Optimal vitamin D spurs serotonin: 1,25-dihydroxyvitamin D represses serotonin reuptake transport (SERT) and degradation (MAO-A) gene expression in cultured rat serotonergic neuronal cell lines
Diminished brain levels of two neurohormones, 5-hydroxytryptamine (5-HT; serotonin) and 1,25-dihydroxyvitamin D3 (1,25D; active vitamin D metabolite), are proposed to play a role in the atypical social behaviors associated with psychological conditions including autism spectrum disorders and depression. We reported previously that 1,25D induces expression of tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway to 5-HT, in cultured rat serotonergic neuronal cells. However, other enzymes and transporters in the pathway of tryptophan metabolism had yet to be examined with respect to the actions of vitamin D. Herein, we probed the response of neuronal cells to 1,25D by quantifying mRNA expression of serotonin synthesis isozymes, TPH1 and TPH2, as well as expression of the serotonin reuptake transporter (SERT), and the enzyme responsible for serotonin catabolism, monoamine oxidase-A (MAO-A). We also assessed the direct production of serotonin in cell culture in response to 1,25D.
Employing quantitative real-time PCR, we demonstrate that TPH-1/-2 mRNAs are 28- to 33-fold induced by 10 nM 1,25D treatment of cultured rat serotonergic neuronal cells (RN46A-B14), and the enhancement of TPH2 mRNA by 1,25D is dependent on the degree of neuron-like character of the cells. In contrast, examination of SERT, the gene product of which is a target for the SSRI-class of antidepressants, and MAO-A, which encodes the predominant catabolic enzyme in the serotonin pathway, reveals that their mRNAs are 51–59% repressed by 10 nM 1,25D treatment of RN46A-B14 cells. Finally, serotonin concentrations are significantly enhanced (2.9-fold) by 10 nM 1,25D in this system.
These results are consistent with the concept that vitamin D maintains extracellular fluid serotonin concentrations in the brain, thereby offering an explanation for how vitamin D could influence the trajectory and development of neuropsychiatric disorders. Given the profile of gene regulation in cultured RN46A-B14 serotonergic neurons, we conclude that 1,25D acts not only to induce serotonin synthesis, but also functions at an indirect, molecular-genomic stage to mimic SSRIs and MAO inhibitors, likely elevating serotonin in the CNS. These data suggest that optimal vitamin D status may contribute to improving behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission.
KeywordsAutism spectrum disorders Depression Neuropsychiatric disorders Social behavior Tryptophan metabolism Vitamin D receptor 5-Hydroxytryptamine
Direct repeat with a spacer of 3 nucleotides
Positron emission tomography
Seasonal affective disorder
Serotonin reuptake transporter
Selective serotonin reuptake inhibitor
Vitamin D receptor
Vitamin D-responsive element
Serotonin, a neurotransmitter derived from the nutritionally essential amino acid tryptophan, executes critical functions in the brain such as control of appetite, energy expenditure, sleep, temperature, mood, and social cognition . Serotonergic neurons innervate vast areas of the brain, with projections arising from cell bodies in the dorsal and median raphe and neighboring nuclei of the lower brain stem. These projections radiate to the hippocampus, amygdala, hypothalamus, nucleus accumbens, and lateral prefrontal cortex, as well as to a widespread array of cortical areas, wherein serotonin modulates a broad range of behavioral actions via metabotropic G protein-coupled and ionotropic, ligand-gated ion channel receptors .
In what constitutes the first step of serotonin production, tryptophan in the CNS is hydroxylated to 5-hydroxytryptophan by the enzyme tryptophan hydroxylase type 2 (TPH2), the rate-limiting step in brain serotonin synthesis. This is followed by subsequent decarboxylation of 5-hydroxytryptophan, catalyzed by aromatic amino acid decarboxylase, to serotonin (5-hydroxytryptamine; 5-HT). Serotonin is then packaged by vesicularization through the action of monoamine transporter isoform 2 in raphe neurons. After the release of 5-HT-containing vesicles from axon terminals, and subsequent reuptake of serotonin from the synapse via a serotonin reuptake transporter (SERT), degradation of serotonin is catalyzed by monoamine oxidase-A (MAO-A) and aldehyde dehydrogenase to the predominant serotonin metabolite, 5-hydroxyindoleacetic acid . Therefore, three important enzymes/transport proteins that determine serotonin concentration and dynamics in the CNS are TPH2, SERT, and MAO-A. It is generally accepted that the amount and activity of these three proteins governs serotonin levels in the CNS .
