- Methodology article
- Open Access
Rapid and ultra-sensitive quantitation of disease-associated α-synuclein seeds in brain and cerebrospinal fluid by αSyn RT-QuIC
- Bradley R. Groveman†1,
- Christina D. Orrù†1,
- Andrew G. Hughson1,
- Lynne D. Raymond1,
- Gianluigi Zanusso2,
- Bernardino Ghetti3,
- Katrina J. Campbell1,
- Jiri Safar4,
- Douglas Galasko5Email author and
- Byron Caughey1Email authorView ORCID ID profile
© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply. 2018
- Received: 16 January 2018
- Accepted: 17 January 2018
- Published: 9 February 2018
The diagnosis and treatment of synucleinopathies such as Parkinson disease and dementia with Lewy bodies would be aided by the availability of assays for the pathogenic disease-associated forms of α-synuclein (αSynD) that are sufficiently sensitive, specific, and practical for analysis of accessible diagnostic specimens. Two recent αSynD seed amplification tests have provided the first prototypes for ultrasensitive and specific detection of αSynD in patients’ cerebrospinal fluid. These prototypic assays require 5–13 days to perform. Here, we describe an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay that has similar sensitivity and specificity to the prior assays, but can be performed in 1–2 days with quantitation. Blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases [12 Parkinson’s and 17 dementia with Lewy bodies] and 31 non-synucleinopathy controls, including 16 Alzheimer’s cases, yielded 93% diagnostic sensitivity and 100% specificity for this test so far. End-point dilution analyses allowed quantitation of relative amounts of αSynD seeding activity in cerebrospinal fluid samples, and detection in as little as 0.2 μL. These results confirm that αSynD seeding activity is present in cerebrospinal fluid. We also demonstrate that it can be rapidly detected, and quantitated, even in early symptomatic stages of synucleinopathy.
- Lewy body
- Cerebrospinal fluid
Many neurodegenerative diseases are related to the accumulation of specific misfolded proteins. These deposits are identified upon post-mortem analysis of brain tissue, allowing definite diagnoses to be made based on specific neuropathological and molecular findings. Less definitive intra vitam diagnoses can be proffered based on specific clinical signs, tissue imaging data, pathological examination of peripheral biopsies, and less-than-specific biomarker levels in the CSF. In particular, early diagnosis can be difficult and discrimination between diseases can be complicated by clinical variability and overlaps in clinical features.
Parkinson’s disease (PD), multiple system atrophy (MSA), dementia with Lewy bodies (DLB) [or Lewy body dementia] are called α-synucleinopathies due to the abnormal accumulation of aggregates of a protein called α-synuclein (αSyn) in the brain. Although the clinical diagnosis of parkinsonism can be relatively simple, the specific diagnosis of PD, especially at early stages, can be difficult. Adler et al. noted that in patients with possible PD (never treated or not clearly responsive to L-dopa) only 26% had autopsy confirmation as PD, while in probable PD (responsive to medications) the diagnostic accuracy was 82% . In DLB, clinical diagnostic criteria for probable DLB predict αSyn pathology with sensitivity of about 80%  but early diagnosis of DLB is less accurate due to the overlapping symptoms with other types of dementia. In addition, in 15–20% of patients with Alzheimer disease (AD) at autopsy, concomitant DLB pathology can be found, with only a minority of patients having exhibited clear diagnostic features of DLB [20, 34]. However, in patients with AD and diffuse Lewy body pathology, disease duration was shortened , indicating that DLB pathology contributes to dementia progression.
Some pertinent tests indirectly measure the effect of α-Syn pathology (e.g., dopamine receptor SPECT or PET scans, and MIBG cardiac scintigraphy), while the sensitivity and specificity of skin, salivary gland and colonic biopsy for PD or DLB has not been established in large scale studies. In these clinical settings of PD and DLB, the presence of a biomarker that indicates that abnormal pathological forms of a αSyn are present would improve diagnostic accuracy not only for prognostic purposes but also for cohort selection in disease-modifying clinical trials for PD. Attempts to determine if cerebrospinal fluid (CSF) levels of total, phosphorylated or oligomeric a-syn are diagnostically useful have been variable and controversial between studies [reviewed in ], and the diagnostic utility of immunoassays for these forms of αSyn in CSF remains unclear [21, 31].
