Fungal Genetics and Biology 70 (2014) 86–93

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Intracellular pH responses in the industrially important fungus Trichoderma reesei Mari Valkonen c,⇑, Merja Penttilä c, Mojca Bencˇina a,b,⇑ a

Laboratory of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia Centre of Excellence EN-FIST, 1000 Ljubljana, Slovenia c VTT Technical Research Centre of Finland, Espoo, Finland b

a r t i c l e

i n f o

Article history: Received 8 May 2014 Accepted 8 July 2014 Available online 19 July 2014 Keywords: Genetically encoded pH sensor RaVC Ratiometric indicator pH homeostasis Filamentous fungus Trichoderma reesei Dual sequential scanning microscopy

a b s t r a c t Preserving an optimal intracellular pH is critical for cell fitness and productivity. The pH homeostasis of the industrially important filamentous fungus Trichoderma reesei (Hypocrea jecorina) is largely unexplored. We analyzed the impact of growth conditions on regulation of intracellular pH of the strain Rut-C30 and the strain M106 derived from the Rut-C30 that accumulates L-galactonic acid—from provided galacturonic acid—as a consequence of L-galactonate dehydratase deletion. For live-cell measurements of intracellular pH, we used the genetically encoded ratiometric pH-sensitive fluorescent protein RaVC. Glucose and lactose, used as carbon sources, had specific effects on intracellular pH of T. reesei. The growth in lactose-containing medium extensively acidified cytosol, while intracellular pH of hyphae cultured in a medium with glucose remained at a higher level. The strain M106 maintained higher intracellular pH in the presence of D-galacturonic acid than its parental strain Rut-C30. Acidic external pH caused significant acidification of cytosol. Altogether, the pH homeostasis of T. reesei Rut-C30 strain is sensitive to extracellular pH and the degree of acidification depends on carbon source. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) is one of the most efficient industrial hemicellulase and cellulase producer (Schuster and Schmoll, 2010). It has significant importance as production host for industrial enzymes in general and for enzyme preparations used in hydrolysis of lignocellulosic raw materials in biorefinery applications. The impact of environmental factors and culture conditions on the production of hydrolytic enzymes by T. reesei has been investigated and it has been seen that the carbon source, temperature, light and external pH (pHex) significantly affect fungal enzyme production (Adav et al., 2011; Aro et al., 2005; Jun et al., 2013; Kubicek et al., 2009; Pakula et al., 2005; Schmoll et al., 2010). Earlier reports have shown that certain xylanases are produced at neutral pH, while acidic pH favors cellulase production (Bailey et al., 1993). Unlike some other filamentous fungi such as Aspergillus niger and A. nidulans, which tolerate broad extracellular pH ranges, T. reesei ⇑ Corresponding authors at: VTT Technical Research Centre of Finland, Espoo, Finland (M. Valkonen); Laboratory of Biotechnology, National Institute of Chemistry, 1000 Ljubljana, Slovenia (M. Bencˇina). E-mail addresses: mari.valkonen@vtt.fi (M. Valkonen), [email protected] (M. Bencˇina). http://dx.doi.org/10.1016/j.fgb.2014.07.004 1087-1845/Ó 2014 Elsevier Inc. All rights reserved.

favors more acidic pH, and its growth and enzyme production is impaired at alkaline pH values of above 6.5 (Adav et al., 2011; Li et al., 2013). Research on pH homeostasis of yeast Saccharomyces cerevisiae has shown that cytosolic pH (pHi) is an important regulator of cell fitness (Aabo et al., 2011; Orij et al., 2012, 2011). A decrease of pHi reduces cell vitality and consequently decreases production yields of S. cerevisiae. Low pHex (reviewed in Mattanovich et al. (2004)) or production of organic acids such as xylonic acid (Toivari et al., 2012) poses pH stress for the cells. Moreover, near neutral pHi has been linked directly to more efficient production of lactic acid (Valli et al., 2006). There appear to be intrinsic differences in pH tolerance between different fungal species. The robustness of pH homeostasis is a trademark of certain industrial strains, e.g. for the citric acid producing strain A. niger. It has been shown that the pHi in the A. niger hyphae can be slightly above neutral even when the pHex is as low as 1.8 (Bagar et al., 2009; Hesse et al., 2002). The pHi measurements with pH-sensitive fluorescent dyes (reviewed in Han and Burgess (2010)) often suffer from problems associated with dye loading and dye sequestration within organelles. The penetration of dyes into cells varies significantly with different dyes and cell types. Fluorescence of pH-sensitive fluorescent proteins (reviewed by Bencˇina (2013)), on the other hand, is