In a recent publication , we proposed that serotonin concentrations are controlled in part by 1,25-dihydroxyvitamin D3 (1,25D), the active, hormonal metabolite of vitamin D. Specifically, we demonstrated that 1,25D dramatically induces TPH2 gene expression in differentiated serotonergic rat raphe RN46A-B14 cells , a finding that is reinforced by in vivo experiments conducted by Jiang et al.  revealing a significant induction of TPH2 mRNA in the prefrontal cortex of rats after chronic 1,25D administration. Central among the plethora of serotonin bioeffects are those that influence executive function, sensory gating, and prosocial behavior; importantly, serotonin levels are often suboptimal in disorders of these actions. Patrick and Ames  have reviewed the observed association between aberrant serotonin concentrations, both during development and in the adult, in a broad range of behavioral illnesses including ADHD, autism, bipolar disorder, depression, and schizophrenia, as well as anti-social, obsessive-compulsive, and suicidal behaviors. They hypothesized that an association between vitamin D insufficiency and low central serotonin concentrations represents a common denominator in a myriad of neuropsychiatric disorders. While it is conceivable that the ability of the vitamin D hormone, 1,25D, to upregulate TPH2 expression is a key factor in maintaining serotonin output by serotonergic neurons, other avenues of serotonin metabolism have yet to be investigated with respect to the effects of vitamin D. In the present communication, we examined the influence of a range of 1,25D concentrations on the expression of SERT, MAO-A/B, and TPH-1/2 to elucidate the mechanism of action underlying the regulation of serotonin reuptake, degradation, and synthesis by vitamin D in serotonergic neurons.
As a negative-control neural gene, MAO-B was evaluated for modulation of expression by 1,25D in RN46A-B14 cells (Fig. 1c). Interestingly, MAO-B was not significantly repressed by 1,25D at any of the tested dosages, indicating that the influence of 1,25D on SERT and MAO-A expression may be selective for biologically relevant serotonin metabolism genes. Furthermore, as a positive control to confirm endogenous expression of a sufficient copy number of biologically functional nuclear vitamin D receptors (VDRs) to mediate transcriptional regulation by 1,25D in RN46A-B14 cells, we examined Cyp24a1 mRNA enhancement in response to the vitamin D hormone. As depicted in Fig. 1d, Cyp24a1 expression is dramatically (38- to 116-fold) augmented by exposure of the cells to 1–100 nM 1,25D, with the pertinent observation that this gene displayed the traditional dose-response curve for induction. Cyp24a1 serves not only as a control for positive modulation of gene expression via VDR in RN46A-B14 cells, but because the RNA probed was isolated from the same treated cells for SERT and MAO-A mRNA assessment, any systematic error in the technology can be eliminated from consideration as an explanation for the unique dose-response results for serotonin catabolic gene expression.
Although recent research has highlighted the possible significance of the relationship between vitamin D and the tryptophan-derived neurotransmitter, serotonin , previous studies focused solely on serotonin synthesis. In the present communication, we have investigated, for the first time, the effects of 1,25D on the regulation of serotonin reuptake via a serotonin reuptake transporter and on the first step of serotonin degradation by monoamine oxidase-A in serotoninergic neuronal cell lines. SERT (SLC6A4, 5HTT), the 5-HT transporter, plays a crucial role in the control of serotonergic neurotransmission by determining the magnitude and duration of the 5-HT synaptic signal and has been an attractive target in rational neuropsychiatric drug design . Fluoxetine (Prozac), citalopram (Celexa), paroxetine (Paxil), and sertraline (Zoloft) represent selective inhibitors of SERT. In the presence of these selective serotonin reuptake inhibitors (SSRIs), 5-HT remains in the extracellular space for an extended duration, allowing prolonged 5-HT receptor activation in the postsynaptic neuron. Thus, monoamine transporters such as SERT constitute the primary mechanism for terminating neurotransmission through removal of the released transmitter from the synaptic cleft. These transporters, including SERT, function as oligomeric complexes in the cell membrane, and are regulated primarily through phosphorylation, protein-protein interactions, and trafficking events . Limited data are available on the control of SERT at the transcriptional level, although the genes encoding mammalian forms have been cloned . The 5-prime flanking region of the SERT (5HTT) human gene contains a cyclic AMP-responsive element (CRE) at − 99 bp plus binding sites for AP-1, AP-2, and SP1, all just upstream of a TATA-like motif. Sequences within − 1.4 kb of the transcription start site confer cell-specific expression, and the basal promoter responds to induction by cAMP . A functional polymorphism (5HTT-LPR) has been identified in the human 5HTT promoter , and evidence has accumulated for an association between alleles of this variation and anxiety traits, affective disorders, as well as autism. Notably, three ChIP-seq databases generated in human immune cells for 1,25D-dependent occupation of vitamin D-responsive elements (VDREs) by VDR yield apparently functional sites at − 64.5 and + 118 kb in relation to the transcription start site of the SERT gene [14, 15, 16]. Thus, 1,25D could potentially modulate human SERT expression in a primary fashion via remote VDREs.
