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10 pags., 4 figs., 2 tabs., Background Photodynamic therapy (PDT) kills target microorganisms via reactive oxygen species (ROS) production. PDT seems to be a good alternative treatment option for onychomycosis. Objective To compare the efficacy of combined therapies based on oral terbinafine (TN) plus adjunctive PDT mediated by methylene blue (MB) (TN + MB/PDT) or methyl aminolevulinate (MAL) (TN + MAL/PDT) in the treatment of onychomycosis. Methods Twenty patients affected by severe dermatophyte onychomycosis in the nails of the big toe (>60% disease involvement of target nail) received oral TN for 12 weeks and concomitantly were randomly allocated to receive nine sessions, separated by 2‐week intervals, of urea (40%) plus a PDT protocol mediated by MB (TN + MB/PDT: group I) or mediated by MAL (TN + MAL/PDT: group II). Clinical and mycological efficacy was evaluated at 16‐, 40‐ and 52‐week follow‐up., Results Both protocols showed a significant decrease in Onychomycosis Severity Index (OSI) scores (P < .05), from 24.2 ± 4.6 to 0.7 ± 0.6 (group I)) and from 18.5 ± 10.1 to 2.1 ± 2.0 (group II). No side effects or complications were reported in any of the combinations used. Mycological cure rates were significantly higher during the last third of the evaluated period of time, reaching 100% and 90% in group I and group II, respectively, at the 52‐week follow‐up. In both modalities, complete cure was achieved in 70% of the patients at the 52‐week follow‐up. Conclusions TN + MB/PDT and TN + MAL/PDT show similar outcomes in the treatment of toenails with severe onychomycosis. PDT is an effective method to accelerate the TN‐mediated healing process., This research was supported by a grant from the Eugenio Rodríguez Pascual Foundation (Madrid, Spain) and by the Spanish Research Project MICINN (Ref.: MAT 2017‐83856‐C3).

5 pags., 3 figs., Background: Although systemic therapies are recommended for severe or recalcitrant cases of pityriasis versicolor (PV), they are not free of important side effects and drug interactions. Photodynamic therapy (PDT) utilizes the action of singlet oxygen and free radicals produced by a light-activated photosensitizer to kill viruses, bacteria, or fungi. In this study, the effect of a PDT mediated by methylene blue (MB) in PV was evaluated. Methods: Five women with PV disseminated on the back and diagnosed by fresh microscopic analysis were treated with a solution of MB (2%) applied to the PV lesions for 3 minutes. Next, a red LED lamp (λ = 630±5 nm, 37 J/cm), placed 100 mm from the skin for 10 minutes, was applied on the dyed PV lesions. Six sessions of MB/PDT were implemented with a 2-week interval in between. Wood's lamp examination was used to monitor fungal infection at each time point. Results: Complete cure was observed in the five women at the 4 weeks post-treatment follow-up. Fluoresce images from PV lesions by Wood's lamp allowed to evaluate whether the lesions were healed or not at each time point. No patient showed relapse at the 6-month follow-up. The patients did not have any adverse effect, and good cosmetic outcome was observed. Conclusions: Six sessions of MB/PDT spaced at 14-day intervals are sufficient for the treatment for PV in healthy patients., Spanish Research Projects MINECO. Grant Number: MAT 2017‐83856‐C3 Fundación Eugenio Rodríguez Pascual