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stable, species-independent, and rarely toxic to cells. A genetically encoded pH sensor can be targeted to subcellular compartments and monitored non-invasively in living cells using fluorescence microscopy, fluorescence spectroscopy, or flow cytometry (Bagar et al., 2009; Maresová et al., 2010; Miesenböck et al., 1998; Orij et al., 2009; Ullah et al., 2012; Valkonen et al., 2013). pH indicators based on pHluorin (Miesenböck et al., 1998) are ratiometric by excitation; the measurement relies on changes in the excitation spectrum upon pH. Therefore, intracellular sensor concentrations and problems encountered by photobleaching do not affect the measurement (O’Connor and Silver, 2007). pHluorin has been used extensively for the analysis of pHi in yeasts (Maresová et al., 2010; Orij et al., 2009; Ullah et al., 2013; Valkonen et al., 2013). Bagar et al. (2009) were the first that used the genetically encoded ratiometric pH sensor in filamentous fungi. They developed and characterized an improved version of the pHluorin, called RaVC, for the analysis of pHi in A. niger and measured pH within individual living hyphae using dual excitation confocal laser scanning microscopy. The current work aims to obtain insight on the pH homeostasis in living hyphae of T. reesei. For improving enzyme production, the pH homeostasis mechanisms should be taken into consideration since many protein synthesis processes are susceptible to pH. In A. niger, a mathematical model has been built in order to predict the behavior of pH homeostasis in citric acid producing conditions (García and Torres, 2011). To our knowledge, the regulation of intracellular pH of T. reesei has not been studied before. We assessed the pHi of the industrial T. reesei Rut-C30 strain and M106 strain that originates from Rut-C30. Due to deletion of L-galactonate dehydrogenase (Dlgd1) (Kuorelahti et al., 2006) the M106 strain accumulates L-galactonic acid, converted from D-galacturonic acid added to the medium (Kuivanen et al., 2012). We also evaluated the impact of lactose and glucose as carbon sources on pHi. In addition, our results showed that there were no differences in the pHi within different regions of hyphae, i.e. pHi gradients were absent, in the living hyphae of T. reesei. The growth conditions, external pH and carbon sources notably influence the pH homeostasis of T. reesei.

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were screened by PCR. Transformants with similar fluorescence levels and morphology were selected for further experiments. Based on determination of dry weight and media acidification after 48 h, differences between the parental Rut-C30 and M106 strains, and the RaVC transformants were not significant. 2.2. Media and growth conditions To analyze the effect of carbon sources on the pHi, the RutC-30 and M106 strain were cultured in a minimal medium (5 g l1 (NH4)2SO4, 15 g l1 KH2PO4, 0.6 g l1 MgSO4, 0.6 g l1 CaCl2, 5 mg l1 FeSO4  7H2O, 1.6 mg l1 MnSO4  H2O, 1.4 mg l1 ZnSO4  7H2O, 3.7 mg l1 CoCl2) (pH 5) (Penttilä et al., 1987) with 20 g l1 glucose, or 20 g l1 lactose as a carbon source. For testing the impact of L-galactonate accumulation on pHi, the fungi were grown in minimal medium with 10 g l1 D-xylose (pH 5) for 24 h, after which 10 g l1 D-galacturonate (sodium salt, Sigma) was added. To determine the influence of extracellular pH (pHex) on pHi, the strains were grown in the Verduyn minimal medium (Verduyn et al., 1992) with 20 g l1 glucose and buffered to pH 4–7 with K-biphtalate or K-phosphate (K-hydrogen phthalate/HCl for pH 4; K-hydrogen phthalate/NaOH for pH 5; K-dihydrogen phosphate/ NaOH for pH 6, 7 and 8). All cultivations were performed in 24-well plates at 30 °C, with shaking, for 24–48 h. Sorbic acid was used for a weak acid stress. The M106 strain was grown in the minimal medium with 20 g l1 glucose and treated with 5 or 10 mM sorbic acid. For exposure to the weak acid stress, samples were analyzed before treatment and for 10 min following treatment. For imaging the spatial distribution of RaVC and analysis of pHi gradient, the T. reesei was grown on the minimal medium with 20 g l1 lactose solidified with 1.5% (w/v) agar. 72-h-old cultures of T. reesei were used for experiments. Agar bearing a leading edge of the colony was cut out and carefully placed, hypha side down, on a glass coverslip. The plasma membrane and vacuoles were stained with SynaptoRed C2 dye (25 lM) for 15–30 min (FischerParton et al., 2000). 2.3. Confocal laser scanning microscopy

