- Open Access
Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis
© The Author(s) 2017
- Received: 14 April 2017
- Accepted: 18 September 2017
- Published: 22 September 2017
Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms.
Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying Km, Vmax and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways.
To our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.
- Kluyveromyces lactis
- KlADH3 promoter
- Multicopy plasmids
- Murine PDE5
Cyclic nucleotides (cNMP) are the components of evolutionary conserved transduction pathways controlling all aspects of cell life  and phosphodiesterases (PDEs) have a central role in the cAMP/cGMP hydrolysis [2, 3]. These enzymes modulate the amplitude and duration of the cAMP/cGMP signal within the cell and ultimately the localization, the composition and the activity of protein kinase A (PKA) and G. To date eleven families of class I PDE isozymes, encoded by 24 distinct genes, have been identified in higher eukaryotes displaying distinct biochemical properties and substrate specificity . PDE5, one of these families containing three isoforms encoded by Pde5a1, Pde5a2 and Pde5a3, has a major role as cGMP-dependent regulator of vascular smooth muscle contraction. This activity is the molecular target of several well-known drugs used to treat erectile dysfunction and pulmonary hypertension .
In Saccharomyces cerevisiae two genes for PDE enzymes that hydrolyse cAMP are present and glucose is the extracellular signal that triggers their expression as well as the cAMP-dependent activation of PKA [6, 7]. Pde1 is a “class II” hydrolysing enzyme with low affinity for cAMP and cGMP [8, 9]. Pde2 has high affinity for cAMP and belongs to class I of the higher eukaryotes superfamily . These two pde mutants, together with fission yeast mutants strains have been successfully used as powerful genetic tools for the expression and cloning of mammalian PDE genes. The activities have been biochemically characterized and brilliant yeast genetic selections were developed for the identification of many specific PDE inhibitors [11–14]. However, to our knowledge, no yeast has been engineered for the quantitative production of mammalian PDEs.
Kluyveromyces lactis harbours two uncharacterized PDE genes closely related to those of S. cerevisiae suggesting their involvement in the regulation of common pathways. K. lactis is a Crabtree–negative GRAS (Generally Recognized as Safe) yeast in which both respiratory and fermentative metabolisms coexist during its growth on glucose . Extensive metabolic and physiological studies have established K. lactis as the fermentative-deficient model equivalent but alternative to the respiratory-deficient S. cerevisiae petite mutants [16–23]. Indeed, K. lactis has been shown to be an attractive host for heterologous protein production , for genetic studies and industrial applications, being one of the few yeast species capable of growing on lactose as a sole carbon source [25–27].
The choice of the promoter is an important element in having an efficient and regulated expression of the gene of interest [24, 28]. In these reviews the authors listed the constitutive/regulated yeast promoters successfully used for the production of recombinant proteins. In this paper, the well-characterized promoter of KlADH3, a gene highly induced by all respiratory carbon sources with the exception of ethanol that specifically mediates its repression [29–32], was used for the construction of pKD1-derived expression vectors. pKD1 is a natural plasmid originally isolated from Kluyveromyces drosophilarum, but stably maintained in K. lactis [33–35]. The use of the KlADH3 promoter and K. lactis for the production of PDE5A isoforms under respiratory growth conditions, has been preferred to the Crabtree–positive S. cerevisiae to avoid/reduce the powerful signaling role of cNMP/PDEs, key components of glucose transduction pathways . For the same reason, KlADH3 has been preferred to commonly used promoters like LAC4, GAL7 and even KlADH4, genes partially induced in glucose-containing medium [18, 19, 27, 28].
These expression vectors were used for the quantitative production of the Mus musculus PDE5 isoforms. We found that the expression of the M. musculus Pde5a1, a2 and a3 variants of the Pde5a gene, obtained from a pCDNA3.1-cloned cDNA embrio library , was well tolerated by K. lactis cells without major growth deficiencies. PDE5A proteins, produced in large amounts, displayed biochemical properties identical to those of the native murine isoforms . This process could potentially be extended for the quantitative production of other higher eukaryotes PDE families.