5 pags., 3 figs., 2 tabs., Background: Although surgical removal is the treatment of choice in Bowen's disease (BD), there are cases in which by age, comorbidities, use of anticoagulants, location, cosmetic result, or size, it is preferable to use other treatments such as cryotherapy, 5-fluorouracil cream, imiquimod 5% cream or photodynamic therapy (PDT). Efficacy of PDT in BD is supported by substantial research and clinical data. Objectives: This study aimed to evaluate the long term effectiveness of methyl aminolevulinate-PDT (MAL/PDT)on a wide range of Bowen lesions in different locations and sizes. Methods: Patients diagnosed with BD were treated in 3 sessions with a 4-week interval in between with MAL/PDT between January 2016 and January 2017 in a private clinic. Clinical response and relevant patient and tumour characteristics were analyzed during the first year after start of the PDT sessions. Results: In total, 21 BD lesions in 18 patients were included in the study. Complete regression (CR)after 3rd PDT session was 87.5% and 100% at the 6-month follow-up. Treatment was well tolerated and local adverse reactions were very scarce. No recurrence was observed at 12-month follow-up. Cosmetic outcome at 12 months was good or excellent in 100% of patients. Conclusions: MAL/PDT is an effective, non invasive and safe treatment modality for BD with excellent cosmesis., This research was supported by a grant from the Eugenio Rodríguez Pascual Foundation (Madrid, Spain) and by the Spanish ResearchProject MINECO (Ref.: MAT 2017-83856-C3).

6 pags., 3 figs., 3 tabs., Background: The increase in the number of basal cell carcinoma (BCC) lesions has prompted use of minimally invasive therapies, including Photodynamic therapy (PDT). . The objective of the present work was to analyze the efficacy of methyl aminolevulinate-mediated photodynamic therapy (MAL-PDT) in patients suffering from superficial or nodular BCCs. Methods: A total of 220 BCC lesions (76 superficial and 144 nodular), clinically diagnosed and confirmed by histopathology analysis, were treated in 174 patients (mean age 72.5). Debulking using curettage was performed before two or three MAL-PDT sessions (λ = 630 nm; 90 J/cm; 23 min) at 4-week intervals. Analyses of clinical clearance and cosmetic outcome were carried out by direct examination, dermoscopy, photographs, as well as by fluorescence diagnosis using a Wood's lamp. Evaluations were carried out at the different PDT sessions and follow-ups over a 3-year period. Results: MAL-PDT was safe and highly tolerated. After an average of 2.6 MAL-PDT sessions, the overall clearance rate at 3-year follow-up was 96.1 % (95 % confidence interval [CI] 100 %–92 %) for superficial BCCs and 95.2 % (95 % [CI] 99 %-92 %) for nodular BCCs after an average of 2.7 sessions. Minimal side effects such as crushing, erythema and edema were reported. All BCC lesions showed excellent or good cosmetic results. Conclusion: The protocol followed in the present study has shown that MAL-PDT is a safe and effective treatment for superficial and nodular types of BCC., This research was supported by a grant from the Eugenio Rodríguez Pascual Foundation (Madrid, Spain) and by the Spanish Research Project MINECO (Ref.: MAT 2017-83856-C3).

8 pags., 3 figs. 3 tabs., Photodynamic therapy (PDT) has emerged as an interesting alternative option for onychomycosis treatment. The impact of a specific photosensitizer (PS) on the final result is an important factor to consider. We conducted a short- and medium-term controlled trial to compare the effectiveness of PDT in the treatment of mild-to-moderate onychomycosis when it is mediated by two different PSs. Twenty patients were randomized to receive nine sessions of PDT distributed over 16 weeks mediated either by methylene blue (MB/PDT group) or methyl aminolevulinate (MAL/PDT group). Onychomycosis severity index (OSI) and nail involvement were checked along the study. Complete cure, treatment success, and clinical improvement were tabulated at 16 and 40-week follow-ups. OSI scores decreased significantly along the study, from 12.1 ± 5.4 to 3.6 ± 3.2 (MB/PDT group) and from 14.8 ± 6.0 to 5.4 ± 4.4 (MAL/PDT group). At 16-week follow-up, only 20% of the patients in the MB/PDT group reached complete cure and none in the group of MAL/PDT. At 40-week follow-up, complete cure rates were 70% and 40% in the MB/PDT group and MAL/PDT group respectively. Both modalities showed good outcomes in treatment of moderate toenail onychomycosis. MB/PDT showed a faster action but with relapse rates slightly higher than MAL/PDT., This research was supported by a grant from the Eugenio Rodríguez Pascual Foundation (Madrid, Spain) and by the Spanish Research Project MICINN (Ref.: MAT 2017-83856-C3)