However, two recent studies have provided evidence that analysis of a distinct feature of disease-associated forms of αSyn (hereafter abbreviated αSynD), namely their amyloid seeding activity, may have substantial diagnostic utility for PD and DLB [7, 35]. The rationale for the seeding activity assays is that the αSynD deposits contain fibrils, or subfibrillar oligomers, that propagate by a seeded polymerization mechanism in which αSynD templates, or seeds, conversion of non-fibrillar αSyn into larger oligomeric or aggregate, fibrillar forms. Mechanistically similar assays called Real-Time Quaking-Induced Conversion (RT-QuIC) have provided ultrasensitive, specific and quantitative diagnostic tests for prion diseases [2, 39]. RT-QuIC assays are multi-well plate-based reactions that can rapidly amplify oligomeric/multimeric prion seeds by as much as a trillion-fold [8, 24, 26, 39]. Prion RT-QuIC assays have been applied successfully to a variety of biological samples including brain [29, 39, 41], cerebrospinal fluid (CSF) [2, 5, 17, 24, 33], whole blood, plasma [26, 38], urine , and nasal brushings [23, 40]. They are being widely implemented for the diagnosis of prion diseases in humans and animals. Notably, our recent studies demonstrated provisional 100% diagnostic sensitivity and specificity in diagnosing human sporadic Creutzfeldt-Jakob disease using CSF and/or nasal swabs .
Green and colleagues adapted the RT-QuIC approach to synucleinopathies and applied it to a total of 137 PD and DLB cases and controls . Their assay (αSyn RT-QuIC) has given 95 and 92% sensitivity for PD and DLB patients, respectively, with 100% specificity. Soto and colleagues developed a similar assay called αSyn protein misfolding cyclic amplification (αSyn-PMCA) which gave 89% sensitivity for PD and 97% specificity in analyses of 173 total cases and controls . In these assays, 5–40 μl aliquots of CSF are added to reactions containing recombinant αSyn (rαSyn). Any αSynD seeds in the sample initiate amyloid fibril formation by the recombinant αSyn which, in turn, enhances the fluorescence of thioflavin T (ThT). The reactions are performed over ~ 5  to 13 days . Sano and colleagues have described an αSyn RT-QuIC assay that detects DLB αSynD seeding activity in brain tissue at extreme dilutions in < 4 days . Bernis and colleagues showed that 10% brain homogenate samples from mice inoculated with human MSA or incidental Lewy body disease brain tissue could seed fibrillization of rαSyn in 1–2 days . Here we report that by using a mutant rαSyn substrate and optimized reaction conditions, αSyn RT-QuIC assays on CSF specimens can be completed within 1–2 days with high diagnostic sensitivity and specificity.
All subjects provided consent to clinical assessment, including longitudinal follow-up, and to lumbar puncture to obtain CSF, under UCSD IRB-approved protocol #080012. All procedures performed in this study were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Some subjects died during the follow-up period, and had consented to their brains being obtained at autopsy.
All subjects underwent a detailed clinical research assessment, including review of outside medical records, history of cognitive and motor symptoms, mental state examination with the Mini-Mental State Exam or Montreal Cognitive Assessment, and detailed neuropsychological testing, structured physical neurological examination, including the Unified Parkinson’s Disease Rating Scale (UPDRS) Part III motor examination. All subjects were enrolled in a research protocol that allowed annual follow-up reassessment and received at least one follow-up assessment after their baseline visit. Neuroimaging (MRI and in some instances FDG PET scan or DaTscan) results were reviewed when available. The research diagnoses were made by consensus of two neurologists who reviewed all of the available clinical information. Research diagnoses followed published criteria: controls had no history of major neurological or psychiatric illness and were normal on cognition and neurological examination; patients with AD met criteria for probable AD (NIA-AA 2011). For PD, criteria proposed by the Movement Disorder Society were used , and research guidelines were applied to diagnose PD-MCI , PD-dementia and DLB (possible and probable DLB were diagnosed according to McKeith ).