Strains, media and culture conditions
The K. lactis CBS2359 (MATa) (http://www.cbs.knaw.nl) strain was used in this work. Cultures were grown under shaking conditions at 28 °C in YP (1% Difco yeast extract, 2% Difco Bacto-peptone) or in minimal medium (6.7 g/L Difco Yeast Nitrogen Base) supplemented by carbon sources at the concentration specified in the text. Geneticin (G418) concentration in selective conditions was 100 μg/mL. Escherichia coli strain DH5α was used for the propagation of plasmid DNA. Cultures were grown at 37 °C in LB medium (0.5% Difco yeast extract, 1% Difco tryptone, 0.5% NaCl, supplemented with 100 μg/mL ampicillin).
Construction of plasmids for the expression of the Pde5a1, a2 and a3 in K. lactis
Construction of pα-PDE5A1
This vector contains the DNA coding for the killer toxin-secretion peptide (α-signal)  fused in frame with the M. Musculus Pde5a1 cDNA gene. The α-signal DNA was synthesized as two short single strands complementary sequences containing at the 5′ and 3′ end the XbaI and SalI sites (Fwd: ctag atg aat ata ttt tac ata ttt ttg ttt ttg ctg tca ttc gtt caa ggt agg ggt gtg ttt cgt cga g; Rev: tc gac tcg acg aaa cac acc cct acc ttg aac gaa tga cag caa aaa caa aaa tat gta aaa tat att cat), respectively, and then annealed. The 2.6 Kbp SalI–SmaI PDE5A1 fragment was purified from p3XFLAG-CMV7 . The two fragments were ligated into the Bluescript KS (Stratagene) plasmid digested with XbaI–SmaI. Transformation and screening of E. coli cells led to the isolation of pKS/αsignal-PDE5A1 containing the α-signal DNA correctly fused at the 5′ end of the Pde5a1 gene. Finally, the recombinant gene, purified as a 2.7 Kbp XbaI-digested fragment from pKS/αsignal-PDE5A1, was cloned in the NheI-digested site of pYG137/1 to harbour pα-PDE5A1 (Fig. 2b).
pYG137/1 (Fig. 2c) is a derivative of pYG132, a multicopy plasmid previously used for the production and secretion of the recombinant human serum albumin under the control of the KlADH4 gene promoter [33, 35, 38]. This vector contains the entire pKD1 plasmid, the kanamycin bacterial gene that confers resistance to the G418 antibiotic in yeast, the promoter of KlADH3 [29–32] and the terminator of the S. cerevisiae PGK1 gene.
pYG137/1 was also used to construct p3XFlagPDE5A1, p3XFlagPDE5A2 and p3XFlagPDE5A3 which contain the M. musculus Pde5a1, a2 and a3 spliced-variants of the Pde5 gene fused at their 5′ end to the short DNA sequence coding for the 3XFLAG peptide (Sigma), an immunogenic peptide for the affinity purification of PDEs from whole cellular extracts. These recombinant vectors were constructed in two stages from pα-PDE5A1 and the p3XFLAG-CMV7 vectors containing either Pde5a1, a2 or a3 in frame with the 3XFLAG coding sequence . To start, pα-PDE5A1 was digested with PmlI, ligated and transformed in E. coli cells. The screening of transformants allowed the isolation of pYG137/1-PDE5Δ0.8, which contains a deletion of 0.8 Kbp of DNA coding for the α peptide and the first 0.75 Kbp of Pde5a1 (Fig. 2b). In the second stage, the p3XFLAG-CMV7 plasmids were digested with the enzymes EcoICRI and PmlI and the 0.7–0.8 Kbp fragments (containing the 3XFLAG fused to the 5′ end of the three genes, respectively) were recovered from each digestion, purified and cloned in pYG137/1-PDE5Δ0.8 digested with PmlI. The screening of E. coli transformed cells led to the isolation of p3XFlagPDE5A1, p3XFlagPDE5A2 and p3XFlagPDE5A3 (Fig. 2b).
