Abstract Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.

Citation: Boyle GM, D'Souza MMA, Pierce CJ, Adams RA, Cantor AS, Johns JP, et al. (2014) Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models. PLoS ONE 9(10): e108887. https://doi.org/10.1371/journal.pone.0108887 Editor: Qiming Jane Wang, University of Pittsburgh School of Medicine, United States of America Received: March 25, 2014; Accepted: September 4, 2014; Published: October 1, 2014 Copyright: © 2014 Boyle et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the National Health and Medical Research Council of Australia Development Grant, Number APP1017676 (PGP, GMB, PWR). MMAD was supported by the University of Queensland International Research Tuition Award (UQIRTA) and a University of Queensland Research Scholarship (UQIRS). GMB was supported by a Smart Futures Researcher-in-Residence fellowship from the Queensland Government, and is currently supported by the Wilson Fellowship for Skin Cancer Research administered by the Perpetual Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors of this manuscript have the following competing interests: GMB was a previous recipient of a fellowship co-sponsored by QBiotics and the Queensland State Government. PGP has received consulting payments and contract research funding from QBiotics Ltd. VAG and PWR have ownership interests (including patents) in QBiotics Ltd. and the compound. No potential conflicts of interest were disclosed by the other authors. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Introduction Surgical excision and ionizing radiation of affected sites have been the mainstay for treatment of cancer patients for decades. Whilst often effective, efficacy of these treatments can be limited by various factors including the condition of the patient, the proximity of adjacent vital tissues, inaccessibility of the tumor and intolerance of normal tissue for repeated courses of treatment. In some of these cases, intra-tumoral treatment may be more appropriate, particularly when surgical intervention is not possible. There have therefore been many attempts to deliver localized therapies, such as injection of anti-cancer agents [1]–[3] and lethal implants [4], aiming for palliation or even cure. Intra-tumoral treatment may have the advantage of allowing for much higher drug concentrations at the tumor site, and potentially less toxicity than systemic agents. However, a limiting factor for greater use of intra-tumoral treatments appears to be lack of suitable agents rather than delivery technologies. The protein kinase C (PKC) family are ubiquitous serine-threonine kinases found in many cell types that translocate to membranes after activation and regulate diverse downstream processes, like proliferation, apoptosis, differentiation, and migration. There are ten main human isoforms that comprise three subgroups divided according to sequence homology and cofactor requirements. The classical PKC subgroup includes -α, -βI and-βII (alternatively spliced from the same gene), and -γ, which require binding of calcium and DAG to activate the enzyme. Members of the novel PKC subgroup include -δ, -ε, -η and -θ, which require DAG for activation but are calcium-independent. The atypical PKCs, -ι and -ζ, are independent of calcium and DAG, but have been shown to be activated by distinct lipids and protein-protein interactions [5], [6]. Inhibition of PKC signaling has been targeted as an anti-cancer treatment as PKC isozymes are known to play roles in cellular proliferation and vasculature formation [7]–[9], important for tumor growth. However, several clinical trials performed with compounds thought to inhibit PKC signaling have had disappointing results [9]. For example, enzastaurin, an orally available specific inhibitor of PKC-β, showed limited efficacy as a single agent in Phase II studies of advanced diffuse large B-cell lymphoma or non-small cell lung cancer [10], [11]. Clinical trials of enzastaurin with additional chemotherapy agents are underway. In contrast, previous work has shown that intravenous administration of the prototypic PKC activating compound phorbol 12-myristate 13-acetate (PMA) to patients suffering from myleocytic malignancies resistant to chemotherapy resulted in remission [12], [13]. We have previously demonstrated that three topical applications of the PKC-activating ingenol ester PEP005 (ingenol mebutate or ingenol 3-angelate) are sufficient for enduring regression of skin cancer lesions (including melanoma) in pre-clinical models [14], [15]. PEP005 has recently been approved for topical treatment of actinic keratoses [16], [17], and shows efficacy in additional models of squamous cell carcinoma [18], [19]. We now describe a novel PKC-activating compound, EBC-46, and demonstrate that a single intra-lesional injection is sufficient for enduring regression and ultimate cure of diverse tumor types in pre-clinical models of cancer.

