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Biochemical and Biophysical Research Communications 472 (2016) 339e345

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Fatty acid esters produced by Lasiodiplodia theobromae function as growth regulators in tobacco seedlings  n Co  rdova-Guerrero c, Carla C. Uranga a, Joris Beld b, Anthony Mrse b, Iva ndez-Martínez a, * Michael D. Burkart b, Rufina Herna a n Científica y de Educacio n Superior de Ensenada (CICESE), Carretera Ensenada-Tijuana 3918, Zona Playitas, 22860 Ensenada, B.C., Centro de Investigacio Mexico b University of California, San Diego, Department of Chemistry and Biochemistry, 9500 Gilman Dr., La Jolla, CA 92093-0358, USA c noma de Baja California (UABC), Calzada Universidad 14418 Parque Industrial Internacional Tijuana, Tijuana, B.C. 22390, Mexico Universidad Auto

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 February 2016 Accepted 23 February 2016 Available online 27 February 2016

The Botryosphaeriaceae are a family of trunk disease fungi that cause dieback and death of various plant hosts. This work sought to characterize fatty acid derivatives in a highly virulent member of this family, Lasiodiplodia theobromae. Nuclear magnetic resonance and gas chromatography-mass spectrometry of an isolated compound revealed (Z, Z)-9,12-ethyl octadecadienoate, (trivial name ethyl linoleate), as one of the most abundant fatty acid esters produced by L. theobromae. A variety of naturally produced esters of fatty acids were identified in Botryosphaeriaceae. In comparison, the production of fatty acid esters in the soil-borne tomato pathogen Fusarium oxysporum, and the non-phytopathogenic fungus Trichoderma asperellum was found to be limited. Ethyl linoleate, ethyl hexadecanoate (trivial name ethyl palmitate), and ethyl octadecanoate, (trivial name ethyl stearate), significantly inhibited tobacco seed germination and altered seedling leaf growth patterns and morphology at the highest concentration (0.2 mg/mL) tested, while ethyl linoleate and ethyl stearate significantly enhanced growth at low concentrations, with both still inducing growth at 98 ng/mL. This work provides new insights into the role of naturally esterified fatty acids from L. theobromae as plant growth regulators with similar activity to the wellknown plant growth regulator gibberellic acid. © 2016 Elsevier Inc. All rights reserved.

Keywords: Trunk disease fungi NMR GC-MS

1. Introduction The Botryosphaeriaceae are a family of fungi that have been found to affect several economically important woody plants around the world and are considered trunk disease fungi. Some of the symptoms these fungi cause include gummosis, wedge-shaped necrotic cankers in tree wood, and stunted growth [1]. Presently, in Vitis vinifera, Lasiodiplodia theobromae (teleomorph Botryosphaeria

Abbreviations: NMR, nuclear magnetic resonance; GC-MS, gas chromatographymass spectrometry; FA, fatty acids; FAE, fatty acid esters; FAME, fatty acid methyl esters; FAEE, fatty acid ethyl esters; LAEE, linoleate ethyl ester; PAEE, palmitate ethyl ester; SAEE, stearate ethyl ester; OAEE, oleate ethyl ester; PA, free palmitate; GA, gibberellic acid. * Corresponding author. E-mail addresses: [email protected] (C.C. Uranga), joris.beld@drexelmed. rdovaedu (J. Beld), [email protected] (A. Mrse), [email protected] (I. Co Guerrero), [email protected] (M.D. Burkart), [email protected] (R. Hern andez-Martínez). http://dx.doi.org/10.1016/j.bbrc.2016.02.104 0006-291X/© 2016 Elsevier Inc. All rights reserved.

rhodina) has been found to be the most virulent [1,2]. However, it can also be found as an endophyte or latent pathogen [3]. Many other Botryosphaeriaceae, including Neofusicoccum parvum, have been isolated from V. vinifera and other plant species [4]. Characterization of the metabolites produced by L. theobromae is critical for understanding the metabolic pathways involved during colonization, as well as for the discovery of novel or interesting compounds. Lipases have an important function in pathogenicity of fungi (triacylglycerol acyl-hydrolases, E.C. 3.1.1.3) [5]. These enzymes are involved in the degradation of cell membranes and storage lipids, and esterification of these with alcohols [6]. Lipases liberate free fatty acids, which are the starting material for many secondary metabolites such as oxylipins, studied in other phytopathogenic fungi, [7e9]. Free fatty acids are also a source of energy and the acetyl CoA necessary for polyketide-type secondary metabolites produced by Botryosphaeriaceae [10]. The objective of this work was to characterize compounds produced or biotransformed by L. theobromae in natural substrates, and assess their effects in plants.

