that, sections were stained with 0.3 mM diamidino-phenyl-indole (Beyotime, Hangzhou, Zhejiang, China) for 15 min, washed with PBS, and the photographed using a fluorescence microscope (Leica, Solms, Germany).
Reverse transcription quantitative polymerase chain reaction (qRT-PCR)
The total RNA was extracted from brain tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into the complementary DNA using PrimeScript RT Master Mix RNA kit. Real-time PCR was conducted using SYBR PreMix Ex Taq (Takara, Tokyo, Japan) and monitored by the Real-time PCR system (ABI 7500). The relative gene expression was quantified using the 2-DDCt method, with GAPDH and U6 as endogenous controls [10]. qPCR primers are listed in Table I.
Western blotting
Upon extraction of the total protein from brain tissues, the protein concentration was determined using Bradford protein analysis kits (Beyotime). The equal amount of protein was isolated from each sample using 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene fluoride membranes. After blockade with 5% milk in PBS-0.05% Tween, membranes were incubated with the primary antibodies at 4°C overnight, washed three times, and then incubated with the secondary antibody goat anti-rabbit IgG (1 : 2000, Abcam) at room temperature for 90 min. With b-actin as the internal reference, immunoblots were visualized with BeyoECL Plus ECL kits (Beyotime). Primary antibodies were as follows: rabbit monoclonal antibody HSF1 (ab242138, 1 : 1000, Abcam); rabbit monoclonal antibody iNOS (ab178945, 1 : 1000, Abcam), rabbit monoclonal antibody COX-2 (ab179800, 1 : 1000, Abcam), rabbit monoclonal antibody BDNF (ab108319, 1 : 1000, Abcam), and rabbit polyclonal antibody b-actin (ab8227, 1 : 1000, Abcam).
Enzyme-linked immunosorbent assay (ELISA)
Contents of interleukin (IL)-1b, IL-6, tumor necrosis factor a (TNF-a), transforming growth factor b1 (TGF-b1), and IL-10 in brain tissues were determined by enzyme-linked immunosorbent assay (ELISA). Briefly, IL-1b, IL-6, TNF-a, TGF-b1, and IL-10 levels were determined using IL-1b-specialized ELISA kit (ab197742, Abcam), IL-6-specialized ELISA kit (ab222503, Abcam), TNF-a-specialized ELISA kit (ab20-8348, Abcam), TGF-b1-specialized ELISA kit (ab119557, Abcam), and IL-10-specialized ELISA kit (ab255729, Abcam), respectively.
Bioinformatics
The binding sites of HSF1 and miR-214-3p were predicted using the Jaspar website (http://jaspar.genereg.net/) [7]. The downstream target genes of miR-214-3p were predicted using Starbase (http://starbase.sysu.edu.cn/) [22], TargetScan (http://www.targetscan.org/vert_72/) [1] and miRDB (http://mirdb.org/) [6] databases. The binding sites of miR-214-3p and NFATc2 were predicted using the Starbase database.
Chromatin immunoprecipitation (ChIP)
According to the manufacturer’s instructions, chromatin immunoprecipitation (ChIP) assay was conducted using SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology, Danvers, MA, USA). Simply put, HEK-293T cells were crosslinked with formaldehyde and lysed in the lysis buffer. After DNA was sheared using ultrasound, the lysates were incubated with rabbit monoclonal antibody HSF1 (ab52757, Abcam), followed by purification of crosslinked DNA released from the protein-DNA complex and analysis of eluted DNA using RT-qPCR. The miR-214 promoter primer sequences: forward primer, 5’-CAAGGTGGCGGGAGTCTTTC-3’; reverse primer, 5’-AACGTGTGCTTCTGTCCAAC-3’.
Dual-luciferase assay
HSF1 fragments containing binding sites of the miR-214-3p promoter (HSF1-WT) and the mutant derivative lacking the binding sites (HSF1-MUT), NFATc2 3’UTR fragments containing binding sites of miR-214-3p (NFATc2-WT), and the mutant derivative lacking the binding sites (NFATc2-MUT) were inserted into pmirGLO-reporter vectors. The above-constructed luciferase reporter plasmids, and mimic NC or miR-214-3p mimic were co-transfected into HEK-293T cells. After 48 h, cells were harvested and lysed, and the luciferase activity was detected according to the instructions of the luciferase detection kit (K801-200; Biovision, Mountain View, CA, USA). All plasmids were provided by GenePharma (Shanghai, China).
