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Anti-Parkinsonian effects of Bacopa monnieri: insights from transgenic and pharmacological Caenorhabditis elegans models of Parkinson’s disease.
Pooja Jadiya1, Asif Khan2, Shreesh Raj Sammi1, Supinder Kaur1, Snober S. Mir2 and Aamir Nazir1*
Abstract
Neurodegenerative Parkinson‟s Disease (PD) is associated with aggregation of protein alpha synuclein and selective death of dopaminergic neurons, thereby leading to cognitive and motor impairment in patients. The disease has no complete cure yet; the current therapeutic strategies involve prescription of dopamine agonist drugs which turn ineffective after prolonged use. The present study utilized the powerful genetics of model system Caenorhabditis elegans towards exploring the anti-parkinsonian effects of a neuro-protective botanical Bacopa monnieri. Two different strains of C. elegans; a transgenic model expressing “human” alpha synuclein [NL5901 (Punc-54::alphasynuclein::YFP+unc-119)], and a pharmacological model expressing green fluorescent protein (GFP) specifically in the dopaminergic neurons [BZ555 (Pdat-1::GFP)] treated with selective catecholaminergic neurotoxin 6-hydroxy dopamine (6-OHDA), were employed for the study. Bacopa monnieri was chosen for its known neuroprotective and cognition enhancing effects. The study examined the effect of the botanical, on aggregation of alpha synuclein, degeneration of dopaminergic neurons, content of lipids and longevity of the nematodes. Our studies show that Bacopa monnieri reduces alpha synuclein aggregation, prevents dopaminergic neurodegeneration and restores the lipid content in nematodes, thereby proving its potential as a possible anti-Parkinsonian agent. These findings encourage further investigations on the botanical, and its active constituent compounds, as possible therapeutic intervention against Parkinson‟s disease.

Keywords: Bacopa Monnieri, Alpha synuclein, Parkinson‟s disease, Caenorhabditis elegans, Neurodegeneration.
Affiliations: 1Laboratory of Functional Genomics and Molecular Toxicology, Division of Toxicology, CSIR-Central Drug Research Institute, 2Department of Biotechnology, Integral University, Lucknow

Abbreviations:
BM - Bacopa Monnieri; 6-OHDA - 6-hydroxy dopamine; PD – Parkinson‟s disease; GFP – Green Fluorescence Protein; DA Dopamine *Corresponding Author: Dr. Aamir Nazir Scientist Division of Toxicology CSIR-Central Drug Research Institute Lucknow, 226001, (U.P.) India Tel: +91-522-2612411 Fax: +91-522-2623405 E-mail: anazir@cdri.res.in

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1.