Despite significant advances have been achieved in the genetic characterization of sporadic colorectal cancer (sCRC), the precise genetic events leading to the development of distant metastasis remain poorly understood. Thus, accurate prediction of metastatic disease in newly-diagnosed sCRC patients remains a challenge. Here, we evaluated the specific genes and molecular pathways associated with the invasive potential of colorectal tumor cells, through the assessment of the gene expression profile (GEP) of coding and non-coding genes in metastatic (MTX) vs. non-metastatic (non-MTX) primary sCRC tumors followed for >5 years. Overall, MTX tumors showed up-regulation of genes associated with tumor progression and metastatic potential while non-MTX cases displayed GEP associated with higher cell proliferation, activation of DNA repair and anti-tumoral immune/inflammatory responses. Based on only 19 genes a specific GEP that classifies sCRC tumors into two MTX-like and non-MTX-like molecular subgroups was defined which shows an independent prognostic impact on patient overall survival, particularly when it is combined with the lymph node status at diagnosis. In summary, we show an association between the global GEP of primary sCRC cells and their metastatic potential and defined a GEP-based classifier that provides the basis for further prognostic stratification of sCRC patients who are at risk of distant metastases., This work has been partially supported by grants from the Instituto de Salud Carlos III (ISCIII; Ministerio de Economía y Competitividad, Madrid, Spain) (PI12/02053-FIS and PI17/01779-FIS), Gerencia Regional de Salud de Castilla y León, Valladolid, Spain (GRS1302/A/16), Consejería de Sanidad (Junta de Castilla y Leon, Valladolid, Spain) (BIO/SA02/13), RTICC and CIBERONC from the ISCIII (RD12/0020/0035-FEDER, RD12/0036/0048-FEDER, CB16/12/00400), Fundación Memoria de Don Samuel Solórzano Barruso, (Salamanca, Spain) and Fundación Eugenio Rodríguez Pascual, (Madrid, Spain). ML Gutiérrez and JM Sayagués are supported by grants: PTA2014-09963-I and CES11/004 from the ISCIII.

[Background]: Cutaneous squamous cell carcinoma (CSCC) is the second most frequent cancer in humans after basal cell carcinoma, and its incidence is dramatically rising. CSCC is rarely problematic, but given its high frequency, the absolute number of complicated cases is also high. It is necessary to identify molecular markers in order to recognize those CSCCs with poor prognosis. There is controversy concerning the role of epidermal growth factor receptor (EGFR) as a marker of prognosis in CSCC. In addition, EGFR-targeted therapies have emerged in recent years and a better understanding of the role of EGFR in CSCC may be of help for some patients in predicting prognosis and guiding curative management. [Objectives]: To evaluate the role of EGFR as a prognostic factor in CSCC. [Methods]: We evaluated clinical and histopathological features, including events of poor clinical evolution, in a series of 94 cases of CSCC. We also analysed EGFR expression by immunohistochemistry, fluorescent in situ hybridization and quantitative polymerase chain reaction. [Results]: We detected EGFR in 85 cases (90%), with overexpression in 33 cases (35%), and aberrant EGFR expression in the cytoplasm in 50 cases (53%). EGFR overexpression in the primary tumours was associated with lymph node progression, tumour–nodes–metastasis stage progression and proliferation (Ki-67 staining) in CSCC. EGFR overexpression and poor grade of differentiation were the strongest independent variables defining lymph node metastasis and progression in CSCC in a logistic regression model. [Conclusions]: We demonstrate that EGFR overexpression has prognostic implications associated with lymph node metastasis and progression in CSCC., J.P.‐L. was partially supported by FEDER and MICINN (PLE2009‐119, SAF2014‐56989‐R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y León (SAN673/SA26/08, SAN126/SA66/09, SA078A09, CSI034U13, BIO/SA31/15), IBSAL (IBY15/00003), the ‘Eugenio Rodríguez Pascual’, the ‘Fundación Inbiomed’ (Instituto Oncológico Obra Social de la Caja Guipozcoa‐San Sebastian, Kutxa) and the ‘Fundación Sandra Ibarra de Solidaridad frente al Cáncer’. C.R.‐C. is funded by Q3718001E (2009‐2010) and GRS 612/A/11 (2011‐2012) and ‘the Fundación Eugenio Rodríguez Pascual’. A.C.‐M. was supported by FIS (PI07/0057) and MICINN (PLE2009‐119).