Materials and Methods Reagents EBC-46 was purified from the kernels of the fruit from Fontainea picrosperma, was provided by QBiotics Ltd. (Yungaburra, Queensland) at greater than 97% purity. Phorbol 12-myristate 13-acetate (PMA), dihydroethidium and bisindolylmaleimide-1 (BIS-1) were purchased from Sigma (St. Louis, MO). Cell lines All cell lines used in this study were purchased from ATCC (SK-MEL-28, B16-F0, HeLa, FaDu, MCF-7, HT-29) except MC-38 [20], [21] and MM649 [22] cells, which were described previously. Cell lines were grown in complete media (RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 3 mM HEPES, 100 U/ml penicillin and 100 µg/ml streptomycin (CSL Ltd, Melbourne, Australia)). Cells were routinely checked by STR profiling, and for mycoplasma infection by Hoechst staining [23] and PCR, and were always negative. Human ethics statement This study was performed in strict accordance with the recommendations in the Australian National Statement on Ethical Conduct in Human Research (2007 - Updated December 2013), of the National Health and Medical Research Council of Australia. All protocols were reviewed and approved by the QIMR Berghofer Medical Research Institute Human Research Ethics Committee (QIMR-HREC), approval number P764. All participants provided written informed consent to donate their blood samples for research. Measurement of reactive oxygen species production in PMN cells PMN cells were isolated from peripheral blood from healthy volunteers. Aliquots of 2×106 PMN cells were stained incubated for 15 min at 37°C with 10 µg/ml dihydroethidium (DHE). 2×106 unstained PMN cells were incubated at 37°C to act as an unstained control. Where appropriate, PMN cells were also incubated with 1 µM bisindolylmaleimide-1. The stained PMN cells were treated with the either PMA or EBC-46 for 15 min, and the change in fluorescence resulting from oxidation of DHE to ethidium bromide measured on a dual beam FACSCalibur (BD Biosciences, Franklin Lakes, NJ) using CellQuest Pro (BD Biosciences) software. PKC-EGFP translocation pPKC-α, -βII, -γ, -θ and –ζ-EGFP were purchased from Clontech (Moutain View, CA). pPKC-βI, -δ, -ε, -η and -ι-EGFP were constructed in-house. PKC isoforms were cloned from Universal Human Tumor cDNA (Life Technologies, Carlsbad, CA) and ligated into pPKC-ζ-EGFP digested with XhoI/SacII. The identity and fidelity of all PKC isoforms was verified by Sanger sequencing. 96-well plates were seeded with 3×104 cells/well of SK-MEL-28 in complete media, and incubated at 37°C, 5% CO 2 , and 95% humidity. After 24 h, cells were transiently transfected with a 1∶3 ratio of pPKC-EGFP vector DNA: Lipofectamine 2000 (Life Technologies) (0.16 µg DNA: 0.48 µl Lipofectamine per well) in Opti-MEM media (Life Technologies). After 24 h incubation, cells were washed with phosphate-buffered saline (PBS) and treated with 100 ng/ml of either PMA or EBC-46 for 1 h. Cells were washed twice with ice-cold PBS, fixed with 2% formaldehyde/0.2% gluteraldehyde in PBS, washed twice again with ice-cold PBS, and overlaid with 100 µl of PBS. Fluorescent cells were examined with an AMG EvosFl (Life Technologies) inverted fluorescence microscope at 40×. Micrographs were taken such that at least 50 fluorescent cells/well were captured, and transmitted light images were overlaid with fluorescence images for control wells to calculate transfection efficiency. Cells were counted in each image using ImageJ (NIH) and classified according to the localization of fluorescence as cytoplasmic, plasma membrane, perinuclear membrane, or other membrane (mitochondria, endoplasmic reticulum, Golgi apparatus, or unknown). A total of three independent experiments were performed for each PKC