Introduction

Neurodegenerative diseases like Alzheimer‟s, Parkinson‟s, Huntington‟s and multiple sclerosis are associated with the process of memory loss and cognitive decline which results from selective degeneration of particular neuronal cells and the accretion of aggregated proteins [1]. Parkinson‟s disease (PD) is predominantly characterized as a movement disorder but non motor symptoms are also involved. It affects dopamine-producing neurons in the brain, and the loss of these neurons induces the symptoms of PD. Since dopamine is associated with motor activity, therefore the progressive loss of dopaminergic neurons leads to muscle rigidity, tremors and bradykinesia as well as cognition, mental, sleeping, personality and behaviour disorders including depression and anxiety [2]. It is an age related disorder because the incidence for PD increases rapidly in the population cohort exceeding 60 years of age [2,3]. At present, the etiology of PD is still not clearly known. Alpha synuclein becomes the major protein in Lewy bodies which has a key role in pathogenesis of both familial and sporadic PD [4]. Currently, there is no cure for PD and the drugs used for treatment are dopamine agonists and monoamine oxidase-B (MAO-B) inhibitors, which provide only symptomatic relief. Considering the promise that several natural products have yielded in treating various ailments, such products as herbs, medicinal plant extracts and other such botanicals are being explored for their therapeutic potentials in neuronal disorders. In this quest, some herbs have been found to be effective neuroprotectants. Bacopa monnieri (BM), commonly known as Brahmi, an ayurvedic medicinal plant acting as antioxidant [5], anti-depressant [6], anti – inflammatory and anti microbial [6,7,8], is the most popular neurotonic and a well known memory booster [9], which, we reasoned, could be tested for its beneficial effects on the neurodegenerative PD. Bacopa monnieri, a member of the Scrophulariaceae family [10] assists in heightening mental acuity and supports the physiological processes involved in relaxation. Extracts of Bacopa monnieri have been reported to exert cognitive enhancing effects in animals [11,12]. Research on anxiety, epilepsy, bronchitis and asthma, irritable bowel syndrome, and gastric ulcers also support the Ayurvedic uses of Bacopa [13]. We employed transgenic Caenorhabditis elegans model to evaluate the effect of BM on Parkinson‟s disease. Since its 6080 % genes are homologous to human [14] and it also has orthologs of PD associated genes, model system C. elegans may help in establishing an insight into therapeutic aspects of PD. Transgenic strain expressing „human‟ α-synuclein, helps in studying the effect of its aggregation on the gross phenotype and provides a robust model to study the effect of any potential therapeutic agent [4,15,16,17,18,19]. The transparent anatomy of the nematode helps in monitoring the aggregation of alpha synuclein protein and degeneration of dopamine neurons. Further, dopaminergic neurodegeneration can easily be induced by neurotoxins such as 6-hydroxydopamine (6-OHDA) [20] thus providing a pharmacological model of PD. Hence, the present study took advantage of the model system C. elegans towards examining the anti-Parkinsonian effects of neuro-protective botanical Bacopa monnieri. Materials and Methods 1.1. C. elegans culture and maintenance Maintenance of C. elegans was carried out as described previously by Brenner [21,22]. Worms were raised on the OP50 seeded standard Nematode Growth Medium (NGM) and grown at 220C. In this study, wild type Bristol N2, transgenic strain NL5901 (Punc-54::alphasynuclein::YFP+unc-119; expressing human alpha synuclein protein with YFP expression in the muscles) and transgenic strain BZ555 (Pdat-1::GFP; bright GFP observable in dopamine neuronal soma and processes) were used. These strains were obtained from the Caenorhabditis Genetics Center (University of Minnesota). 1.2. Treatment of worms with Bacopa monnieri

Concentrated mother tincture of Bacopa monnieri extract, which is used as a homeopathic medicine in India, was obtained (SBL Private Limited, Uttaranchal, India). The objective of using the mother tincture was to study this homeopathic drug „as is‟, so as to evaluate the anti-Parkinsonian effects of the whole mother tincture of the botanical. Any encouraging observations will lead to detailed studies on various active constituents of the extract. The mother tincture was diluted tenfold in OP50 before seeding onto NGM plates. The plates were incubated overnight for optimum growth of bacteria OP50 following which, age synchronized worms were grown on the plates, for further studies.

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1.3. Treatment of worms with 6 hydroxy dopamine (6 – OHDA) and 6 –OHDA - BM To generate selective degeneration of dopaminergic neurons, worms were exposed to 6-hydroxy dopamine (6-OHDA; Sigma, St. Louis, MI; catalogue no - H4381) at concentrations of 25mM [20]. Briefly, 6-OHDA was mixed with OP50 and seeded onto NGM plates. To prepare 6-OHDA- BM treatment plates, BM was added along with OP50 at this stage. Plates were then incubated overnight. Embryos of BZ555 strain of C. elegans were then transferred onto these prepared treatment plates for 48 hours at 22ºC. Bacopa extract is known to be stable, as studies have reported beneficial effects of the extract over long periods after extraction. 1.4. Assay for analysis of alpha synuclein protein aggregation Aggregation of alpha synuclein protein was observed in control and BM treated NL5901 strain of C. elegans. After 48 hrs. of treatment, worms were washed thrice with M-9 buffer to remove adhering bacteria and transferred to agar padded slides (2% agarose) and sealed with a cover slip. Worms were immobilized with 100mM sodium azide (Sigma, cat no. 71289). Imaging of live (immobilised) worms using confocal microscopy (Carl Zeiss) was carried out to monitor the aggregation of alpha synuclein protein and aggregation was quantified using image J Software (Image J, National Institutes of Health, Bethesda, MD) by measuring florescence intensity. Statistical analysis was carried out using Graph Pad software package; calculation of statistical significance between various groups was carried out employing Student‟s t test. 1.5. Assay for analysis of dopaminergic neurodegeneration Study of dopaminergic neurodegeneration was carried out by exposing worms with 6-OHDA and 6-OHDA-BM as described previously in section 1.3. Any adhering bacteria were removed after 48 hrs of treatment by washing the worm pellet three times before mounting the worms onto agar padded glass slide using 100mM sodium azide. Imaging of live (immobilised) worms was carried out to monitor the dopaminergic neurodegeneration in control and experimental conditions using laser scanning confocal microscope (Carl Zeiss). Fluorescence intensity was quantified using Image J software (Image J, National Institute of health, Bethesda, MD). 1.6. Nile red staining of lipid deposits