Examining the influence of 1,25D on SERT mRNA expression in rat raphe RN46A-B14 cells, we observed a repression of SERT mRNA by 59% when the cells were treated with 10 nM 1,25D (Fig. 1a). This repression was selective for serotonergic neuronal cells in that it was not found in U87 human glioblastoma cells (Fig. 2a). The initial conclusion is that 1,25D, analogous to SSRIs, suppresses serotonin reuptake, possibly potentiating serotonergic neurotransmission. However, this conclusion must be reached with caution, because neither 1 nor 100 nM 1,25D treatment of RN46A-B14 cells significantly represses SERT mRNA expression (Fig. 1a), generating an unusual, biphasic dose-response curve. Nevertheless, this dose response is reminiscent of the U-shaped curves generated when plotting circulating 25-hydroxyvitamin D levels versus disease in humans, most recently reported in a study of autism risk in 3-year-old Chinese children as a function of circulating 25-hydroxyvitamin D levels at birth . In other words, there appears to exist an optimal level of circulating vitamin D for health, and this may correspond empirically to 10 nM 1,25D in the case of RN46A-B14 cell treatment.
However, the cellular and molecular mechanism that accounts for the observed effects of various concentrations of 1,25D in RN46A-B14 cell treatment experiments requires careful consideration. The authors favor the concept that limited, but more physiologically relevant, concentrations of 1,25D signal biologic actions such as cell proliferation, whereas higher levels of the hormone cause the opposing responses, namely inhibition of cell growth and apoptosis. We first identified this phenomenon when studying osteosarcoma cells in culture, observing that low concentrations of 1,25D stimulated cell growth, whereas higher levels drastically curbed proliferation . Therefore, we propose that the data in Fig. 1a can be explained if one assumes that the minimum effective concentration to stimulate proliferation (and other effects such as altered transcription factor profile and epigenetic modifications) in RN46A-B14 cells is 10 nM 1,25D, conditioning the neuronal cells to exhibit a repression of SERT expression, then the lack of response to treatment with 1 nM is appropriate. If one then assumes that 100 nM 1,25D, analogous to the action of this level in many cell types, promulgates an inhibition of cell proliferation with attending alteration of transcription factor profile and epigenetic modifications, then the lack of responsiveness of SERT expression to this higher dose of 1,25D of these growth-inhibited RN46A-B14 cells is also readily explainable. Nevertheless, the observations, in vitro, that higher concentrations of 1,25D are less effective than 10 nM hormone must be viewed with some skepticism and it will ultimately need to be determined if data from cell lines can be directly equated to the U-shaped dose-response curves, in vivo, because cell growth aspects and cell culture conditions (i.e., interaction with other media components) could be responsible for the results with high 1,25D concentrations. However, very recent data obtained in autistic children suggest that maintaining circulating 25-hydroxyvitamin D levels in an optimal range elicits maximal therapeutic efficacy in vitamin D-responsive children . Regardless of the mechanistic nuances, we provide herein more support for the vitamin D-serotonin hypothesis in the pathophysiology of neuropsychiatric disease that has been professed by Patrick and Ames [1, 6]. Consistent with the relevance of SERT modulation by 1,25D is a report  of increased concentrations of SERT protein (monitored by positron emission tomography (PET) brain scans) in patients with seasonal affective disorder (SAD). SERT levels in SAD patients were 5% higher in winter, when vitamin D generated by sunlight is deficient, than in summer when sunlight is at a maximum; control subjects without SAD manifest no change in SERT levels with season. It is accepted that sunlight is mood-elevating, and the 1,25D effect to repress SERT reported herein may represent a facet of this benefit.