We also constructed p-PDE5A1 (Fig. 2b) which contains Pde5a1 under the control of the KlADH3 promoter. This vector was obtained by cloning the 0.8 Kbp PmlI–EcoICRI fragment, containing the 5′ end of PDE5A1 recovered from pcDNA3.1 , in the PmlI-digested pYG137/1-PDE5Δ0.8.
Preparation of cellular extracts and PDE activity assay
Kluyveromyces lactis cells harbouring the PDE5A plasmids (Fig. 2b) were grown under shaking conditions at 28 °C for up to 5 days in 5 mL of YP supplemented with glucose (YPD) or glycerol (YPG) at the concentration of 2%. Harvest cells were collected by centrifugation, washed twice with distilled water and resuspended in 300 μL of lysis buffer (20 mM Tris–HCl buffer pH 7.2, 0.2 mM EGTA, 5 mM MgCl2, 5 mM β-mercaptoethanol, 0.1% v/v Triton X-100, 2% v/v antiprotease cocktail (Sigma Aldrich, CA, USA), 1 mM PMSF) and broken with glass beads (∅ 0, 5 mm). The lysate was recovered by centrifugation at 14,000g for 30 min at 4 °C, ready for further analyses. The recovered pellets were resuspended in 200 μL of lysis buffer and analysed for PDE activity.
PDE activity was measured at 30 °C with the two-step method described by  using [3H]cGMP (Perkin Elmer, MA, USA). Aliquots of extracts were incubated in 60 mM HEPES pH 7.2 assay buffer containing 0.1 mM EGTA, 5 mM MgCl2, 0.5 mg/mL bovine serum albumin, 30 µg/mL soybean trypsin inhibitor, in a final volume of 0.15 mL (PDE assay buffer). The reaction was started by adding tritiated substrate at a final concentration of 1 μM [3H]cGMP and stopped by adding 0.1 M HCl. The Sildenafil used in enzymatic inhibition experiments was a generous gift from Pfizer.
Quantitative purification of the 3XFlagPDE5A1, A2 and A3 isoforms
100 mL of YPG medium were inoculated with the CBS2359 strain, harbouring the 3XFlagPDE5A plasmids, and grown for 4 days under shaking conditions. All purification steps were carried out at 4 °C. Cells were harvested and broken in 10 mL of lysis buffer, as described above and the lysate centrifuged twice at 20,000g for an hour. The supernatant was collected, diluted 20 times in the purification buffer (50 mM HEPES pH 7.5, 75 mM NaCl, 37.5 mM MgCl2) and loaded for three times in a chromatography column with an ANTI-FLAG M2 affinity gel (Sigma-Aldrich) equilibrated with the purification buffer. The column was washed with 20 mL purification buffer. The elution of the 3XFlagPde5A proteins was performed by competition with the 3XFLAG peptide (Sigma-Aldrich) according to instructions. The eluted fractions were analyzed by western blotting and for PDE activity.
SDS-PAGE and western blotting
The purity and integrity of proteins were assessed using SDS-PAGE. Protein samples were boiled for 3 min and subjected to 8% SDS-PAGE and then visualized by 0.1% Coomassie Brilliant Blue R-250 staining. For western blotting analysis, after the electrophoresis, the proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Blots were incubated overnight at 4 °C with rabbit polyclonal anti-PDE5 antibody (1:1000; Santa Cruz Biotechnology). Alkaline phosphatase conjugated anti-rabbit IgG (1:5000; Sigma-Aldrich) was used as a secondary antibody to reveal the immune-complexes. Immunoreactive bands were stained with nitroblue tetrazolium (0.3 mg/mL) in the presence of 5-bromo-4-chloro-3-indolyl-phosphate (0.15 mg/mL).
In-gel native ADH staining assay and PDE activity in gel slice
Cell extract preparation, native polyacrylamide gel electrophoresis (PAGE), electrophoresis conditions and ADH staining assay were carried out as previously described . PDE activities were recovered from the native gel slices with 100 μL of PDE assay buffer by over night-incubation on ice. The samples were then centrifuged at 14,000g for 15 min at 4 °C and aliquots of the eluates assayed for cNMP hydrolysing activity.