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2. Materials and methods Two isolates were kindly provided by Dr. Douglas Gubler from the University of California at Davis, (USA), L. theobromae UCD256Ma (isolated in Madera County, California, USA) [11]; and an isolate of N. parvum, (UCD646So, isolated in Sonoma County). Isolates used belonging to CICESE at Mexico are: L. theobromae MXL28, Fusarium oxysporum f. sp. lycopersici, and Trichoderma asperellum isolated from grapevine, tomato and carnation plants, respectively. 2.1. Induction of secondary metabolism in L. theobromae Media for induction of metabolism of both L. theobromae isolates consisted of 25 g ground oatmeal powder and 50 mL Vogel's salts solution, autoclaved twice. Isolates were inoculated with one 1 cm mycelial disc of the fungi grown in potato dextrose agar (PDA). As a negative control, oatmeal without fungus was used. Three biological replicates for the negative controls and the experimental conditions were set. Samples were incubated at room temperature (RT) in the dark for a total of 60 days. To assess the production of compounds of interest in different carbon sources, fungal isolates were incubated in 50 mL Vogel's minimal media supplemented with 5% glucose, 5% grape seed oil, 5% glucose þ5% grape seed oil, or 5% fructose. These were incubated in triplicate for 20 days at 25  C in the dark. 2.2. Solvent extraction of fungal incubations Before extraction, samples were frozen at 80  C then lyophilized for 48 h. A modified Folch extraction [12] was done using a solvent mixture of 75 mL dichloromethane (DCM), 75 mL methanol and 0.01% butylated hydroxytoluene (as antioxidant), and extracted overnight at 4  C. The samples were placed in a separating funnel to separate and collect each phase. The organic phases (DCM) were evaporated with a rotovapor (Buchi R-114) at 45  C and the remaining oils aliquotted to Eppendorf tubes and stored at 20  C until analysis. Thin layer chromatography (TLC) on silica gel sheets (Merck) with a fluorescence indicator was performed on the crude oil extracts and developed using 5% ethyl acetate (v/v) in hexane with two sequential chromatographic developments. The chromatograms were stained with vanillin/H2SO4. The oil extract was separated on silica gel (Unisil) using step elutions consisting of different concentrations of ethyl acetate (0, 5, and 10% EtOAc) in hexane. Fractions with the compounds of interest (eluted in 100% hexane) were evaporated and re-fractionated by preparative chromatography with a C-18 column (Phenomenex), using a gradient of 100% H2O containing 0.1% formic acid to 100% acetonitrile containing 0.1% formic acid. The compound of interest eluted with 100% acetonitrile/0.1% formic acid, was collected, evaporated and repurified using preparative TLC with a solvent system of 1% ethyl acetate in hexane. Pure compound, monitored by TLC, was used for mass spectrometry analysis, GC-MS, proton and carbon NMR. 2.3. Nuclear magnetic resonance (NMR) and mass spectrometry In order to determine the molecular weight and formula of the purified compound of interest, high resolution mass spectrometry was performed on an Agilent 6230 ESI-TOF MS. Proton (1H) and carbon (13C) NMR analyses were obtained from a Varian 500 MHz instrument equipped with an XSens 2-channel NMR cold probe optimized for direct observation of 13C. Data was analyzed with the program ACD/NMR processor Academic Edition [13].