isoform. Immunofluorescence HeLa cells were treated with either 175 nM (100 ng/ml) PMA or EBC-46, or vehicle alone for 1 h. PKC-α (Cat. No. 2056; Cell Signaling Technologies) was detected by immunofluorescence as described by the manufacturer. Images were acquired using a Leica TCS Inverted Fluoresence Microscope with a Nikon DS-Fi1C camera. PKC kinase assay HeLa cells were treated with 175 nM (100 ng/ml) or 17.5 µM (10 µg/ml) of either PMA or EBC-46 for 1 h. Cells were lysed and stored at −80°C, before the level of PKC-specific kinase activity was measured in 30 µg HeLa lysate using the PKC Kinase Activity Assay Kit (AbCam, Cambridge, U.K.; Cat. No. ab139437) as described by the manufacturer. Assays were performed in triplicate with the mean ± SD shown. Cell growth assay Cells were seeded at sub-confluence (5,000 cells/well) in 96-well microtitre plates. All drugs and inhibitors were diluted in complete media. Controls were treated with vehicle alone. Cells were treated with PMA or EBC-46 the day after seeding and cultured for 4 days. To measure inhibition of cell growth, attached cell lines were assayed using sulforhodamine B (SRB) [24], [25]. The experiments were repeated at least twice and the mean ± SD was determined in Prism 6 (GraphPad Software, San Diego, CA). Animal ethics statement This study was performed in strict accordance with the recommendations in the Australian Code for the Care and Use of Animals for Scientific Purposes 8th Edition (2013), of the National Health and Medical Research Council of Australia. All protocols were reviewed and approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee (QIMR-AEC), approval numbers A0106-042M and A0404-606M. All mice were housed in a specific pathogen free (SPF) facility, with 12 hours light/dark cycle and continual access to food and water. All mice were monitored daily and tumor volume measured at least twice weekly, recorded using digital calipers and expressed as mm3 according to the formula A×b×b×0.5 where A the length and b the measured breadth of the tumor. Mice were also assessed for clinical signs according to a QIMR-AEC approved clinical score sheet for distress during the period of the experiment to determine whether the treatments (i.e. tumor burden and effects of drugs) were causing distress to the mice to a degree and to where they should be euthanased (Table 1). Scores for each parameter were summed to give a possible total of 8. Less than 3 was considered a mild clinical score, between 3 and 6 was considered a moderate clinical score, and over 6 was considered a severe clinical score. The experiment was ceased when an unacceptable clinical score (>6) was reached, or the cumulative tumor burden of the mouse exceeded 1,000 mm3. Mice were humanly euthanized by asphyxiation at the end of the experiment. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Clinical scoring for Drug Treated and Tumor Bearing Mice. https://doi.org/10.1371/journal.pone.0108887.t001 EBC-46 treatment of tumors in mice SK-MEL-28, MM649, FaDu (2×106) or B16-F0 (1×105) cells were injected (two tumors per mouse) on the hindquarter of 5 week old immunocompromised BALB/c Foxn1nu mice or C57BL/6J mice. When the tumors reached approximately 50 mm3 (SK-MEL-28 and MM649) or 100 mm3 (FaDu and B16-F0), mice in the control group were treated with vehicle (20% propylene glycol in water, 50 µl), and the treatment group received 50 nmol (30 µg) EBC-46 in vehicle, via a single intra-tumoral injection. Mice were euthanized when the cumulative tumor burden per mouse exceeded 1,000 mm3 or at the end of the experiment. Pharmacokinetic study of EBC-46 in tumor and non-tumor-bearing mice Nine BALB/c Foxn1nu mice were injected with 