Lumbar puncture and CSF handling
Lumbar punctures (LPs) were performed in the early morning, after a fast of at least 8 h. Subjects were either sitting or lying, and LPs were performed with sterile technique using an atraumatic needle. CSF (15–20 mL) was withdrawn into a polypropylene tube and a sample was sent for analysis of cell count, total protein and glucose to a local laboratory. The remaining CSF was gently mixed, centrifuged at 1500 g for 10 min, then aliquotted in 500 μL fractions into polypropylene cryotubes, flash frozen and stored at − 80 °C.
Autopsy brain analysis
Procedures at autopsy at the UCSD Alzheimer’s Disease Research Center are as follows: the brain is divided sagittally and the left hemibrain is fixed in 10% buffered formalin while the right hemibrain is sectioned coronally and then frozen at − 70 °C in sealed plastic bags. Routinely, tissue blocks from the right hemibrain of the midfrontal, inferior parietal, and superior temporal cortices, primary visual cortex in the occipital cortex, hippocampus, basal ganglia, substantia nigra and cerebellum are removed and placed in 2% paraformaldehyde for subsequent thick sectioning by vibratome. Tissue blocks adjacent to the ones described above are stored at − 70 °C for subsequent immunoblot analysis for synaptic proteins and Aβ species (soluble and oligomers). Vibratome sections (40 μm thick) are stored in cryoprotective medium at − 20 °C for subsequent immunochemical studies. The formalin-fixed left hemibrain is serially sectioned in 1 cm slices and tissue blocks from the regions described above are processed for histopathological examination by H&E, and Thioflavin-S (Thio-S) to detect tau and β-amyloid deposits. Lewy body pathology is evaluated using phosphorylated α-synuclein immunoreactivity with a mouse monoclonal antibody at 1:20,000 (BioLegend Cat# 825701 RRID:AB 2564891). Pathological diagnoses of AD and DLB are made using National Institute on Aging-Alzheimer’s Association (NIA-AA) guidelines .
K23Q rαSyn expression vector preparation
DNA sequences coding for human α-synuclein sequence (Accession No. NM_000345.3) amino acid residues 1–140 (wildtype) were amplified and ligated into the pET24 vector with an N-terminal His-tag (EMD Biosciences) and sequences were confirmed. The α-synuclein K23Q mutation  was engineered using Q5 Site-Directed Mutagenesis (NEB) using the primers CCACACCCTGTTGGGTTTTCTCAG and CAGAAGCAGCAGGAAAGAC. The plasmids were transformed into BL21(DE3) Escherichia coli (EMD Biosciences).
rαSyn protein purification
Five ml of LB media containing 50 μg/mL kanamycin were inoculated from a glycerol stock of E. coli bacteria containing vectors either for wildtype (WT) or K23Q rαSyn protein expression. Following 4–5-h incubation with continuous 225 rpm agitation at 37 °C, 1 L of the auto-induction media  also containing 50 μg/mL kanamycin was prepared and the 5 mL starter culture was added. The cells were grown in a shaking incubator at 37 °C, 225 rpm, overnight. The next day cells were harvested by splitting the 1 L culture into four 250 ml conical tubes and centrifuging at 3273×g, 4 °C, 10 min.