2.4. In vitro FAE production (Fischer-Speier esterification) of oat and grapeseed oil Knowing the nature of the compound, a positive control consisted of an in vitro Fischer-Speier esterification [14] of the oat oil fraction extracted, and the grape seed oil used in the incubations. Briefly, 2.5 mL of oil was mixed with 1 mL of ethanol or methanol, to which five drops of H2SO4 were added as a catalyst. The samples were placed in sealed glass vials and heated to 100  C for 30 min. Saturated sodium bicarbonate was added to neutralize the acid, and the phases containing FAE were collected and analyzed via GC-MS. 2.5. Gas chromatography-mass spectrometry of crude extracts All samples, including the positive controls, were analyzed for naturally produced fatty acid ethyl esters by GC-MS. A standard curve was created from octadecadienoate (Z, Z) ethyl ester (LAEE) standard (Cayman Chemical) from which concentrations of unknowns were calculated. The standard was diluted from 625 mg/L to 40 mg/L in hexanes. Ten mL of all unknowns were dissolved in 1 mL hexanes without esterification and analyzed by analytical GC-MS on an Agilent 7890A GC system, connected to a 5975C VL MSD quadrupole MS (EI), using helium as the carrier gas and a 60 m DB23 column, with a gradient of 110  Ce200  C at 15  C/min followed by 20 min at 200  C and 20 min at 240  C. All compounds were identified via NIST library searches, and where applicable, co-injection of standard and comparison with a 37 FAME mix (SigmaeAldrich). LAEE, OAEE ((Z)-9-oleate ethyl ester), SAEE (stearate ethyl ester) and PAEE were purchased as purified standards (Cayman Chemical). 2.6. Effects of FAE on tobacco seed germination and hypocotyl growth With the aim to test the effect of the isolated compounds in planta, we chose tobacco (Nicotiana tabacum), a well-studied plant model [15]. Seeds were surface-sterilized in 50% household bleach (8.25% NaOCl) for 1 min and rinsed 3 times with sterile water before use. Approximately 100 ml packed volume of seeds were placed in Murashige and Skoog salts (with Gamborg vitamins), 0.8% agar, 3% sucrose and the antifungal Plant Preservative Mix (PPM, Plant Cell Technology Inc.), containing 200 mg/mL of either LAEE, PA, PAEE, OAEE and SAEE emulsified in 0.08% kolliphor-188. All experiments, including negative controls were done in triplicate under natural lighting conditions. The length of the hypocotyl was measured after 7e10 days post-sowing by pictures taken with a calibrated Olympus stereo microscope (SZX12) at 7x magnification, using Image J software [16]. Seedling lengths (N ¼ 30) from cotyledon tip to root tip were measured for each experimental condition. Morphology was assessed and documented 45 days post-dosing and sowing. Concentration dependence was then studied in Murashige and Skoog (MS)þ3% sucrose using a concentration range of 3.1 mg/mLe 98 ng/mL for SAEE and LAEE, in triplicate, including negative controls. Finally, a germination experiment was done using 1 mg/mL of each FAE, including the known plant growth regulator gibberellic acid (GA, “Supergrow” from Consolidated Chemical) as a positive control, using MS without sucrose to resemble field conditions. A one-way ANOVA followed by a TukeyHSD post-hoc analysis was performed on the data with p values < 0.05 considered significant, using XLSTAT statistical analysis software. Graphs were generated with Graphpad Prism software. Time-lapse video (Lapse It 2.5 pro) during germination was taken of a negative control and N. tabacum exposed to 98 ng/mL SAEE under continuous white light. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2016.02.104.