2×106 MM649 melanoma cells, one tumor per mouse. Tumors were monitored until they reached approximately 100 mm3. Mice were then treated by injecting 50 nmol (30 µg) EBC-46 either into the tumor (tumor bearing mice) or into normal skin (sub-cutaneously, 9 tumor-free mice). Blood (maximum of 150 µl) was collected from the tail vein by nicking at the base of the tail at 30 min, 1, 2, 4, 8 and 24 h post-treatment (3 animals at 30 min and 4 h, 3 animals at 1 and 8 h, 3 animals at 4 and 24 h) into a lithium heparin Microvette CB300 blood collection system (Sarstedt, Numbrecht, Germany), and processed to plasma by centrifugation at 2,000 g for 5 min at 20°C until separation occurred. Plasma was frozen at −80°C until analysed. Samples were analyzed using a specifically developed HPLC method to detect EBC-46 in mouse serum against a spiked standard curve. Erythema and oedema were rated using a five point scale (0 to 4; none to severe) 24 h after injection. Weight of animals was determined immediately prior to, and 24 h following treatment. Ex vivo analysis of tumor cell survival SK-MEL-28 or FaDu cells were injected (two tumors per mouse) on the hindquarter of 5 week old immunocompromised BALB/c Foxn1nu mice. When the tumors reached approximately 100 mm3, mice in the control group were treated with 20% propylene glycol in water, and the treatment group received 50 nmol (30 µg) EBC-46 via a single intra-tumoral injection. Mice were euthanized at time of injection, 1, 2, 4, 8 and 24 h post-treatment with vehicle or EBC-46, and tumors were harvested. Tumors were dissected, briefly dissociated with collagenase A, and finally resuspended in culture medium. Serial 3-fold dilutions of the cell suspension were cultured in vitro for 6 days, and the SRB assay used to compare the growth of viable EBC-46-treated tumor cells with that of vehicle treated controls. EBC-46 treatment in neutrophil-depleted mice SK-MEL-28 cells (2×106) were injected (two tumor sites per mouse) into the flanks of thirty 5- to 6-week old male BALB/c Foxn1nu mice (n = 5 mice and n = 10 tumors/group). When tumors had reached >50 mm3, ten mice were given i.p. injections of rat anti-neutrophil targeting antibody mLy-6G (100 µg in PBS; clone 1A8, Cat. # BE0075-1) or rat IgG2a isotype control (clone 2A3, Cat. # BE0089) from BioXCell (West Lebanon, NH). The antibodies were injected on days −2, 0, and 2, relative to initiation of vehicle or EBC-46 (25 nmol, 15 µg) treatment on day 0. An additional ten control mice received no antibody. The tumors on a total of 15 mice (5 each of no antibody, IgG2a isotype control or anti-mLy-6G antibody) were treated with 25 nmol EBC-46 per site (15 µg in 20% propylene glycol in water at 300 µg/ml, 50 µl injection), while the tumors on the remaining 15 mice were treated with 50 µl 20% propylene glycol in water only. Blood was taken from tail tips on Days −2, 0 and 2, and then twice weekly, smeared, and air dried on glass slides before being stained with Quick Dip (Fronine Laboratory Supplies, Rivertone, NSW, Australia). Tumor size was measured with calipers twice weekly. Mice were euthanized when the cumulative tumor burden per mouse exceeded 1,000 mm3 or at the end of the experiment. In vitro permeability assay HUVEC cells (Invitrogen/Life Technologies) were grown as described by the manufacturer and used at passage 4 to 6. Media and supplements (M200 [Cat. No. M200PRF500] and Low Serum Growth Supplement [Cat. No. S-003-10] respectively, Life Technologies) were prepared as directed. The In vitro Vascular Permeability Kit was from Millipore (Billerica, MA; Cat. No. ECM642). All assays were performed as described by the manufacturer. Assays were performed in at least triplicate wells.