When examining MAO-A modulation by 1,25D in rat raphe RN46A-B14 cells, we observed a repression of MAO-A mRNA by 51% when the cells were treated with 10 nM 1,25D (Fig. 1b). The 1,25D dose-response profile was equivalent for MAO-A and SERT repression, and the effect on MAO-A was also selective for serotonergic neuronal cells as it was not found in U87 human glioblastoma cells (Fig. 2b). Thus, the same arguments can be applied in discussing the control of MAO-A by 1,25D that were elaborated above for SERT repression. However, there exists a caveat to the observed repression of MAO-A by 1,25D in cultured rat serotonergic cells, in that Jiang et al.  demonstrated a significant induction of MAO-A mRNA in the prefrontal cortex of rats after chronic 1,25D administration. This conflict between the in vitro and in vivo results with respect to MAO-A could be because Jiang et al.  analyzed the entire prefrontal cortex, whereas the cell culture data are selective to serotonergic neuronal cells. Notably, MAO-A also catalyzes the degradation of dopamine and norepinephrine, as well as serotonin, implying a broader array of functions beyond serotonin catabolism in the cortex, perhaps explaining the differences between cell culture results and those obtained in rodent models.
MAO-A is a gene of interest that has been given the moniker “warrior gene” as it has been linked to aggression in observational and survey-based studies . In addition, MAO-A knockout mice are more aggressive than their wild-type littermates or mice null for MAO-B, which degrades a different set of neurotransmitters . The transcriptional regulation of MAO-A has not been fully characterized but, in a recent computational and experimental analysis of Mao-A expression, Gupta et al.  identified critical roles for a conserved proximal promoter domain (− 71/− 40 bp) in cis, and transcription factors Sp1/Gata2/Tbp in trans, to govern mouse Mao-A gene basal expression in a coordinated manner. We have located, in silico, a near-consensus candidate VDRE, consisting of a direct repeat with a spacer of three nucleotides (DR3), CGGACActgAGGTCA (antisense), at + 776 in relation to the start of transcription of the human MAO-A gene. Furthermore, Tuoresmaki et al.  have reported two VDR ChIP-seq target regions (roughly 200 bp in size) in the human MAO-A gene, the first of which overlaps the candidate DR3 VDRE at + 776 bp cited above and the second, located approximately 350 bp 3-prime to the first, which contains two additional candidate (antisense)VDREs: GGTGCAggaGGTTCT and GGTGCAggaGGTGCT. It is not yet known whether these putative VDREs are involved in MAO-A control by 1,25D, but their proximity to the core regulatory module at − 71/− 40 bp is suggestive of a possible cis regulatory module spanning the proximal promoter and first exon of the MAO-A gene. If this concept is verified, yet another avenue may exist whereby 1,25D could not only benefit patients with behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission, but also impact other behavioral traits and neurological disorders, including Alzheimer’s dementia, aggression, and attention deficit hyperactivity disorder .
The present results support the vitamin D-serotonin hypothesis both experimentally and mechanistically. In fact, when we assessed the direct production of serotonin in neuronal cell culture in response to treatment with 10 nM 1,25D for 72 h, serotonin concentrations were significantly enhanced (two- to threefold) in the medium (Fig. 5), consistent with the concerted induction of TPH2 (Fig. 4b) and repression of SERT (Fig. 1a) and MAO-A (Fig. 1b) by the empirically optimum level of 1,25D in cultured serotonergic neurons. Taking all of these results together, it seems highly likely that vitamin D can amplify serotonin in the central nervous system, making vitamin D a candidate for the treatment of neuropsychiatric disorders in which vitamin D and/or serotonin are implicated. Indeed, vitamin D supplementation has been reported to improve inattention, hyperactivity, and impulsivity in children and adults with ADHD .