Statistical analysis
Data statistical analyses and graphing were processed with the help of SPSS21.0 software (IBM Corp, Armonk, NY, USA) and GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). Measurement data were presented as mean ± standard deviation (SD) and conformed to normal distribution and homogeneity of variance. The t test was appointed for pairwise comparisons, one-way or two-way analysis of variance (ANOVA) was appointed for multi-group comparisons, and Tukey’s multiple comparison test was appointed for the post-test. The p value was obtained from two-sided tests, a value of p < 0.05 indicated statistical significance, and a value of p < 0.01 indicated extreme statistical significance.
Results
HSF1 overexpression attenuated motor dysfunction and nerve injury in PD mice
To explore the regulatory role of HSF1 in neuroinflammation in PD mice, we established PD mouse models using MPTP treatment and found that HSF1 was significantly downregulated in brain tissues (p < 0.05, Fig. 1A, B).
Then, we injected LV-oe-HSF1 (oe-HSF1) into brain tissues of PD mice to upregulate HSF1 expression (p < 0.05, Fig. 1A, B). The behavioural test showed that, relative to control mice, the latency period of falling off the rotating lever was shortened (p < 0.05, Fig. 1C), the number of passing grids was reduced (p < 0.05, Fig. 1D), and the moving speed in the open field was slowed (p < 0.05, Fig. 1E) in MPTP-treated mice, indicating that MPTP treatment resulted in deficits in motor coordination and locomotor activity; whereas HSF1 overexpression alleviated these deficits (p < 0.05, Fig. 1C-E). TH staining showed that DA neurons were lost and TH-positive neurons in SNpc tissues were significantly decreased upon MPTP treatment, which was alleviated by HSF1 overexpression (p < 0.05, Fig. 1F). Above all, HSF1 overexpression attenuated motor dysfunction and nerve injury in PD mice.
HSF1 overexpression repressed microglia activation
To explore the role of HSF1 in PD-induced microglia activation, we assessed microglia activation via immunofluorescence and found that IBA-1-positive microglial cells were significantly increased upon MPTP treatment and significantly decreased as a response to HSF1 overexpression (p < 0.05, Fig. 2A). Next, we detected cytokine levels and found that pro-inflammatory IL-1b, IL-6, and TNF-a levels were significantly augmented (p < 0.05, Fig. 2B-D) and anti-inflammatory TGF-b1 and IL-10 levels were significantly declined (p < 0.05, Fig. 2E, F), protein levels of iNOS and COX-2 were significantly elevated and protein level of BDNF was significantly lowered (p < 0.05, Fig. 2G) after MPTP treatment, while HSF1 overexpression reversed the above changes of cytokine levels (p < 0.05, Fig. 2B-G). Above all, HSF1 overexpression repressed microglia activation.
HSF1 bound to the miR-214-3p promoter to improve miR-214-3p expression
Through the Jaspar website, we predicted that HSF1 bound to the miR-214-3p promoter region (Fig. 3A, B). The binding relationship between HSF1 and the miR-214-3p promoter was further testified via the dualluciferase assay (p < 0.05, Fig. 3C, D). Through the ChIP assay, HSF1 was found to be enriched more in the miR-214-3p promoter region (p < 0.05, Fig. 3E). Then, we detected miR-214-3p expression and found that miR-214-3p was significantly lowered in brain tissues of MPTP-treated mice and significantly increased as a response to HSF1 overexpression (p < 0.05, Fig. 3F). Above all, HSF1 bound to the miR-214-3p promoter to improve miR-214-3p expression.
miR-214-3p downregulation reversed the inhibition of HSF1 overexpression on neuroinflammation and microglia activation
To explore the regulatory role of miR-214-3p in PD-induced neuroinflammation and microglia activation, we injected antagomiR-214-3p (antago) into brain tissues of PD mice to downregulate miR-214-3p level (p < 0.05, Fig. 4A) and performed a collaborative experiment with LV-oe-HSF1 (oe-HSF1). It was observed that upon silencing miR-214-3p, the latency period of falling off the rotating lever was shortened (p < 0.05, Fig. 4B), the number of passing grids was reduced (p < 0.05, Fig. 4C), and moving speed in the open field was slowed (p < 0.05, Fig. 4D), TH-positive neurons were significantly decreased (p < 0.05, Fig. 4E), IBA-1-positive microglial cells were significantly increased (p < 0.05, Fig. 4F), IL-1b, IL-6, and TNF-a levels were significantly augmented (p < 0.05, Fig. 4G-I), TGF-b1 and IL-10 levels were significantly declined (p < 0.05, Fig. 4J, K), protein levels of iNOS and COX-2 were significantly elevated, and protein level of BDNF was significantly lowered (p < 0.05, Fig. 4L). Above all, miR-214-3p downregulation reversed the inhibition of HSF1 overexpression on neuroinflammation and microglia activation.