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3. Results 3.1. Metabolite identification Thin layer chromatography (TLC) of the oil portion from extraction of L. theobromae incubated in oatmeal revealed a prominent band that stained dark green/blue with vanillin/H2SO4. This compound was present in all three biological replicates incubated with L. theobromae and absent in the negative controls (Fig. 1A). High resolution mass spectrometry of the compound detected an M þ H ion at 309.278 m/z, the most probable molecular formula being C20H36O2 (Dataset A in Ref. [17]). Proton and carbon nuclear magnetic resonance (NMR) spectra (Fig. 1 in Ref. [17]) identified this compound as LAEE. The relative chemical shifts agree with those published in the literature for LAEE [18,19]. The (Z, Z)-configuration is evident by the average coupling constant of multiplet 5.27e5.46 (6.1 Hz), and confirmed to be within the range for cis hydrogen coupling in double bonds [20,21]. From L. theobromae incubated in 5% glucose þ5% grape seed oil, TLC results revealed a compound with the same Rf as LAEE. This was confirmed using GC-MS by co-injection of LAEE standard with the crude samples. Further GC-MS analysis demonstrated the presence of a variety of ethyl esters (Table 1) in both isolates of L. theobromae incubated in oatmeal, not detected in the negative controls. Chromatograms may be found in Fig. 2,3,4,5 in Ref. [17]. A four-point standard curve using (Z, Z)-9, 12-octadecadienoate ethyl ester standard with an R2 value of 1.00 was obtained with the linear equation y ¼ 3.53E þ 08x  1.43Eþ06, from which the unknowns were calculated. Using oat as substrate, LAEE was the major FAE produced by L. theobromae. The incubation of L. theobromae for 60 days yielded 20.1 ± 1.3 g/L in UCD256Ma and 28.7 ± 7.1 g/L in MXL28 for LAEE. LAEE was also detected in strain UCD256Ma

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incubated in 5% glucose þ5% grape seed oil, producing 2.4 ± 0.7 g/L (Fig. 2), as well as in other isolates (Table 1) after 20 days. No FAE were produced by L. theobromae UCD256Ma when incubated in 5% fructose. Ethyl palmitate (PAEE) and 1H-2Benzopyran-1-one, 3, 4-dihydro-8 hydroxy-3-methyl- (mellein) were detected in 5% glucose as the sole carbon source (Table 1, Fig. 3 in Ref. [17]) indicating de novo production of PAEE by the fungus. FAE identified by GC-MS, 37 FAME standard comparison and the NIST library are listed in Table 1, several of which have not been reported previously to be produced by these fungi. In grapeseed oil only, metabolism shifted towards the production of 9-octadecenoate methyl ester (OAME) (44%) and LAEE (40.9%) (Table 1 in Ref. [17]) indicating ethanol production in the absence of glucose. SAEE and PAEE were also produced in the absence of glucose. 3.2. Tobacco seed germination and growth in FAE Statistically significant inhibitory effects on seed germination were observed by all FAE compounds tested at 0.2 mg/mL as compared to the negative controls except for free palmitate (PA) and PAEE in MS without sucrose (Fig. 2A and B). In MS without sucrose, LAEE, OAEE, SAEE and the crude oil caused seedling growth inhibition. In MSþ3% sucrose, LAEE, PAEE, OAEE, SAEE and the crude extract inhibited growth. However, both LAEE and SAEE induced growth at lower concentrations in MSþ3% sucrose (Fig. 2C and D). The effect was clear for LAEE, which increased seedling length at 98 ng/mL. SAEE induced growth at the lowest concentrations tested, with more variability between concentrations. Time-lapse video shows a faster germination rate in N. tabacum exposed to 98 ng/mL SAEE under continuous white light (Video file 1), indicating light to be a factor in this process. Leaf morphology of tobacco seedlings germinated in FAE in

Fig. 1. Identification of the compound isolated and characterized from L. theobromae. A: TLC of the negative control (C-) and the compound of interest (Cþ, black arrow). B: GC-MS of compound isolated from L. theobromae. C: NIST library standard match of the unknown to LAEE. D: Graph of LAEE quantification from incubations in oatmeal or 5% glucoseþ 5% grapeseed oil, error bars represent standard error of the mean.

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Table 1 Fatty acid esters (FAE) from isolates of Lasiodiplodia theobromae, Neofusicoccum parvum, Fusarium oxysporum and Trichoderma asperellum, identified via GC-MS, 37 FAME standard and NIST library comparison. Average areas under the curve values from triplicates are shown for the incubations with fungal isolates. ChEBI identifications are shown where applicable. Compound