Mammalian cell culture
Two mammalian cell lines were employed in this study: embryonic rat medullary raphe (RN46A-B14; obtained from Dr. Robert J. Handa/Dr. John Neumaier) cultured with neurobasal A (Gibco/Life Technologies, Grand Island, NY) medium with B-27 supplement (Invitrogen Corporation) and l-glutamine (Gibco) and human brain glioblastoma/astrocytoma (U-87 MG, U87; obtained from the American Type Culture Collection) grown in DMEM/high glucose + l-glutamine + pyruvate (Hyclone, Logan, Utah). All cells were passaged in the above-indicated media supplemented with 10% FBS and penicillin/streptomycin (pen-strep) under a humidified atmosphere of 5% CO2 in air at 37 °C. RN46A-B14 cells in culture were utilized in their native state, i.e., not transfected with any expression vector. However, U87 cells were transfected with a VDR overexpression plasmid to ensure that they consistently expressed biologically functional levels of the nuclear receptor. The PolyJET transfection reagent (SignaGen, Rockville, MD) was utilized to transfect U87 cells according to the manufacturer’s protocol. Briefly, each well was transfected with 15 μL PolyJET reagent and 500 ng of pSG5-hVDR (plasmid-expressing human VDR).
The clonal cell line, RN46A-B14, was isolated following transfection of the gene encoding rat brain-derived neurotrophic factor (BDNF) into RN46A cells . RN46A-B14 cells synthesize and secrete biologically active BDNF in vitro and synthesize serotonin following partial membrane depolarization. To optimize the growth conditions and neuronal morphology of adherent, rat serotonergic B14 cells in culture, three different media formulations were examined: media 1: neurobasal A medium, B-27 supplement, l-glutamine, and pen-strep (serum free); media 2: media 1 plus 10% FBS; media 3: DMEM/high glucose + l-glutamine + pyruvate, 10% FBS, and pen-strep.
Quantitative real-time PCR
U87 and RN46A-B14 cells were plated at 500,000–800,000 cells per well in a 6-well plate, and dosed with 1–100 nM 1,25D (see Figs. 1, 2, 3 and 4) or ethanol vehicle control for 24 h. Total RNA was isolated from each well using an Aurum Total RNA Mini kit (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. The RNA quantity and quality were assessed using A260/280 spectrophotometry. DNase-treated RNA (1 μg) was reverse transcribed using an iScript cDNA Synthesis kit (Bio-Rad) to produce 20 μL of first-strand cDNA. For real-time PCR, 2 μL of cDNA was used in a 10 μL PCR reaction containing 5 μL FastStart Universal SYBR Green Master Mix + Rox (Roche Applied Science, Indianapolis, IN) and forward and reverse primers. Reactions were performed in 96-well PCR plates in a Bio-Rad CFX96 instrument using a standard 40-cycle profile. Data were analyzed using the comparative ΔΔCt method as means of relative quantitation, normalized to an endogenous reference (GAPDH) and relative to a calibrator (normalized Ct value from vehicle-treated cells) and expressed as 2-ΔΔCt according to Applied Biosystems’ User Bulletin 2: Rev. B, “Relative Quantitation of Gene Expression.” Primer sets for the PCR were as follows:
Rat GAPDH (forward 5′-AGGTCGGTGTGAACGGATTTG-3′, reverse 5′-CATTCTCAGCCTTGACTGTGC-3′)
Rat Cyp24a1 (forward 5’-AACGAAGCCTACGGGTTGATG-3′, reverse 5’-AGAAAGTCAGCCAAGACCTCA-3′)
Rat SERT  (forward 5′-TCTGAAAAGCCCCACTGGACT-3′, reverse 5′-TAGGACCGTGTCTTCATCAGGC-3′)
Rat MAOA  (forward 5′-CCACAGCCAGAGCGTTCAG-3′, reverse 5′-TGAGAGCCTTTGCCCAGATTG-3′)
Rat MAOB  (forward 5′-CAGTGGAAGCAGAGGAGAG-3′, reverse 5′-TGCTGCCATACCTGAGATG-3′)
Rat TPH1  (forward 5′-GCTGAACAAACTCTACCCAAC-3′, reverse 5′-TTCCCGATAGCCACAGTATT-3′)
Rat TPH2  (forward 5′-CTCCAAGCTTCGCATCACAG-3′, reverse 5′-AGCACTTCAGGAAGCGTACC-3′)
Human GAPDH (forward 5′-ACAACTTTGGTATCGTGGAAGGAC-3′, reverse 5′-CAGGGATGATGTTCTGGAGAGC-3′)
Human CYP24A1 (forward 5′-CAGCGAACTGAACAAATGGTCG-3′, reverse 5′-TCTCTTCTCATACAACACGAGGCAG-3′)
Human SERT  (forward 5′-TTCTCCCCTCCAAGTGAATG-3′, reverse 5′-GGGGAATCCATGGAAATAAG-3′)
Human MAOA  (forward 5′-TAAATGGTCTCGGGAAGGTG-3′, reverse 5′-CCCAGGGCAGTTACTGATGT-3′)
Human MAOB  (forward 5′-GCTCTCTGGTTCCTGTGGTATGTG-3′, reverse 5′-TCCGCTCACTCACTTGACCAGATC-3′)