Lipid specific dye- Nile Red (Cat. no - N1142, Invitrogen) was mixed with E.coli before seeding it onto the NGM plates [23]. Nile Red stock solution was prepared by dissolving 0.5mg Nile Red dye in 1 mL of acetone. Nile Red was fed to the worms by mixing it with E. coli OP50 in the ratio 1: 250 as described previously [23].The synchronous aged embryos were transferred onto the Nile-red containing plates and grown for 48 hrs at 22 oC. Worms were washed off and mounted onto 2% agarose pads using sodium azide. The extent of fat staining was assessed using fluorescence upright microscope (Nikon). 1.7. Life span study Life span assay was performed by transferring adult worms every other day, from control as well as BM treatment condition, to a fresh control or treated plate. This was done to avoid mixing of multiple generations. The number of live, missing and dead worms was counted each day until the last worm was dead. Analyses were carried out by two different investigators. Survival curves were plotted using the product-limit method of Kaplan and Meier; statistical analyses was performed using SPSS software [24]. 2. Results and Discussion

2.1. Bacopa monnieri reduced alpha synuclein protein aggregation Worms of untreated and Bacopa monnieri treated groups were observed under confocal microscope for assaying their alpha synuclein aggregation pattern. Phenotypically, the worms appeared normal and optimally fed. We observed a marked and significant reduction in the aggregation of alpha synuclein in case of NL5901 worms treated with Bacopa monnieri (Figure 1B) as compared to that of control group (Figure 1A). We quantified the images for fluorescence intensity of alpha synuclein aggregation using Image J software. Treatment of worms with BM showed significantly reduced fluorescence

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intensity of aggregation as compared to that of untreated worms. The mean fluorescence (GFP) intensity was 8.950 ± 0.6195 arbitrary units in control worms and 2.550 ± 0.2500 arbitrary units in BM treated subjects (Fig. 1C).

Figure 1: Alpha synuclein aggregation in NL5901 strain of C. elegans fed on OP50 (A, B), Bacopa monnieri (C, D). Images A and C are fluorescent images, B and D are images grabbed using Differential Interference Contrast (DIC) optics. Scale bar, 50 µm. Fig 1E is Graphical representation for fluorescence intensity of the nematodes as quantified using Image J software *p < 0.05.

A 3.5 fold reduction (p<0.05) in alpha synuclein protein aggregation was observed in BM exposed worms. This preventive effect on α-synuclein aggregation could be as a result of the effect of BM on protein aggregation mediated by expression of stress proteins within the system. It is very well known that a suite of proteins within the living systems, called stress proteins, buffer the cells from harm under stressful conditions [25,26]. Bacopa monnieri, has previously been reported to induce such chaperoning protein called Hsp-70 [27]. Such an effect of BM on stress proteins, might be leading to disaggregation of α-synuclein deposits or the unfolding of mis-folded proteins that otherwise are the part of toxic aggregates. The protective effect of Bacopa could also be attributed to the antioxidant properties of the botanical, as it has been reported that Bacopa scavenges free radicals and reduces lipid peroxidation in tissues [5,28]. Studies have also reported enhancement of antioxidant enzyme levels as a result of treatment with Bacopa [5,28]. These protective properties of Bacopa monnieri make it tempting for us to speculate that the extract might be decreasing the aggregation of α-synuclein thereby reducing its toxic outcome in the cells, which, in case of our in vivo studies employing whole organismic environment of model system C. elegans, makes the studies more relevant in overall disease condition. 2.2. 6 – OHDA induced selective degeneration of dopaminergic neurons was prevented with Bacopa monnieri The significance of using C. elegans as a model to study neuronal disorders lies in the fact that its nervous system is simple, comprising of precisely 302 neurons wired by 7000 synapses, each neuron can be individually identified and the entire connectivity of the nervous system has been reconstructed. Most significantly, all gene families involved in neuronal function in mammals are present in the worm. Of importance to the present study, C. elegans contains precisely 8 dopaminergic neurons (Fig 2A, 2B); two pairs of CEP neurons and one pair of anterior deirid (ADE) neurons in head region. In a posterior lateral position, it has another pair of posterior deirid (PDE) neurons. Selective degeneration of these dopaminergic neurons was achieved through exposure to 6 –OHDA [20,29]. We found that processes of CEP and ADE