Positive control, fischer esterification Oat oil

Methyl hexadecanoate ChEBI:69187 Ethyl hexadecanoate (PAEE) ChEBI:84932 Hexadecanoate, 2-methylpropyl ester 9-Octadecenoate (Z)- methyl ester ChEBI:27542 Octadecanoate ethyl ester (SAEE) ChEBI:84936 9-Octadecenoate (Z), ethyl ester (OAEE) ChEBI:84940 9-Octadecenoate (E) ethyl ester 9,12-Octadecadienoate (Z,Z)-, methyl ester ChEBI:69080 9,12-Octadecadienoate (Z,Z) ethyl ester (LAEE) ChEBI:31572 9,12,15-Octadecatrienoate (Z,Z,Z)-ethyl ester) ChEBI:84851 2H-1-Benzopyran, 3,4-dihydro(R ± mellein)

L. theobromae incubation 5% in oatmeal 60 days Glucose

Grape seed UCD 256 oil Ma

MXL28

UCD 256Ma

5% grape-seed Incubation in 5% glucoseþ5% grape oil seed oil, 20 days UCD 256Ma

RT

UCD256Ma N. parvum F. ox UCD646So

T. as

6  106 1.4  106 3.2  107 1.9  106

1.4  106 5.2  107

2.6  106 6.6  107

N/D 1.7  105 9.1  104 1  105

N/D 2.6  106

N/D 2.3  106

N/D 7  105

N/D 12.7 9.2  104 13.3

N/D N/D 9.8  106 2.4  107

9.5  104 1.1  106

1.2  106 1.1  106

N/D N/D

N/D 2.8  106

N/D 3.1  105

N/D 2  106

N/D N/D

N/D 15.7 6.9  105 17.0

1.8  106 7.9  105

1.8  106

2.2  106

N/D

1.3  104

1.6  106

7.9  105

7.7  104 N/D

5.3  107 3.2  107

3.1  107

4.2  107

N/D

N/D

3.0  107

2.3  107

2.4  105 2.3  106 18.1

1.1  106 2.5  105 2.3  107 7  106

1.5  106 2  106

3.4  106 3.1  106

N/D N/D

N/D 6  105

3.9  105 2.7  104

3.3  105 5.7  105

N/D N/D

1.1  108 8.9  106

7  107

1  108

N/D

2.6  106

6.9  106

9.1  106

5.5  105 2.3  105 19.6

2.0  106 1.7  105

1.3  106

2.2  104

N/D

7.6  104

7.4  105

2.8  105

N/D

N/D

21.7

N/D

N/D

6.9  105

4.5  105 N/D

N/D

N/D

N/D

N/D

23.6

N/D

N/D N/D

17.3

18.3 18.4

N/D: not detected; F.ox: Fusarium oxysporum; T.as: Trichoderma asperellum; RT: Retention time. Negative controls did not yield FAE, therefore are not included in the table. See Fig. 2A in Ref. [17].

Fig. 2. Concentration dependence of FAE on tobacco seed germination rates. Seedling length 7e8 days post-planting, N ¼ 30 for each condition, using a one-way ANOVA and a posthoc Tukey-HSD analysis, p-value < 0.05. A; 0.2 mg/mL FAE or crude oil extract in MS only. B; 0.2 mg/mL FAE or crude oil extract in MSþ3% sucrose. C; LAEE 3.1 mg/mL98 ng/mL in MS-3% sucrose. D; SAEE 3.1 mg/mL- 98 ng/mL in MSþ3% sucrose. Letters above graphs indicate statistically significant differences or similarities between experimental conditions.

MSþ3% sucrose was also affected. Leaves showed abnormal elongation and bifurcation when exposed to the crude extract or 0.2 mg/mL SAEE, and expanded cotyledons with abnormal elongation of the first true leaf at lower SAEE concentration (3.1 mg/mL).

Tobacco seedlings exposed to LAEE, PAEE, SAEE and the crude extract during germination showed stunted growth and chlorosis (Fig. 3). More examples of effects may be found in Fig. 8, 9 in Ref. [17].

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Fig. 3. Morphology of N. tabacum germinated with LAEE, SAEE, PAEE or crude extract of L. theobromae 45 days post-sowing and dosing. A; Negative control. B; 0.2 mg/mL crude extract from L. theobromae incubated in 5% glucoseþ5% grapeseed oil. C; 0.2 mg/mL LAEE: D; 0.2 mg/mL PAEE. E; 0.2 mg/mL SAEE. F; 3.1 mg/mL SAEE.