Cells were lysed using an osmotic shock protocol modified from Paslawski et al. . Using a 25 mL serological pipette, the cell pellets were gently resuspended in 10% volume of room temperature osmotic shock buffer, (25 mL per 250 mL of cell culture before centrifugation) and incubated at room temperature for 10 min. The suspension was centrifuged at 9000×g, 20 °C, 20 min. The supernatant was discarded and the pellet was gently resuspended in 10 mL of ice-cold water per pellet, using a 25 mL serological pipette. The cell suspensions were pooled into two 50 mL tubes to 20 ml each. 20 μL of saturated MgCl2 was added to each 20 mL suspension. The suspension was then mixed and incubated on ice with mild rocking for 3 min. Next, the suspension was centrifuged at 9000×g, 4 °C, 30 min. The supernatant was collected in a 100 ml glass beaker that contained a stir bar for rapid continuous mixing while being careful not to incorporate air bubbles. The pH was reduced to pH 3.5 by adding a bolus of ~ 800 μl 1 M HCl followed by additional 25 μl increments as necessary while continuously monitoring the pH. A large amount of white precipitate was generated. The suspension was then incubated with gentle stirring at room temperature for 10 min, avoiding the formation of air bubbles. The tubes were centrifuged at 9000×g, 4 °C, 30 min, and the supernatant was collected in a fresh 100 ml beaker using continuous agitation with a stir bar. The pH was adjusted to 7.5 with an ~ 800 μl bolus of 1 M NaOH followed by 25 μl increments as necessary. The protein extract was then filtered through a 0.45 μm filter. Next, the extract was loaded onto a 5 ml Ni-NTA column (Qiagen) on an Äkta Pure chromatography system (GE) and washed with 20 mM Tris, pH 7.5 at room temperature. The column was further washed with 50 mM imidazole, 20 mM Tris, pH 7.5 which generated a peak that was not collected. A linear gradient up to 500 mM imidazole in 20 mM Tris, pH 7.5, was performed and a peak was collected between 150 and 375 mM imidazole (Additional file 1). This peak was then loaded onto a Q-HP column (GE) and washed with 20 mM Tris, pH 7.5. The column was further washed with 100 mM NaCl, 20 mM Tris, pH 7.5. A linear gradient up to 500 mM NaCl in 20 mM Tris pH 7.5 was performed and a peak was recovered between 300 and 350 mM NaCl. The protein was filtered through a 0.22 μm filter and dialyzed against water overnight at 4 °C using a 3 kDa MWCO dialysis membrane. The next day, the protein was moved into fresh water for another 4 h dialysis. The protein concentration was determined with a UV–VIS spectrophotometer using a theoretical extinction coefficient at 280 nm of 0.36 (mg/mL)− 1 cm− 1. The protein was lyophilized in aliquots and stored for a final concentration of ~ 1.0 mg/ml once resuspended in 500 μL of 40 mM phosphate buffer (pH 8.0). These aliquots were stored at − 80 °C until further use.
For comparative studies, human recombinant full-length (1–140 aa) WT αSyn was also purchased from Stratech, (Cambridge, UK).
Endotoxin concentration levels for rαSyn protein purifications was determined using a ToxinSensor Endotoxin Detection kit (GenScript) according to manufacturer’s protocol.
Samples were prepared in 2× sample loading buffer (final concentration: 62.5 mM Tris–HCl, pH 6.8, 5% glycerol, 3 mM EDTA, 5% SDS, 0.02% bromophenol blue, 4 M urea, 4% β-mercaptoethanol) and boiled for 5 min. Proteins were separated by gel electrophoresis using 10% Bis-TrisNuPAGE gels (Invitrogen). For total protein analysis, gels were stained with GelCode Blue Safe Protein Stain (Thermo Scientific, 24,594) according to the manufacture’s protocol.
Preparation of synthetic wildtype rαSyn fibrils
A 100 μL solution of 1 mg/ml WT rαSyn in PBS was subjected to continuous 1000 rpm shaking at 37 °C for 3 days in a 1.5 mL tube. The products were ThT-positive, indicating the presence of amyloid, and the OD280 and Western blot comparisons of supernatants before and after centrifugation indicated that > 30% of the total rαSyn was insoluble and therefore aggregated.
Brain homogenate preparations
Brain homogenates (BH; 10% w/v) were prepared by homogenizing the tissue in PBS using a Bead Beater (Biospec Products; 11079110z) for 1 min at maximum speed. The homogenate was then spun at 2000 xg for 2 min at room temperature and the supernatant was transferred to a new tube and stored at − 80 °C for αSyn RT-QuIC analysis. For αSyn RT-QuIC testing, BHs were serially diluted in PBS.
αSyn RT-QuIC protocol
In the case of CSF seeded reactions, wells pre-loaded with 6 glass or silica beads were given 85 μL of a reaction mix adjusted to give final reaction concentrations of 40 mM phosphate buffer (pH 8.0), 170 mM NaCl, 0.1 mg/ml rαSyn, 10 μM thioflavin T (ThT) and 0.0015% sodium dodecyl sulfate (SDS) and then 15 μL CSF, or dilutions thereof in normal pooled CSF. The reaction plates were subjected to shake-rest cycles as for BH samples above and the reactions classified as RT-QuIC-positive or -negative based on criteria similar to those previously described for RT-QuIC analyses of brain specimens [23, 39]. Briefly, a ThT fluorescence threshold was calculated as the average fluorescence for all samples within the first 10 h of incubation, plus three Standard Deviations (SD). A sample was considered positive overall when at least two of four replicate wells crossed this calculated threshold. When only one of the quadruplicates crossed the threshold, the analysis was repeated.