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neurons showed complete GFP loss with moderate reduction in GFP expression in PDE neurons (Fig. 2C, 2D). When worms were fed on 6 – OHDA- BM plates, significant protection was found in dopaminergic neurons with CEP, ADE and PDE neurons exhibiting an enhanced expression of GFP; (Fig. 1E, 1F). We further quantified the images for fluorescence intensity in DA neurons using Image J. In control worms, the mean fluorescence (GFP) intensity was 8.158 ± 0.2380 arbitrary units whereas it was reduced to 3.050 ± 0.05000 arbitrary units in 6-OHDA treated subjects (a 2.7 fold reduction (p< 0.05) as compared to that of untreated worms). Worms raised on 6 – OHDA- BM plates exhibited a fluorescent intensity of 6.900 ± 0.1000 arbitrary units (a 2.3 fold increase (p< 0.001) as compared to 6-OHDA treated subjects (Fig 2 G). This neuroprotective role of BM in 6 –OHDA induced dopaminergic neurodegeneration is probably associated with its antioxidant [5,30] and anti apoptotic activity [31]. Since dopaminergic neurons are more susceptible for ROS and oxidative stress plays an important mediator role in neuronal cell death and it consequently induces apoptosis. Although the contribution of ROS and apoptosis in 6-OHDA-induced neuronal cell death have been fully elucidated [32,33]. BM may be able to suppress oxidative stress of these neuronal cells. Furthermore, neuroprotective effect of BM against rotenone, pharmacological model of PD, has also been reported in Drosophila melanogaster model [34]. It is also well known that the central components of apoptotic pathways are the proteases of the caspase family. Caspase-3 is a potent effector of apoptosis triggered via several different pathways in a variety of mammalian cell types, and is one of the most important caspases in the cytochrome c-dependent apoptosis pathways [35]. Activation of caspase-3 appears to be a key event in apoptosis. Furthermore, it has also been postulated that mitochondrial dysfunction triggered by 6-OHDA induces release of cytochrome c, and the activation of caspase-3 [32,36,37]. The protective effect of Bacopa monnieri could probably be associated with such events as activation of bcl-2, maintaining the stability of MMP, and decreasing the activation of caspase-3 through the mitochondria-dependent pathway; however such mechanistic aspects need to be explored in greater detail.

Figure 2: GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain BZ555. Control (A, B), 6OHDA treated (C, D), 6-OHDA treated worms raised on BM (E, F). Images A, C and E are fluorescent images; B, D and F are Differential Interference Contrast (DIC) images. Scale bar, 50 µm. Fig 2G is Graphical representation for fluorescence intensity of GFP expression pattern in dopaminergic neurons of transgenic C. elegans strain as quantified using Image J software *p < 0.05,

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2.3.