DNA manipulation, plasmid engineering and other techniques were performed according to standard procedures. Yeast transformation was performed by electroporation with a Biorad Gene-Pulser apparatus. Protein concentration was determined according to the Bradford method .
Analysis of the KlAdh3 activity by native PAGE ADH staining
Engineered plasmids for the production of the murine PDE5A1, A2 and A3 isoforms
The aim of this work was to express Pde5a1 and secrete its product in the extracellular medium. Therefore, the DNA sequence coding for the killer toxin α-peptide was fused in frame in front of the Pde5a1 gene. This peptide, engineered for production of human serum albumin, has proved to be an efficient targeting export signal for many heterologous proteins [24, 34, 35, 41]. The final construct, containing the chimeric gene in pYG137/1, was called pα-PDE5A1 (Fig. 2b). pYG137/1 was also used to express the native murine Pde5a1 and Pde5a1, a2 and a3 alternative spliced mRNA variants of the Pde5 gene only differing in their N-terminal coding products (Fig. 2a)  in frame with the 3XFLAG DNA. This sequence encodes an immunogenic peptide used for the affinity purification of the PDE5A isoforms (Fig. 2b). The latter plasmids, called pPDE5A1, p3XFlagPDE5A1, p3XFlagPDE5A2 and p3XFlagPDE5A3, were constructed for the intracellular quantitative production of the native and recombinant PDE5A isoforms (see below).
Use of the KlADH3 promoter for the production of PDE5A1
pα-PDE5A1 was introduced by electroporation in the K. lactis CBS2359 strain and transformants were grown for up to 5 days in YP medium containing glycerol, a carbon source that induces KlADH3 .
The glycerol- and glucose-extracts from the transformed strain (Fig. 3a, lanes 7–12) were also analysed by immuno blot to determine the intracellular accumulation levels of the α-PDE5A1 protein (Fig. 3c). The PDE5 antibody revealed the presence of at least two bands, a faint one of 100 kDa mainly visible at 2 days of growth (Fig. 3c, lane 2 and 5) and a slower migrating band of about 130 kDa accumulated at higher levels in extracts from respiratory carbon source grown-cultures (lanes 1 and 6–8). In contrast, cells grown in YPD showed very low levels of activity (Fig. 3c, lanes 2–4). The lactate-grown culture accumulated higher amounts of putative PDE5A1 than glycerol and acetate-grown ones (Fig. 3c, lane 8 vs. lanes 1 and 7) but, as stated earlier, with a lower biomass yield.
The α-signal is incapable of promoting the secretion of PDE5A1
To prove that the slower migrating bands visualized in the immuno blot (Fig. 3c, lanes 1 and 6–8) and the cGMP-hydrolysing activities, accumulated by the pα-PDE5A1 transformed strain (Fig. 3b, days 1–5), are PDE5A1, we assayed both soluble and pellet fractions of the extracts in the presence of Sildenafil, the specific inhibitor of PDE5. Indeed, as reported in Fig. 3d, whole extracts showed highly reduced levels of activity in the presence of Sildenafil in both soluble and pellet fractions confirming its identity. However, the amounts of PDE5 measured in the pellet fraction were ten times higher than in the soluble fraction. In contrast, a very small amount of activity was measured in the pellet fraction of the parental untransformed strain and at even lower levels than in the soluble fraction (Fig. 3d).
Unexpectedly, a very small amount of PDE (0.85 pmol cGMP hydrolised/min/mL) was measured in the medium supernatant of the transformed glycerol-grown strain but only after it was concentrated 20 times (Fig. 3e).