Rapid detection of αSynD by αSyn RT-QuIC
In developing our αSyn RT-QuIC, we focused primarily on a recombinant 6× histidine-tagged K23Q mutant of αSyn as a soluble rαSyn substrate for αSynD-induced fibrillization. Recombinant K23Q αSyn was reported to fibrillize with kinetics similar to wild-type (WT) αSyn when seeded with preformed synthetic WT αSyn fibrils, but was slower to spontaneously fibrillize in the absence of preformed seeds . This latter characteristic, we hypothesized, might improve the sensitivity of an αSyn RT-QuIC assay by enhancing the kinetic distinction between reactions seeded with samples from synucleinopathy cases versus controls. We iteratively optimized the K23Q purification protocol and αSyn RT-QuIC reaction conditions. The best purification protocol to date involves lysis by osmotic shock followed by acid precipitation and sequential metal-ion affinity and ion exchange chromatography steps . No protein impurities were observed by SDS-PAGE analyses of our K23Q mutant rαSyn, our similar preparation of a histidine-tagged WT αSyn, or a commercial wild-type αSyn preparation (without a 6× histidine tag) (WT*) that was used for the previously described αSyn RT-QuIC assay  (Additional file 1). However, because lipopolysaccharide (LPS) can contaminate bacterially derived protein preparations and might influence fibrillization, we assayed the three rαSyn preparations and found that whereas our WT and K23Q rαSyn preparations were negative for LPS in this assay (< 0.25 EU/ml), the WT* preparation had ≥ 0.25 EU/ml LPS.
Blinded analysis of CSF from synucleinopathy cases and controls
Demographic data and cognitive impairment at the time of lumbar puncture (LP) in studied subjects
Age at onset (years)
Age at LP (years)
Mean interval between onset and LP (years)
Dementia with Lewy Bodies
69.6 ± 7.8
73.8 ± 7.8
23.0 ± 4.6
63.1 ± 12.0
66.0 ± 12.9
28.9 ± 1.1
69.9 ± 9.1
73.9 ± 9.1
22.9 ± 3.3
71.3 ± 7.0
28.8 ± 1.2
65.7 ± 11.4
67.7 ± 10.7
20.5 ± 8.1
Overall, the results from this blinded panel indicated diagnostic sensitivities, i.e. the percentage of cases giving positive RT-QuIC responses, of 93% for both PD and DLB. None of the non-synucleinopathy or AD controls met criteria to be considered positive RT-QuIC responses resulting in an apparent specificity of 100%.
Relative αSyn seeding activities in CSF and brain tissue from PD and DLB cases
Analytical sensitivity using synthetic αSyn fibrils
The ability to detect αSynD as a causative pathological biomarker for syncleinopathies has important implications in diagnostics, the development of therapeutics, and fundamental studies of αSynD-based pathogenesis. Recent studies have demonstrated diagnostic utility for αSyn RT-QuIC and closely related αSyn PMCA assays using CSF specimens [7, 35]. Here we present an αSyn RT-QuIC assay with similar diagnostic accuracy but much reduced assay time, i.e. 1–2, rather than 5–13 days. Sano and colleagues detected αSyn seeding activity of DLB brain in 3–4 days , but as brain has much higher concentrations of αSynD seeding activity than CSF (Fig. 4), it is unclear how well their αSyn RT-QuIC assay would perform with CSF specimens. In any case, our reduced assay time markedly enhances the cost effectiveness and practicality of the αSyn RT-QuIC analyses of CSF. Most of the CSF specimens that we analyzed were collected relatively early in the disease course of the given synucleinopathy. The early detection of αSynD is particularly helpful, firstly, because the accuracy of diagnoses based on other clinical indices is poorest in the earlier phases of disease, and, secondly, because the earlier the diagnosis, the earlier that any appropriately targeted therapies can be initiated before further tissue damage is done.