Discussion Activation of specific PKC isoforms in vascular endothelial cells, particularly the PKC-β isoforms, has previously been shown to induce permeability [30], [31]. Here we show that intra-lesional treatment with EBC-46, a novel PKC-activating compound with apparent specificity for PKC-β isoforms, induces permeability of endothelial cell monolayers in vitro, as well as vascular swelling and apparent disruption of vessel morphology in vivo. Further, sub-cutaneous injection of EBC-46 into normal skin led to significant levels of the drug found in the peripheral circulation. In contrast, intra-lesional injection of EBC-46 resulted in greatly reduced levels detected in the peripheral blood of mice. Additionally, the erythema and oedema observed following EBC-46 administration was significantly higher in mice with tumors compared to mice with normal skin. These results suggest a specificity of vascular damage within tumor sites compared to normal skin leading to anti-cancer efficacy. This specificity may reflect the disorganization and inherent “leakiness” of the vasculature within a solid tumor [32]. We hypothesize that the damage to the tumor vasculature prevents EBC-46 from entering the circulation. There is also a direct effect on tumor cell survival, as no viable tumor cells were evident four hours after injection by ex vivo culture. This is supported by in vitro data which showed treatment with EBC-46 was capable of inhibiting cell survival. Previous studies elegantly showed that another PKC-activating ingenol ester, Ing3A (ingenol 3-angelate, ingenol mebutate, also known as PEP005) was able to penetrate deeper into the dermis following topical application compared to PMA, as it is a substrate for MDR1/P-glycoprotein (P-gp)/ABCB1 [29]. The authors showed that penetration of PMA after topical application was restricted to the epidermis of the skin, thereby sparing the vasculature in the sub-epidermal compartments and resulting in a lack of anti-tumor activity. Further, Li and colleagues also demonstrated that Ing3A bound to and inhibited P-gp whereas PMA did not [29]. Our results presented here additionally show that direct intra-tumoral injection of PMA only led to a transient reduction of tumor growth followed by a rapid relapse. In contrast, intra-tumoral injection of EBC-46 had an enduring anti-tumor effect. We were unable to definitively demonstrate that neutrophils contribute significantly to the anti-tumor efficacy of EBC-46 by intra-lesional injection. Our conclusion was that neutrophils play a minor role in overall efficacy of EBC-46 from the experiments presented in this study. This is in contrast with previous work with other PKC activators (Ing3A/PEP005), where neutrophils were required for anti-tumor efficacy [15], [29]. However, the method of delivery used in this study (intra-tumoral injection) is very different compared to that used in the previous studies with the PEP005 (topical application). Further, the anti-neutrophil antibody used here was from a different, more neutrophil-specific hybridoma clone [33] than that used in previous studies. Nevertheless, in the current study approximately 6% of the cells in the peripheral WBC counts were found to be neutrophils. It is important to note that intra-lesional injection of the prototypic PKC-activator PMA did not lead to a cure of tumors in the current study, but rather an initial shrinking of the tumor followed by a rapid relapse. In contrast, treatment with EBC-46 led to a rapid ablation of the tumor. In greater than 70% of cases, the response and cure was enduring and long term, as demonstrated by the lack of relapse of MM649 tumors over a period of 12 months. PKC activation must clearly play an important role in the action of EBC-46, since pre-treatment with the wide-spectrum PKC inhibitor bisindolylmaleimide-1 resulted in a loss of efficacy in vivo, and prevented endothelial cell dye uptake in vitro. However, we cannot rule out other known targets of diacylglycerol analogues other than PKC isoforms. Recent work has identified that Ing3A/PEP005 binds to and activates members of the RasGRP family of Ras activators, and that this activation may be in part responsible for the anti-cancer activity of the compound [34]. Future studies will investigate if EBC-46 similarly activates molecules in addition to PKC isoforms. In summary, we have identified and characterized EBC-46 as a novel PKC-activating compound. We demonstrate that a single intra-lesional bolus injection is sufficient for the short term regression and ultimate cure of multiple different cancer types in pre-clinical models. EBC-46 is currently being prepared under GMP conditions for use in an upcoming Phase I clinical trial.