To test whether the α-signal is suited for the secretion of PDE5A1 in the medium, the cellular extracts and the medium fractions were concentrated and subjected to immuno blot analysis. The PDE antibody showed the presence of three putative bands of PDE5A1 in the cellular extract (Fig. 3e, lanes 2) indicating that large amounts of the chimeric protein are blocked inside the cell and unable to be secreted in the medium supernatant. In fact, only one of these bands corresponds to the faint one detected in the concentrated growth medium and to the PDE5A1 control band fractioned from the N18TG2 cells extract (Fig. 3e, lanes 4 and 5). N18TG2 is a murine neuroblastoma cell line with very high levels of PDE5A1 [42, 43].
Kluyveromyces lactis produces high levels of native PDE5A1 and recombinant 3XFlagPDE5A1 activities
Finally, we compared the amounts of PDE5A1 produced in the strains transformed with either pα-PDE5A1, p-PDE5A1 or with p3XFlagPDE5A1. As shown by the arrow in the Coomassie-stained SDS-PAGE (Fig. 5c, lanes 1–3) and by the histograms (Fig. 5d), the activities determined in the intracellular soluble fractions of the three strains suggested that p-PDE5A1 and p3XFlagPDE5A1 are good vectors for the quantitative production of the PDE5A1 isoform. In contrast, the pα-PDE5A1 strain was confirmed to be unable to accumulate significant amounts of intracellular PDE5A1 (Fig. 5d).
Growth curves and viability of pα-PDE5A1 and p3XFlagPDE5A1 transformed strains
The strains transformed with 3XFlagPde5A2 and 3XFlagPde5A3 showed growth curves and viabilities almost identical to that of the p3XFlagPDE5A1 strain (not shown). These results confirmed that overexpression of the murine genes in K. lactis leads to a slower adaptation of the cultures, as compared to the parental untransformed strain (Fig. 6a).
Finally, extracts prepared from the p3XFlagPDE5A1-transformed strain, grown for 2–4 days in rich glucose or glycerol medium, were compared for the production of the 3XFlagPDE5A1 isoform by immuno blot using a PDE5 antibody. As shown in Fig. 6c, a small amount of 3XFlagPDE5A1 was visible in the extracts from glucose-grown cells and these levels were independent from the days of growth (lanes 2–4). On the contrary, high levels of 3XFlagPDE5A1 were produced in glycerol-containing medium and greatly increased at 3 and 4 days of growth (Fig. 6c, lanes 5–7). The amounts of 3XFlagPDE5A1 accumulated in the two conditions coincide with the levels of KlAdh3 produced in the same conditions in the parental and transformed strains (Fig. 3a).
Production and purification of the 3XFlagPDE5 isoforms
PDEs are cyclic nucleotides hydrolysing enzymes grouped into two classes of proteins with distinct/dual substrate hydrolysing specificity for cAMP and cGMP. Class I groups together more than hundred higher eukaryote isoforms into eleven PDE families (PDE1 to PDE11) . PDE5, one of these families, has three isoforms (A1, A2 and A3) (Fig. 2a) encoded by the a1, a2 and a3 alternative-spliced variants of the Pde5a gene. PDE5 has a major role as cGMP-dependent regulator of vascular smooth muscle contractions and it has been identified as the target of several well-known drugs . The large profit generated by the commercialisation of these drugs led their exploitation for other pathologies expanding the study of PDE5 to other organs. Recently, PDE5 has been shown to be involved in learning/memory processes, in heart failure, in cardio-vascular diseases and in human breast and thyroid cancers [49–53]. Despite the great commercial relevance of these drugs, productions of large amounts of PDE5 isoforms for the development of new specific inhibitors or for the crystal structure determination of the entire PDE5 proteins have either not been carried out or if so, not published. At the moment, crystallographic models have only been constructed for the single catalytic/regulatory domains .