Bacopa monnieri restored lipid content in alpha synuclein expressing model of C. elegans

PD is known to be associated with altered levels of fatty acids and lipid content. We assayed the lipid levels in alpha synuclein expressing worms, vis-a-vis normal (wild type) worms and worms treated with BM. Nile red staining was carried out to fluorescently label the lipids within nematodes. The fluorescent intensity across various groups of worms was analysed by fluorescent microscopy and quantified using Image J software. Worms of control group exhibited an optimum level of lipids (Fig 3A), the mean fluorescent intensity of the group, as quantified by Image J, being 39.06 ± 4.158 arbitrary units. The lipid content in alpha synuclein expressing worms was reduced (Fig 3B) as the fluorescent intensity of nile red staining was found to be 17.77 ± 1.804 arbitrary units, thereby exhibiting a 2.2 fold reduction (p<0.05) as compared to the control group. When the alpha synuclein expressing worms, were treated with Bacopa monnieri, they showed an increased staining pattern for nile red along the entire body of worms (Fig 3C) depicting an increase in lipid content of the worms; the content in this group of worms appeared to be similar as seen for wild type worms; the mean fluorescence intensity being 45.37 ± 4.676 arbitrary units, which is a 2.6 fold increase (p< 0.05) with respect to that of untreated NL5901 worms. The reduction of lipid content in alpha synuclein expressing worms is because of disturbed lipid composition caused as a result of alpha synuclein toxicity within the worms. Further, the toxic nature of these protein aggregate species tends to increase the lipid peroxidation in the tissues via increase in burden of reactive oxygen species [38]. The enormous abundance of lipid molecules in the central nervous system (CNS) suggests that their role is not limited to be structural and energetic components of cells. Some lipids in the CNS are known to function in neurotransmission. Perturbations in cellular signalling have been reported in virtually every neurodegenerative disease. Spatial and temporal features of cellular signalling are in part controlled by lipid components that can regulate protein location and scaffolding events through a dynamic modulation of membrane microdomains. This protective effect of BM could be for the known antioxidant properties of the botanical that leads to reduction in reactive oxygen species thus resulting in a reduced lipid peroxidation [39]. Such a protective effect, thus, mediates the prevention of disturbed combination of lipids thereby resulting in a rather efficient cellular signalling.

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Figure 3: Nile Red staining of wild type C. elegans fed with OP50 (A), NL5901 strain of C. elegans treated with OP50 (B) , NL5901 strain of C. elegans fed on Bacopa monnieri (C). Scale bar, 50 µm. Fig. 3D is graphical representation for fluorescence intensity of the nematodes as quantified using Image J software *p < 0.05 2.4. Bacopa monnieri increased the life span of wild type worms

The effect of Bacopa monnieri on the longevity of worms was studied. We observed that Bacopa monnieri increased the life span of wild type worms marginally whereas the alpha synuclein expressing worms did not cause any significant effect on the longevity of worms. The cumulative survival patterns, as calculated by Kaplan-Meier survival analysis of each group are depicted in Figure 4.

Figure 1: Alpha synuclein aggregation in NL5901 strain of C. elegans fed on OP50 (A, B), Bacopa monnieri (C, D). Images A and C are fluorescent images, B and D are images grabbed using Differential Interference Contrast (DIC) optics. Scale bar, 50 µm. Fig 1E is Graphical representation for fluorescence intensity of the nematodes as quantified using Image J software *p < 0.05

The mean survival for N2-BM group was 13.57 ± 0.03 days vs. 12.2 ± 0.17 days of N2-OP50 condition (significant at p<0.05). However the life span in NL5901-BM group was 12.72 ± 1.54 days which was statistically insignificant at the studied concentration as compared to that of control group. The subtle effect induced by BM on the longevity of nematodes could be attributed to their antioxidant and anti-stress properties. Parkinson‟s disease is associated with ageing and in the