2. Experimental section 2.1. Strains and vector construction A RaVC gene (Bagar et al., 2009) was cloned into an expression vector pMV119 using yeast recombinational cloning (Oldenburg et al., 1997). Initially, the RaVC gene was amplified by polymerase chain reaction (PCR) from a pMOJ009 vector with 30 bp overlapping ends to an A. nidulans gpdA, glyceraldehyde-3-phosphatase, promoter and a trpC, tryptophan synthase terminator. The forward and reverse primers were: 50 -GACTAACAGC TACCCCGCTT GAGCAGACAT CATGGTGAGC AAGGGCGAGG AGCTGTTCAC CGGGGTG and 50 -CAGTAACGTT AAGTGGATCC CCGCGGACTA GTTTATTTGT ATAGTTCATC CATGCCATGT GTAATCC, respectively. An A. nidulans acetamidase (amdS) gene (within the same transforming cassettes as the RaVC gene) was used as a selection marker for T. reesei transformation. The RaVC expression construct was targeted to a specific locus using 1.5 kbp sequences flanking the hfb2 gene. For transformation into the T. reesei, pMV119 plasmid DNA was digested with PmeI and a fragment containing the RaVC expression cassette, the amdS marker and the hfb2 flanking sequences, was used for transformation. The RaVC was transformed into the T. reesei strains M106, with deleted L-galactonate dehydratase (Dlgd1) (Kuorelahti et al., 2006) and the strain, RutC-30 (Montenecourt and Eveleigh, 1979). The transformation was performed according to Penttilä et al. (1987). The transformants, where the hfb2 gene was replaced by the RaVC expression cassette,

A Leica TCS SP5 laser scanning microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany) equipped with an HCX PL APO 63 (NA 1.4) oil immersion objective was used for imaging. For pH indicator RaVC recording, a method described by Bagar et al. (2009) was adopted. For sequential excitation, a 50 mW 405-nm diode laser and a 476-nm line of a 25 mW argon laser were used. Laser powers of 3% and 10% were used for the diode laser and the argon laser, respectively. Successive images excited at 405 and 476 nm were captured. The fluorescence emission was detected at 500–550 nm. 2.4. pH measurements The pHi was measured using the pH-sensitive fluorescent protein RaVC. Fluorescence intensities, after excitation at 405 and 476 nm, were recorded and converted to pH values using a custom written software program incorporating Eqs. (1) and (2) (Bagar et al., 2009).

Ri ¼ ½Fð405 nmÞi  Fð405 nmÞbackground =½Fð476 nmÞi  Fð476 nmÞbackground 

ð1Þ

where F(405 nm)i and F(476 nm)i are the fluorescence intensities of the region of interest. F(405 nm)background and F(405 nm)background denote the average background fluorescence intensities.

pH ¼ pK a  log10

h . i Rmin Ri  Rmax pH pH  Ri

ð2Þ

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max where Ri is the emission ratio at a given pH, and Rmin pH and RpH are limits for the ratio at extreme acidic (pH 5.2) and alkaline (pH 8.5) pH, respectively. These limits were individually determined for each max set of experiments described in next section. The Rmin pH and RpH were obtained by incubating mycelia in buffer solutions with the pH values of 5.2 and 8.5 with freshly prepared 50 lM nigericin for approx. 30 min.