miR-214-3p inhibited NFATc2 transcription
To further analyse the regulatory mechanism of miR-214-3p in PD-induced neuroinflammation and microglia activation, we predicted downstream target genes of miR-214-3p on Starbase, TargetScan, and miRDB databases and identified intersections using Venn diagram (Fig. 5A). The existing literature reported that NFATc2 is potent to activate microglia in neurodegenerative diseases and is upregulated in PD patients [18,26]. The potential binding site of miR-214-3p and NFATc2 was predicted on the Starbase database (Fig. 5B), and the binding relationship between miR-214-3p and NFATc2 was testified via the dual-luciferase assay (p < 0.05, Fig. 5C). The mRNA level of NFATc2 in brain tissues was upregulated by MPTP treatment, downregulated as a response to HSF1 overexpression, and declined again by a combination of HSF1 overexpression and miR-214-3p inhibition (p < 0.05, Fig. 5D). Above all, miR-214-3p inhibited NFATc2 transcription.
NFATc2 overexpression reversed the inhibition of HSF1 overexpression on neuroinflammation and microglia activation
Lastly, to explore the role of NFATc2 in PD-induced neuroinflammation and microglia activation, we injected LV-oe-NFATc2 (oe-NFATc2) in brain tissues of PD mice to upregulate NFATc2 level (p < 0.05, Fig. 6A) and performed a collaborative experiment with LV-oe-HSF1 (oe-HSF1). It was observed that upon silencing miR-214-3p, the latency period of falling off the rotating lever was shortened (p < 0.05, Fig. 6B), the number of passing grids was reduced (p < 0.05, Fig. 6C), and moving speed in the open field was slowed (p < 0.05, Fig. 6D), TH-positive neurons were significantly decreased (p < 0.05, Fig. 6E), IBA-1-positive microglial cells were significantly increased (p < 0.05, Fig. 6F), IL-1b, IL-6, and TNF-a levels were significantly augmented (p < 0.05, Fig. 6G-I), TGF-b1 and IL-10 levels were significantly declined (p < 0.05, Fig. 6J, K), protein levels of iNOS and COX-2 were significantly elevated, and protein level of BDNF was significantly lowered (p < 0.05, Fig. 6L). Above all, NFATc2 overexpression reversed the inhibition of HSF1 overexpression in neuroinflammation and microglia activation.
Discussion
During PD, DA neuronal death is accompanied by microglia activation and overproduction of inflammatory cytokines and chemokines, leading to neuroinflammation [21]. Shreds of evidence shed light that transcription factors interact with miRNAs to play a synergic role in neurodegenerative diseases [8,15,31]. HSF1 as a transcription factor of heat shock proteins protects neurons against neurodegeneration [19]. In the current study, our findings highlighted that HSF1 binds to the miR-214-3p promoter to promote its expression and inhibits NFATc2 transcription, thereby attenuating microglia activation and neuroinflammation in PD mice.
Initially, we established PD mouse models using MPTP treatment and found that HSF1 was downregulated in PD mice, in line with prior studies demonstrating that HSF1 exhibits abnormal degradation in neurodegenerative diseases, including Huntington’s disease, Alzheimer’s disease, and Lafora disease [4,9,33]. To explore the role of HSF1 in PD, we overexpressed HSF1 in PD mice via injection of LV-oe-HSF1. Our results found that HSF1 overexpression prolonged the latency period of falling off the rotating lever, augmented the number of passing grids and moving speed in the open field, reduced loss of DA neurons, and increased TH-positive neurons in the substantia nigra. Consistently, the HSF1 agonist plays a therapeutic role in PD as manifested by increased dopamine levels and neuronal aggregates [2,35]. Besides, activation of HSF1 is beneficial for cognitive performance by maintaining synaptic fidelity [13]. Collectively, HSF1 overexpression alleviated deficits in motor coordination and locomotor activity and loss of DA neurons to play a therapeutic role in MPTP-induced PD mice.