In this paper, we showed that the α-peptide signal, in contrast with previous proteins-secretion reports, is unable to target PDE5A1 in the extracellular space (Fig. 3e). In fact, native PAGE, immuno blot analysis and PDE determinations from the protein extracts of the pα-PDE5A1 strain showed that a large amount of the enzyme was located in the insoluble pellet fraction (Figs. 3d, 4 fraction 1) and only a very small amount of PDE5 was detected in the medium (Fig. 3e, lane 4). Moreover, the intracellular accumulated activity displayed PDE5 bands with MWs that vary according to dilutions and/or concentrations of the protein extract (Fig. 3c, e, lane 2). In addition, the pα-PDE5A1 strain enters into a stationary phase at only 36 h of growth showing a reduced vitality, as compared to the other strains (Fig. 6a, b). Since PDEs are components of intracellular signaling pathways, we speculate that the bands of Fig. 3c–e and the activity of the pellet fraction accumulate as unprocessed α-PDE5A1 suggesting the presence of domains conflicting with the secretory apparatus.
On the contrary, the expression cassettes lacking the α-signal allowed the intracellular accumulation of large amounts of the PDE5A1, A2 and A3 isoforms (Figs. 6c, 7). PDE5 yields, nearly 1 mg/g wet weight biomass (30 mg/L culture), are well tolerated by K. lactis cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways.
These results were made possible by the choice of the host organism, by the use of the KlADH3 promoter expression vectors and by the growth conditions. K. lactis is a Crabtree-negative yeast in which both respiratory and fermentative metabolisms coexist during its growth on glucose. Respiratory growth could be an ideal neutral condition to produce PDE5 isoforms avoiding interferences with glucose transduction pathways [6, 7]. Conversely, KlADH3 is a gene highly induced during the growth under respiratory growth conditions and pYG137/1 is a multicopy natural pKD1-derived vector. Finally, K. lactis transformed strains grow to high yields in rich glycerol medium with only a slightly delayed growth up to early stationary phase (Fig. 6a). A delay probably due to the gradual adaptation of the cells to the synthesis of PDE5A1 accumulating at constant rates during growth phases (Figs. 3b, 6c).
In prospective, the large amounts of the three purified proteins produced could help to define their macromolecular structure by small-angle X-ray scattering (Saxs)/crystallographic studies, to compare the differences between the three isoforms with known structures of other mammalian PDEs [54–56] and for the development of new inhibitors for clinical and therapeutic applications.
In this paper we report the KlADH3 promoter-dependent production of the M. musculus PDE5A isoforms in K. lactis. The use of this promoter was the appropriate choice for the production of PDE5 isoforms as it proved to be well tolerated by K. lactis cells. To our knowledge this is the first time that the entire family of PDE5 isoforms containing both regulatory and catalytic domains has been produced in a heterologous eukaryotic organism. The three purified proteins will be used to define their macromolecular structure by crystallographic studies and to compare putative differences between the three isoforms and other mammalian PDEs. These results established K. lactis as a model organism for production of mammalian PDE and, in prospective, as a platform to increase their yields by the control of the growth parameters, the use of alternative carbon sources/media and the genetic manipulation of the host.
SC performed most of experimental analysis and critical reading of the manuscript. MG expert of mammalian PDEs, purification of PDEs, design of the project, reviewed the manuscript, FN expert of mammalian PDEs, provided the murine PDE5 plasmids, commented and reviewed the manuscript. FM contributed to the design of the project, biochemical analysis of PDEs. SB contributed to the design of the project, data analysis, reviewed the manuscript. MS expert of K. lactis biology, construction of plasmids for the manipulation of K. lactis, design of the project, data analysis, wrote the manuscript. All authors read and approved the final manuscript.
We would like to thank Dr. Federica Barbagallo of the Dept. of Experimental Medicine, for the detailed information on PDE5 murine vectors previous to their publication  and Dr. Angela Tramonti of the Dept. Scienze Biochimiche “Rossi Fanelli”, of the Sapienza Università di Roma for critical reading of the manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The material and data supporting their findings can be found in the main paper and available from the authors upon reasonable request.
Ethics approval and consent to participate
This work was funded by “Progetti Ateneo 2014”—Sapienza University of Rome and FIRB “Accordi di Programma 2010” (RBAP109BLT_003). S.C. is a recipient of a Post-Doctoral Fellowship of “Progetti Ateneo 2014”.
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