Cholangiocarcinoma (CCA) and pancreatic adenocarcinoma (PDAC) may lead to the development of extrahepatic obstructive cholestasis. However, biliary stenoses can also be caused by benign conditions, and the identification of their etiology still remains a clinical challenge. We performed metabolomic and proteomic analyses of bile from patients with benign (n = 36) and malignant conditions, CCA (n = 36) or PDAC (n = 57), undergoing endoscopic retrograde cholangiopancreatography with the aim of characterizing bile composition in biliopancreatic disease and identifying biomarkers for the differential diagnosis of biliary strictures. Comprehensive analyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (1H-NMR) in all patients. MS analysis of bile proteome was performed in five patients per group. We implemented artificial intelligence tools for the selection of biomarkers and algorithms with predictive capacity. Our machine-learning pipeline included the generation of synthetic data with properties of real data, the selection of potential biomarkers (metabolites or proteins) and their analysis with neural networks (NN). Selected biomarkers were then validated with real data. We identified panels of lipids (n = 10) and proteins (n = 5) that when analyzed with NN algorithms discriminated between patients with and without cancer with an unprecedented accuracy., This research was funded by: Instituto de Salud Carlos III (ISCIII) co-financed by Fondo Europeo de Desarrollo Regional (FEDER) Una manera de hacer Europa, grant numbers: PI16/01126 (M.A.A.), PI19/00819 (M.J.M. and J.J.G.M.), PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129 (J.M.B.); Fundación Científica de la Asociación Española Contra el Cáncer (AECC Scientific Foundation), grant name: Rare Cancers 2017 (J.M.U., M.L.M., J.M.B., M.J.M., R.I.R.M., M.G.F.-B., C.B., M.A.A.); Gobierno de Navarra Salud, grant number 58/17 (J.M.U., M.A.A.); La Caixa Foundation, grant name: HEPACARE (C.B., M.A.A.); AMMF The Cholangiocarcinoma Charity, UK, grant number: 2018/117 (F.J.C. and M.A.A.); PSC Partners US, PSC Supports UK, grant number 06119JB (J.M.B.); Horizon 2020 (H2020) ESCALON project, grant number H2020-SC1-BHC-2018–2020 (J.M.B.); BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia, grant numbers BIO15/CA/016/BD (J.M.B.) and BIO15/CA/011 (M.A.A.). Department of Health of the Basque Country, grant number 2017111010 (J.M.B.). La Caixa Foundation, grant number: LCF/PR/HP17/52190004 (M.L.M.), Mineco-Feder, grant number SAF2017-87301-R (M.L.M.), Fundación BBVA grant name: Ayudas a Equipos de Investigación Científica Umbrella 2018 (M.L.M.). MCIU, grant number: Severo Ochoa Excellence Accreditation SEV-2016-0644 (M.L.M.). Part of the equipment used in this work was co-funded by the Generalitat Valenciana and European Regional Development Fund (FEDER) funds (PO FEDER of Comunitat Valenciana 2014–2020). Gobierno de Navarra fellowship to L.C. (Leticia Colyn); AECC post-doctoral fellowship to M.A.; Ramón y Cajal Program contracts RYC-2014-15242 and RYC2018-024475-1 to F.J.C. and M.G.F.-B., respectively. The generous support from: Fundación Eugenio Rodríguez Pascual, Fundación Echébano, Fundación Mario Losantos, Fundación M Torres and Mr. Eduardo Avila are acknowledged. The CNB-CSIC Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0001 (F.J.C.). Comunidad de Madrid Grant B2017/BMD-3817 (F.J.C.).