Neuroinflammation exerts neurotoxic effects to facilitate PD progression and neuroinflammatory pathways rely on microglia activation [16,29]. IBA-1, iNOS, COX-2, IL-1b, IL-6, and TNF-a are positively associated with microglia activation and neuroinflammation, while BDNF, TGF-b1, and IL-10 counteract the effects of microglia activation and neuroinflammation [27,40]. Subsequent results indicated that HSF1 overexpression reduced IBA-1-positive microglial cells, IL-1b, IL-6, and TNF-a expressions and iNOS and COX-2 protein levels, while it increased TGF-b1 and IL-10 expressions and BDNF protein levels, indicating that HSF1 overexpression repressed microglia activation and neuroinflammation. In favour of our results, HSF1 deficiency increases activated microglia to promote aging-related brain pathology [12]. Similarly, HSF1 is druggable by senkyunolide I to suppress the secretion of pro-inflammatory cytokines by BV-2 microglia, thus attenuating stroke-induced neuroinflammation [14]. Altogether, our results initially demonstrated that HSF1 suppresses microglia activation to attenuate neuroinflammation in PD mice.
A prior study has reported that miR-214 is a direct downstream target of HSF1 [5]. Among the miR-214 family members, miR-214-3p plays a protective role in MPTP and 1-methyl-4-phenylpyridinium-induced PD mouse and cell models [41]. Accordingly, the binding between HSF1 and miR-214-3p was predicted on the Jaspar website and testified by the dual-luciferase assay, and HSF1 was found to be enriched more in the miR-214-3p promoter through ChIP assay. Besides, decreased miR-214-3p was found in brain tissues upon MPTP treatment and elevated by HSF1 overexpression, suggesting that HSF1 bound to the miR-214-3p promoter to enhance miR-214-3p expression. Then, to evaluate the role of miR-214-3p in PD, we silenced miR-214-3p in PD mice and performed a collaborative experiment with oe-HSF1. Our results indicated that silencing miR-214-3p aggravated deficits in motor coordination and locomotor activity and loss of DA neurons, increased levels of positive markers of microglia activation and neuroinflammation, and decreased levels of negative ones. Similarly, epigenetic depletion of miR-214-3p increases reactivity of astrocyte (another type of neuroglia), contributing to neuroinflammation and neuropathic pain [24]. Altogether, our results elicited that HSF1 suppresses microglia activation to attenuate neuroinflammation in PD mice by upregulating miR-214-3p. Nevertheless, the role of miR-214-3p in microglia activation has not been discussed before, which highlighted the novelty of our study.
Thereafter, we probed the downstream mechanism of miR-214-3p. The database predictions and intersections of predicted results directed our focus to NFATc2. NFATc2 transcriptional activity is increased by PD and regulates cytokines and chemokines to construct a neurotoxic inflammatory environment [37]. The targeting relationship between miR-214-3p and NFATc2 was testified via the dual-luciferase assay. Besides, the mRNA level of NFATc2 was found to be increased in brain tissues of PD mice, declined by HSF1 overexpression, and diminished again by a combination of HSF1 overexpression and miR-214-3p downregulation. To probe the role of NFATc2 in PD, we overexpressed NFATc2 and performed a collaborative experiment with oe-HSF1. Subsequent results indicated that NFATc2 overexpression aggravated deficits in motor coordination and locomotor activity and loss of DA neurons, increased levels of positive markers of microglia activation and neuroinflammation, and decreased levels of negative ones. In tandem with our results, NFATc2 also promotes inflammatory chemotaxis of microglia in other neurodegenerative disorders, such as synucleinopathy and Alzheimer’s disease [18,26]. Overall, our results elicited that HSF1 suppresses microglia activation to attenuate neuroinflammation in PD mice by promoting miR-214-3p and inhibiting NFATc2.
To conclude, our study is the first of its kind to verify the therapeutic role of HSF1 in PD-induced microglia activation and neuroinflammation via regulating the miR-214-3p/NFATc2 axis and may provide a novel theoretical reference for HSF1-mediated molecular targeted therapy for PD. However, since we only discussed the roles of HSF1, miR-214-3p, and NFATc2, whether other transcription factors, miRNAs, and downstream targets of miR-214-3p exert function on PD-induced microglia activation and neuroinflammation remains unknown. In the next step, we will continue to investigate the effects of other transcription factors, miRNAs, and downstream targets of miR-214-3p in PD.
Acknowledgements
We would like to thank all the participants for their time and effort.
Funding
This work was supported by Hainan Provincial Key Research and Development Program under Grant number ZDYF2019196 and Huanggang Science and Technology Bureau under Grant number XQYF2021000005.
Ethical approval
The protocol of animal experiments was approved by the Animal Ethics of Huanggang Central Hospital and followed the Guidelines for the care and use of laboratory animals [17].
Disclosure
The authors report no conflict of interest.
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