2.5. In situ calibration An in situ calibration curve, as described by Bagar et al. (2009) with some modifications, was used to calculate pKa. Briefly, mycelia were washed and resuspended in buffer solutions with a pH range of 5.2–8.5. The ionophore nigericin (50 lM) was added and the cells were incubated at least 30 min to equilibrate pHi with pHex. The calibration buffer contained 50 mM MES, 50 mM HEPES, 50 mM KCl, 50 mM NaCl, 200 mM ammonium acetate, 10 mM NaN3, 10 mM 2-deoxyglucose, and 50 lM nigericin. The pH was adjusted with NaOH or HCl (described by Brett et al. (2005)). After mycelia had been treated for 30 min with nigericin, fluorescence intensities after excitation at the 405 and 476 nm were recorded and ratios were calculated using Eq. (1). The mean ratio was converted to pH using Eq. (2). 2.6. Image processing and statistical analysis A representative pseudocolored image of T. reesei hypha— calculated from sequentially acquired raw images—was selected from a set of at least five pseudocolored images at given conditions. Different colors were assigned to defined pH values in accordance with the in situ calibration curve calculated with Eqs. (1) and (2) (blue, alkaline; red, acidic). Each point of ratio/pH on a graph was calculated from mean fluorescence intensities of region of interest from a pair of two images collected at the 405- and 476nm excitations. All microscopic data are shown as the mean with a standard deviation calculated from at least ten images per experiment. Each set of experiments was independently repeated three times. Graphs were prepared with GraphPad Prism 5.0 software package (http://www.graphpad.com/) that was also used for statistical analysis. Student’s unpaired two tailed t-test (assumed Gaussian distribution of values) was used for statistical comparison between groups and p values of 0.05.

The genetically encoded pH-sensitive fluorescent proteins have proven immensely valuable for studies of pHi of yeast (Maresová et al., 2010; Orij et al., 2009; Ullah et al., 2013), filamentous fungi (Bagar and Bencˇina, 2012; Bagar et al., 2009), and higher eukaryotes (Bencˇina, 2013; Miesenböck et al., 1998). Therefore, we used the RaVC protein for live-cell imaging of pHi within the hyphae of the filamentous fungus T. reesei. RaVC is genetically encoded, which avoids cell manipulations to introduce the pH indicator into a cell that is needed when fluorescent dyes are used. Like pHluorin and pHluorin2, RaVC has bimodal absorbance/fluorescence spectral characteristics (Bagar et al., 2009; Mahon, 2011; Miesenböck et al., 1998), which enables the quantification of pHi with high spatial and temporal resolution within the pH range from 5.5 to 8.0 (Fig. 1). The RaVC protein expressed in T. reesei hyphae distributes within the cell similarly to what was observed in A. niger (Bagar

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Fig. 5. Effect of pHex on the pHi. (A) The M106 and (B) Rut-C30 strains were grown in a buffered liquid medium (pH 4, 5, 6, and 7) for 48 h. The pHi values calculated from fluorescence intensities ratios, and the representative pseudocolored ratio images (below) from growing hyphae expressing RaVC, are depicted. The pHex that the cultivations reached during the experiment is indicated below the graphs. (C) The effect of sorbic acid on pHi of the M106 strain. The pHi of hyphae grown in minimal liquid medium with glucose (20 g l1) for 48-h before and 10 min after treatment with the sorbic acid (10 mM). Each point on a graph presents a intracellular pH value (pHi) calculated from a ratio (Ri) from average fluorescence intensities of selected regions collected at 405- and 476-nm excitations. Lines indicate pH values 6.0 and 6.5; ns stands for not significant p > 0.05.