Supporting Information Figure S1. Translocation of PKC isoforms induced by EBC-46 in SK-MEL-28 cells. PKC-EGFP isoform translocation in transiently transfected SK-MEL-28 cells following 1 h treatment with either 175 nM (100 ng/ml) PMA or EBC-46. Data was obtained from assessment of at least 50 cells per well from each of triplicate transient transfection experiments. Error bars - standard deviation. a – no data available due to mitochondrial location prior to and after treatment; b – no data available as isoform was toxic to SK-MEL-28 cells. https://doi.org/10.1371/journal.pone.0108887.s001 (TIF) Figure S2. Cell survival assays following treatment with EBC-46. Dose response for cell killing by EBC-46 compared to PMA. Cells were treated with the indicated doses of either EBC-46 (blue) or PMA (red) for 4 days, before assay for cell survival using the sulforhodamine B assay. Data shown are mean ± SD from triplicate readings from three independent experiments, n = 3. https://doi.org/10.1371/journal.pone.0108887.s002 (TIF) Figure S3. Treatment of FaDu tumors with 30 µg EBC-46 or vehicle alone. 2×106 FaDu tumor cells were injected were injected (two tumors per mouse) on the hindquarter of 5 week old immuno-compromised BALB/c Foxn1nu mice. When the tumors had reached approximately 100 mm3, mice in the control group were treated with vehicle (20% propylene glycol in water, 50 µl) and the treatment group received 50 nmol (30 µg) EBC-46 in vehicle via a single intra-tumoral injection. Figure shows tumor appearance prior to treatment, 1 h following treatment, and 2, 5 and 11 days post treatment of tumors treated with vehicle alone (left) or 50 nmol (30 µg) EBC-46 (right). Also shown are ablated tumors 21 days following treatment with 50 nmol (30 µg) EBC-46. No vehicle only control tumors are shown due to the animals being euthanized at day 12 following treatment due to excessive tumor volume. https://doi.org/10.1371/journal.pone.0108887.s003 (TIF) Figure S4. EBC-46 Efficacy against head and neck or colon cancer tumors. A. Tumor volume of FaDu HNSCC line in BALB/c Foxn1nu mice. B. Kaplan Meier plot of HT-29 tumor volume reaching greater than 100 mm3 in BALB/c Foxn1nu mice. C. Kaplan Meier plot of MC-38 tumor volume reaching greater than 100 mm3 in C57BL/6J mice. Grey - vehicle (20% propylene glycol in water); Black – 50 nmol (30 µg) EBC-46 (in vehicle). https://doi.org/10.1371/journal.pone.0108887.s004 (TIF) Figure S5. Effect of EBC-46 on normal skin. Normal skin of BALB/c Foxn1nu mice was treated with either A. 50 µl vehicle (20% propylene glycol in water) or B. 50 nmol (30 µg) EBC-46 in vehicle. Arrows indicate examples of dilated blood vessels. Scale bar = 100 µm. https://doi.org/10.1371/journal.pone.0108887.s005 (TIF) Figure S6. Effect of EBC-46 on normal skin vasculature. Normal skin of BALB/c Foxn1nu mice was treated with either A. 50 µl vehicle (20% propylene glycol in water) or B. 50 nmol (30 µg) EBC-46 in vehicle. Arrows indicate examples of intact blood vessels. Scale bar = 100 µm. https://doi.org/10.1371/journal.pone.0108887.s006 (TIF)

Acknowledgments We thank TetraQ Pty. Ltd. for performing the chromatographic assay to determine the level of EBC-46 in the plasma (under a fee-for-service agreement with QBiotics Ltd.).

Author Contributions Conceived and designed the experiments: GMB VAG PWR PGP. Performed the experiments: GMB MMAD CJP RAA ASC JPJ LM PGP. Analyzed the data: GMB MMAD CJP RAA ASC JPJ LM PGP. Contributed reagents/materials/analysis tools: GMB MMAD CJP RAA ASC JPJ LM VAG PWR PGP. Wrote the paper: GMB VAG PWR PGP.