Improvements in the early diagnosis of synucleinopathies should also aid in the selection of suitable patients and controls for therapeutic trials. Furthermore, the ability to serially measure relative levels of αSynD in treated and untreated cohorts may provide an alternate means of monitoring the effects of treatments, especially those aimed at reducing the burden of αSynD in the brain. Here we have used end-point dilution analysis for quantitation by αSyn RT-QuIC, an approach that has been helpful in many studies using prion RT-QuIC [23, 24, 39]. A potential alternative approach to quantitation from RT-QuIC assays is the comparison of lag phases or times-to-threshold [13, 36] but further studies will be required to document its utility in αSyn RT-QuIC analyses of various specimen types.
Consistent with previous findings for αSyn PMCA , αSyn RT-QuIC is capable of detecting sub-femtogram amounts of rαSyn fibrils that are orders of magnitude less than those required for more conventional assays such as Western blotting and ELISA, with typical detection limits for total levels of αSyn, in the ng and pg range, respectively. The αSyn RT-QuIC, however, detects only the forms of αSynD capable of seeding further αSyn misfolding. How our αSyn RT-QuIC sensitivity for synthetic rαSyn amyloid seeds relates to its absolute sensitivity for any given form of αSynD in tissues is difficult to determine accurately because different forms of αSynD may have different seeding capacities per unit mass. Nonetheless, based on the average kinetics of seeding using PD or DLB CSF in Fig. 3, one might estimate femtogram levels of seeding capable αSynD may exist in the CSF of PD or DLB patient when compared to the synthetic rαSyn amyloid seeding kinetics in Fig. 5. Furthermore, the difference in the average kinetics of seeding using PD or DLB CSF may be indicative of a “strain” difference, similar to what has been observed from CSF samples from different types of CJD cases in prion RT-QuIC reactions [8, 25]. While the difference in the average kinetics is statistically significant (Welch’s t-test, p < 0.02) between the two seed types, at this point we do not know the basis for this difference, or whether it might be diagnostically useful. It could be due to differences in average seed concentration or seed characteristics, such as potential structural differences between PD and DLB seeds, that contribute to kinetic differences in the αSyn RT-QuIC responses. Testing of much higher numbers of PD and DLB samples is required to discern whether the difference in kinetics is maintained and if it is of diagnostic or mechanistic importance.
As our assay evolved, we saw evidence that several factors, such as sample volume, SDS concentration, temperature, and silica beads, strongly influenced the performance of the assay. However, at present we cannot be sure of the relative importance of these factors individually in allowing the assay to be substantially faster than previously described αSyn RT-QuIC or PMCA assays. Further testing will be necessary to better understand i) the breadth of synucleinopathies that are detected and/or discriminated by αSyn RT-QuIC assays, ii) the diagnostic sensitivities and specificities in clinical settings, iii) the extent to which levels of αSyn seeding activity in CSF or other diagnostic specimens correlates with disease prognosis, and iv) the relevance of αSynD seeding activity as a biomarker in therapeutic trials.
Compared to previously described aSynD seed amplification assays, the use of the raSyn substrate preparation, RT-QuIC reaction conditions, and end-point dilution assays that we describe here allowed for much more rapid detection and quantitation of aSyn seeding activity in CSF of patients with PD and DLB without reductions in diagnostic sensitivity or specificity.
This work was supported in part by the Intramural Research Program of the NIAID, NIH (BC), NIH grant AGO5131 (DG), the Parkinson’s and Movement Disorder Foundation (BRG), the Shiley-Marcos Alzheimer’s Disease Research Center at UCSD (DG), and PHS P30-AG010133 (BG). BRG, CDO, AGH and BC are inventors on a related U.S. Patent pending No. 62/567,079. The authors thank Dr. Daniel Otzen and Cagla Sahin, Aarhus University, Denmark for advice on purification, and Dr. Suzette Priola, Dr. Cathryn Haigh, and Michael Metrick for critical evaluation of this manuscript. We also thank Dr. Christina Sigurdson for establishing contact between BC and DG that made this project possible.
BC and DG oversaw the project. BRG, CO, LR and AH designed, performed, and helped interpret experiments. DG, GZ and BG provided clinical specimens. BC, BRG, CO, and DG drafted the initial manuscript. All authors read, helped to edit, and approved the final manuscript.
BRG, CO, AH, LR and BC are named as inventors on a US provisional patent application related to the technology described herein (see Acknowledgements). The other authors declare that they have no other competing interests.
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