et al., 2009). As in A. niger, it is sequestered within tubular and spherical organelles. These organelles are more acidic than the surrounding cytoplasm, and they are in T. reesei more acidic than in A. niger (pH 6.2–6.5) (Bagar et al., 2009; Hesse et al., 2002). The hallmark of filamentous fungi is polarized hyphal growth. Seiler and Plamann (Seiler and Plamann, 2003) identified many proteins that contribute to the polarized growth of Neurospora crassa and divided them in four groups regulating the (i) cytoskeleton, (ii) cell wall and membrane biosynthesis, (iii) signal transduction pathways, and (iv) secretory pathway. Existence of ion gradients in polarized growing cells has been proposed as a mechanism for establishing and maintaining cell polarity. For filamentous growth of Candida albicans, a long range gradient of phosphoinositide bisphosphate has to be generated and maintained by the tip localized PI(4)P-5-kinase (Vernay et al., 2012). Whether the polarized tipgrowth requires a proton gradient has not been confirmed. For example, Robson et al. (1996) showed an alkaline pHi gradient at the extending hyphal tip of N. crassa using a ratiometric pH sensitive dye. On the other hand, experiments carried out both with A. niger (Bagar et al., 2009) and N. crassa (Parton et al., 1997) failed to show any pHi gradients longitudinal or across the apical regions of actively growing hyphae, which is in agreement with our results (Fig. 2, Movie S1). These results support the idea that a cytoplasmic pH gradient is not a general feature of cells growing by tip extension and argue against a central regulatory role of cytoplasmic pH gradients in supporting polarized tip growth. 4.2. External growth conditions affect the degree of intracellular acidification As far as we know there is only one previous analysis of pHi after growth on different carbon sources in filamentous fungi. Hesse et al. (2002) have shown that A. niger can maintain the pHi values within a narrow range in the presence of various carbon sources. In yeast, the intracellular acidification is directly associated with glucose starvation (Karagiannis and Young, 2001; Orij et al., 2009; Valkonen et al., 2013). Peters et al. (2013) identified pHi as a specific cellular signal involved in glucose sensing that manages the kinetics of proteosome storage granules’ formation and dissociation. In our experimental set up, there was still approximately 50% of the carbon source left in the cultures after 48-h of growth, so the intracellular acidification that we observed in this study is not triggered by starvation. We studied pH homeostasis of the Rut-C30 and M106 strains grown in minimal medium

containing glucose or lactose. Lactose is a known inducer for production of hydrolytic enzymes in T. reesei. Glucose on the other hand, represses expression of these enzyme genes. The Rut-C30 strain is mutated in the glucose repressor gene cre1, which reduces glucose repression, but the strain is not fully derepressed (MachAigner et al., 2010; Parajó et al., 1998; Schaffner and Toledo, 1991; Warzywoda et al., 1983). Consequently, cellulase expression levels remain lower on glucose than on inducing media. The analysis of cytosolic pH using the RaVC sensor revealed that the carbon source in the cultivation medium affects the degree of intracellular acidification of T. reesei (Fig. 3). The pHi of mycelia grown in a medium with lactose was below the calculated value of 5.5, showing an unexpectedly strong effect on intracellular acidification. The M106 strain was derived from the parent strain Rut-C30 by deletion of L-galactonate dehydrogenase (Dlgd1) (Kuorelahti et al., 2006). The M106 strain, converts D-galacturonic acid – the main monomer of pectin and an attractive substrate for bioconversions – to L-galactonic acid, which is excreted to the culture medium and also accumulates inside the cells (Kuivanen et al., 2012). With respect to the pHi, the reference strain Rut-C30 was more sensitive to the presence of the D-galacturonic acid in the medium than the M106 strain (Fig. 4). L-Galactonic acid, due to high pKa (12.6 taken from ECMDB data base id no.: ECMDB00565), is undissociated and diffuses from the cell with no apparent impact on the pHi of M106 as shown in this study. The Rut-C30 strain, on the other hand, converts D-galacturonic acid to pyruvate and L-glyceraldehyde, which both are readily metabolized. After 48 h of cultivation, the pHex of the cultures on different carbon sources decreased from 5 to around 3. The decrease of pH of culture medium during cultivation has been observed also in other studies (Bailey and Viikari, 1993; Bailey et al., 2002). We examined whether acidic pHex has an effect on pH homeostasis and consequently on the pHi of T. reesei. The pHi is a tightly regulated physiological parameter in all cellular systems. For instance, A. niger can withstand substantial shifts in pH without loss of viability during citric acid production. Besides, A. niger can grow at pHex values ranging from 2.5 to 8.5, while pHi remains unaffected (Bagar et al., 2009). The pH homeostasis of the studied T. reesei strains is not as robust as that of A. niger. At pHex above 4, the pHi of Rut-C30 is maintained reasonably well but clear acidification of pHi was observed in the medium with initial pH 4 compared to pH 7 (Fig. 5). Different pHi responses on low pHex was determined for yeast (Orij et al., 2009; Valli et al., 2005; Valkonen et al., 2013). Thus, it is likely that the intracellular acidification is