To measure serotonin (5-HT) concentrations, colorimetric competitive 5-HT enzyme-linked immunosorbent assay (ELISA) experiments were performed utilizing the Serotonin Research ELISA kit (Rocky Mountain Diagnostics, Inc., Colorado Springs, CO). RN46A-B14 were plated at 700,000 cells per well in a six-well plate in media 2 (see above for details) and incubated in a humidified atmosphere of 5% CO2 at 37 °C overnight. The following day, media 2 was aspirated and the cells were gently washed with PBS to remove residual serum. Media 1 (see above for details) was added, and the cells were incubated overnight to generate a serum-deprived cell state. Next, the cells were treated with either ethanol vehicle control or 10 nM 1,25D for 0–72 h in media 1. Samples of media/culture supernatant (200 μL) were removed from each well (without disrupting the adherent cells) at 0, 24, 48, or 72 h and assayed for 5-HT content in accordance with the manufacturer’s protocol. Briefly, the diluent, wash buffer, six standards, and two positive controls were prepared at the time of the assay, and 100 μL of the diluted standards, controls, and experimental samples were acylated for 30 min at room temperature. Next, 100 μL of the acylated standards, controls, and experimental samples were placed into the appropriate wells of the serotonin/5-HIAA microtiter strips, and 25 μL of the 5-HT antiserum were added to each well and incubated for 15–20 h at 4 °C. The wells were washed, and 100 μL of the enzyme conjugate were added and allowed to incubate for 30 min followed by additional washes and incubation with 100 μL of the substrate. After the final incubation, 100 μL of the stop solution were added and the absorbance was read within 10 min using a microplate reader set to 450 nm and a reference wavelength between 620 and 650 nm. The calibration curve was obtained by plotting the absorbance readings measured for the standards (linear, y axis) against the corresponding standard concentrations (linear, x axis, ng/mL). A logarithmic trendline was fitted to the data for the standards and the corresponding equation was utilized to determine the serotonin concentrations in the experimental samples. The mean of three biological replicates, each with duplicate treatment groups, was utilized in the analysis.
Data are expressed as means ± SD. All data are presented as fold-effects, with transcription of the gene in question (i.e., mRNA level) in the absence of 1,25D (i.e., ethanol vehicle) set at 1.0-fold. Thus, all results are normalized to basal transcription and presented graphically and numerically as fold-effect of the single tested variable, i.e., 1,25D. Because the design of the experiments was a simple motif in which the fold-ability of 1,25D to enhance transcription over basal (i.e., ethanol vehicle) was assessed, statistical differences between two groups were determined initially by a two-tailed Student’s t test. However, because variance was high in several experiments, further statistical analyses were performed using GraphPad Prism 7 to generate ANOVA data, followed by a post hoc Dunnett test that compares every mean to a control mean and takes into account the scatter of all the groups. In each figure illustrating the fold-effect of 1,25D, exact P values (unless < 0.0001) are listed above the bar for concentrations of 1,25D versus EtOH control, except when the difference from the vehicle control is not significant (NS); a P value less than 0.05 is considered significant.
This research was supported in part by NIH grants DK033351 to MRH and CA140285 to PWJ and a grant from the University of Arizona, Department of Basic Medical Sciences to GKW.
MSS contributed to the study design, data collection, data analysis/interpretation, and developing a preliminary version of the manuscript in poster format. MRH, the corresponding author, contributed via conception/design of the work and wrote the initial version, as well as the final revisions of the manuscript. SM, IK, and DAL contributed to the data collection/analysis and to the revision of this manuscript. CAH and GKW contributed the critical experimental insight and interpretations, as well as to the independent revision of the article. PWJ, the senior investigator directing the study, contributed via conception/design of work, data interpretation, and critical revision of the article. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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