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process of aging, accumulation of free radicals, cognitive impairment and memory loss has been proposed as the causal factor. Further, the beneficial effects of Bacopa were observed in the control group too, which proves that the effects are indeed as a result of absorption of Bacopa into the system and could not be because of any artifactual interaction in the culture media. In Ayurvedic materia medica, Bacopa monniera is commonly mentioned as a rasayana which manifests its effect to prevent ageing, increase longevity, impart immunity and improve mental functions. However, BM has already been utilized for its anti-aging action [40]. Our studies provide strong evidence that Bacopa monnieri exhibits a significant ameliorative potential against alpha synuclein effects. The degeneration of dopaminergic neurons, induced by 6-OHDA, is also prevented by BM, thus warranting further studies regarding the identification of effective constituents of the botanical and regarding the study on its mechanistic aspects by which it affords the amelioration. Our studies encourage further investigations on BM as a possible therapeutic intervention against Parkinson‟s disease. 3. Acknowledgments Confocal Microscopy facility of CDRI is acknowledged for their assistance in imaging; in particular Mr. Manish Singh is gratefully acknowledged for his technical assistance. Nematode strains used in this work were provided by the C. elegans Genetics Center (CGC) University of Minnesota, MN, USA, which is funded by the NIH National Center for Research Resources (NCRR). CDRI communication no: 187/2011/AN References [1] A. Trancikova, D. Ramonet, D.J. Moore, Genetic mouse models of neurodegenerative diseases, Prog Mol Biol Transl Sci 100 (2011) 419-482. [2] L.M. Bekris, I.F. Mata, C.P. Zabetian, The genetics of Parkinson disease, J Geriatr Psychiatry Neurol 23 (2010) 228242. [3] C.M. Tanner, Early intervention in Parkinson's disease: epidemiologic considerations, Ann Epidemiol 6 (1996) 438441. [4] A.D. Gitler, A. Chesi, M.L. Geddie, K.E. Strathearn, S. Hamamichi, K.J. Hill, K.A. Caldwell, G.A. Caldwell, A.A. Cooper, J.C. Rochet, S. Lindquist, Alpha-synuclein is part of a diverse and highly conserved interaction network that includes PARK9 and manganese toxicity, Nat Genet 41 (2009) 308-315. [5] S.K. Bhattacharya, A. Bhattacharya, A. Kumar, S. Ghosal, Antioxidant activity of Bacopa monniera in rat frontal cortex, striatum and hippocampus, Phytother Res 14 (2000) 174-179. [6] K. Sairam, M. Dorababu, R.K. Goel, S.K. Bhattacharya, Antidepressant activity of standardized extract of Bacopa monniera in experimental models of depression in rats, Phytomedicine 9 (2002) 207-211. [7] S. Channa, A. Dar, S. Anjum, M. Yaqoob, R. Atta Ur, Anti-inflammatory activity of Bacopa monniera in rodents, J Ethnopharmacol 104 (2006) 286-289. [8] P.K. Chaudhuri, R. Srivastava, S. Kumar, Phytotoxic and antimicrobial constituents of Bacopa monnieri and Holmskioldia sanguinea, Phytother Res 18 (2004) 114-117. [9] U.S. Vollala VR, Nayak S, Effect of Bacopa monniera Linn. (brahmi) extract on learning and memory in rats – a behavioral study, Journal of Veterinary Behavior: Clinical Applications and Research 5 (2010) 69–74. [10] R.N. Chopra, Nayar, S.L. and Chopra, I.C., Glossary of Indian medicinal plants, New Delhi: CSIR (1956) 32. [11] C. Stough, J. Lloyd, J. Clarke, L.A. Downey, C.W. Hutchison, T. Rodgers, P.J. Nathan, The chronic effects of an extract of Bacopa monniera (Brahmi) on cognitive function in healthy human subjects, Psychopharmacology (Berl) 156 (2001) 481-484. [12] N. Uabundit, J. Wattanathorn, S. Mucimapura, K. Ingkaninan, Cognitive enhancement and neuroprotective effects of Bacopa monnieri in Alzheimer's disease model, J Ethnopharmacol 127 (2010) 26-31. [13] C.S. Paulose, F. Chathu, S.R. Khan, A. Krishnakumar, Neuroprotective role of Bacopa monnieri extract in epilepsy and effect of glucose supplementation during hypoxia: glutamate receptor gene expression, Neurochem Res 33 (2008) 1663-1671. [14] T.W. Harris, N. Chen, F. Cunningham, M. Tello-Ruiz, I. Antoshechkin, C. Bastiani, T. Bieri, D. Blasiar, K. Bradnam, J. Chan, C.K. Chen, W.J. Chen, P. Davis, E. Kenny, R. Kishore, D. Lawson, R. Lee, H.M. Muller, C. Nakamura, P. Ozersky, A. Petcherski, A. Rogers, A. Sabo, E.M. Schwarz, K. Van Auken, Q. Wang, R. Durbin, J. Spieth,

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