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at least partially linked to a decrease in pHex, and the type of carbon source only delineates a degree of pHi decrease. In this respect, lactose would be the least favored carbon source since the pHi drops below 5.5. A lactose-associated cytosolic acidification could be caused by different speed and metabolic routes for carbon utilization (reviewed by Seiboth et al. (2007b), Ivanova et al. (2013) and Seiboth et al. (2007a, 2005)), which might contribute to the energy pool to maintain an optimal pHi. The slower growth of T. reesei on lactose compared to glucose containing media indicates a slow utilization and conversion of lactose (Fekete et al., 2008), which might explain the observed behavior of pH homeostasis in the presence of lactose. The cytosolic acidification of T. reesei could be associated with influx of protons during the uptake of nutrients as has been shown for yeasts (Guimarães et al., 2008). In addition, the excretion of organic acids during growth acidifies growth medium (Bailey and Viikari, 1993; Bailey et al., 2002; Olejníková et al., 2011). As we have shown here, low pHex at least partially shapes pHi of T. reesei. For A. niger, on the other hand, the pHi remains unchanged also at acidic pHex (Bagar et al., 2009). For yeast S. cerevisiae, carbon starvation initiates significant decrease in pHi (Orij et al., 2009; Valkonen et al., 2013). It seems that the pHi regulation of A. niger, yeasts and T. reesei differs; nevertheless a direct comparison is difficult since growth conditions differ between organisms. The acidification is normally counteracted by H+-ATPases (reviewed for A. niger (Bencˇina et al., 2009)) draining protons against a proton gradient into the organelles, or into the medium. A transcriptomic analysis of Trichoderma viride revealed that acidification of medium triggers the repression of P-type H+-ATPases (Trushina et al., 2013). It has been shown that during cultivation and at the acidification of media, H+-ATPase activity decreases (Simkovic et al., 2007; Trushina et al., 2013) probably leading to acidification of the cytosol. Direct correlation between H+-ATPases activities and cytosolic acidification remains to be established. Taken together, the external growth parameters—carbon source and pHex—affect the pHi of the hydrolase-hyperproducing strains, Rut-C30 and M106. The differences observed for T. reesei when compared to A. niger and yeast S. cerevisiae may at least partly be due to the fact that the RutC-30 strain is a heavily mutagenised strain. We could not exclude that mutations in the genome of this industrially important strain affect the pHi regulation. The effect of mutations on pHi could be elucidated with detailed analysis pH homeostasis of T. reesei wild type strains. The importance in sustaining optimal pHi has been demonstrated with yeast in connection with lactic acid production; higher pHi was associated with better productivity (Valli et al., 2006) and faster growth (Orij et al., 2012). For T. reesei, it has been shown that fed-batch and pH-controlled fermentations give better cellulase yields (Bailey and Tähtiharju, 2003) than batch fermentations where the nutrient depletion and the medium acidification occur. So far, however, data on pHi in filamentous fungi in different culture conditions is limited and the maintenance of pHi might be important to ensure optimal cellular activity during fermentations. It would be valuable to study the pHi of T. reesei (and other fungi) industrial strains in production conditions. Therefore, further analysis will be necessary to evaluate a link between intracellular pH and productivity of the Trichoderma. Acknowledgments This research was supported by the Slovenian Research Agency and the EN-FIST Centre of Excellence, the Academy of Finland under the Finnish Centre of Excellence in White Biotechnology— Green Chemistry program (Grant 118573). The authors wish to thank Dr. Marilyn Wiebe, Dr. Juha-Pekka Pitkänen, and MSc Yvonne Nygård for their help in the design of the experiments.

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