In the final germination experiment, all FA and FAE were found to induce germination at 1 mg/mL to varying degrees (Fig. 4). SAEE and LAEE induced germination similarly to gibberellic acid. 4. Discussion Fatty acids and modified fatty acids are important molecules during colonization of plants by pathogenic fungi, serving diverse functions such as energy-sources, signaling, and virulence factors [8]. L. theobromae naturally produces a variety of FAE in plantderived triglycerides. This is the first report of their production in L. theobromae, and the other fungi studied. Two Botryosphaeriaceae were found to be able to produce a wider variety and higher quantities of FAE than the rest of the tested fungi. FAE production in T. asperellum was lower than L. theobromae and N. parvum. The FAE that affect growth regulation in tobacco were produced in higher abundance by the trunk disease

Fig. 4. N. tabacum exposed to 1 mg/mL FAE during germination in MS without sucrose. Gibberellic acid (GA) serves as a positive control for comparison. Letters above graphs indicate statistically significant differences or similarities between experimental conditions.

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fungi, and barely by the other tested fungi. For example, SAEE was not produced by T. asperellum, and F. oxysporum produced more PAEE than T. asperellum. These differences in fatty acid metabolism may be a factor that differentiates trunk disease fungi from other phytopathogenic fungi, or from non-phytopathogens such as T. asperellum. FAE may act in a variety of plant growth processes. FAE are known to have various functions in eukaryotes, activating steroid hormone receptors in humans [22], or inducing apoptosis [23]. LAEE and SAEE have been extracted from plants such as Allium sativum (garlic) [18], the purple shamrock Oxalis triangularis [19], and the medicinal plant Moringa oleifera [24]. The biosynthetic machinery leading to these compounds in the plant may involve pyruvate decarboxylase, alcohol dehydrogenase and lipases [25,26]. Plants are able to ferment glucose for energy production during flower and pollen development [27e29], and oxygen levels at the ovary are known to be at zero [30]. Hypoxia is part of dormancy and dormancy release in V. vinifera [25] and other fruiting trees, with the production of cyanogenic glucosides and starch breakdown linked to the onset of flowering [31,32]. Hypoxia is also induced in agriculture in grapevines and other trees to artificially stimulate bud break and increase agricultural yields with the use of hydrogen cyanamide [33]. Another cause of low oxygen levels in the plant is excess watering of roots [34]. Since L. theobromae has an endophytic phase in the plant, both natural dormancy periods and chemically induced hypoxia in V. vinifera or other trees may provide the fungus with the habitat that promotes anaerobic fermentation, resulting in the production of ethanol and other alcohols required for fungal lipases to esterify these to free fatty acids. Fungi may be using FAE to manipulate plant growth. The ability of fungi to affect plant growth has been observed in the fungus Gibberella fujikuroi, which is known to produce gibberellic acid, as well as in the fungus Botrytis cinerea, which produces abscisic acid [35,36]. In this work it was shown that Botryosphaeriaceae are able to produce higher quantities and a wider variety of FAE as compared to the other fungi studied. LAEE, and SAEE were found to have significant physiological effects in tobacco, acting as growth regulators during germination and early growth, on par with gibberellic acid at 1 mg/mL. Although much work remains to be done to understand the detailed physiological routes affected in the plant, it is proposed that fatty acid esters be considered plant growth regulators due to their ability to affect tobacco germination and early growth.

[2] [3]

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Acknowledgements [24]

Thanks to CONACyT and UCMEXUS, who provided a doctoral stipend for Carla C. Uranga. Thanks to Dr. Yongxuan Su from the small molecule mass spectrometry department at UCSD, Eduardo Morales and Dr. Manuel Segovia from CICESE, special thanks to Dr. Katrin Quester from the UNAM in Ensenada for instrumentation support. Thanks to Dr. James Nowick from the University of California, Irvine for his support with NMR analysis in this work, and special thanks to Claudio Espinosa de los Monteros for help with figure graphic design.

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Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.02.104.

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