Colorectal cancer (CRC) is the third most common cancer worldwide. The standard treatment in locally advanced rectal cancer is preoperative radiation alone or in combination with chemotherapy, followed by adjuvant chemotherapy. Rectal cancer is highly lethal, with only 20% of patients showing a complete remission (by RECIST) after standard treatment, although they commonly show local or systemic relapse likely due to its late detection and high chemotherapy resistance, among other reasons. Here, we explored the role of PAI1 (Serpin E1) in rectal cancer through the analyses of public patient databases, our own cohort of locally advanced rectal cancer patients and a panel of CRC cell lines. We showed that PAI1 expression is upregulated in rectal tumors, which is associated with decreased overall survival and increased metastasis and invasion in advanced rectal tumors. Accordingly, PAI1 expression is correlated with the expression of (Epithelial-to-Mesenchymal Transition) EMT-associated genes and genes encoding drug targets, including the tyrosine kinases PDGFRb, PDGFRa and FYN, the serine/threonine kinase PIM1 and BRAF. In addition, we demonstrate that cells expressing PAI1 protein are more sensitive to the PIM inhibitor AZD1208, suggesting that PAI1 could be used to predict response to treatment with PIM inhibitors and to complement radiotherapy in rectal tumors., This research was funded by Ministerio de Ciencia, Innovación y Universidades (MCIU) Plan Estatal de I+D+I 2018, Agencia Estatal de Investigación (AEI) and Regional Development European Funds (FEDER, European Union): RTI2018-097455-B-I00 (MCIU/AEI/FEDER, UE); grants from AEI-MICIU/FEDER (RED2018-102723-T; CIBER of Cancer (CB16/12/00275); Consejería de Salud of the Junta de Andalucía (PI-0397-2017); Consejeria of Economía, Conocimiento, Empresas y Universidad of the Junta de Andalucia (P18-RT-2501); Fundación AECC and Fundación Eugenio Rodriguez Pascual. SMG was funded by a Sara Borrell grant from ISCIII (CD16/00230) and the Fundación AECC.

9 pags., 3 figs., 5 tabs., Background: Photodynamic therapy (PDT) or intense pulsed light (IPL) are efficient therapeutic methods in the treatment of superficial skin infections, and thus, they could be good options for onychomycosis treatment, the most common nail disorder. Methods: Forty patients, affected with different diagnosed types of onychomycosis in nails of the first toe, were randomly divided into two groups of 20 patients to be treated by PDT or IPL. Nail plates were softened with urea 40% by occlusive dressing for 12 hours during 3-7 days before treatments. Then, eight sessions separated by an interval of 2 weeks of a PDT protocol mediated by methylene blue (MB) and red laser diode (Periowave, λ = 670 nm, 200 mW) or an IPL protocol based on 10 pulses/cm (Dye-VL-F module, Alma Lasers, λ = 500-600 nm, 10 J) were applied. Results: Both treatments reduced significantly the Onychomycosis Severity Index (OSI) (P, Ministerio de Economía, Industria y Competitividad MINECO, Grant/Award Number: MAT 2017-83856-C3-1-P and MAT2015-68837-REDT; Fundación Eugenio Rodríguez Pascua

Caloric restriction (CR) ameliorates cardiac dysfunction associated with obesity. However, most of the studies have been performed under severe CR (30-65% caloric intake decrease) for several months or even years in aged animals. Here, we investigated whether mild (20% food intake reduction) and short-term (2-weeks) CR prevented the obese cardiomyopathy phenotype and improved the metabolic profile of young (14 weeks of age) genetically obese Zucker fa/fa rats. Heart weight (HW) and HW/tibia length ratio was significantly lower in fa/fa rats after 2 weeks of CR than in counterparts fed ad libitum. Invasive pressure measurements showed that systolic blood pressure, maximal rate of positive left ventricle (LV) pressure, LV systolic pressure and LV end-diastolic pressure were all significantly higher in obese fa/fa rats than in lean counterparts, which were prevented by CR. Magnetic resonance imaging revealed that the increase in LV end-systolic volume, stroke volume and LV wall thickness observed in fa/fa rats was significantly lower in animals on CR diet. Histological analysis also revealed that CR blocked the significant increase in cardiomyocyte diameter in obese fa/fa rats. High resolution magic angle spinning magnetic resonance spectroscopy analysis of the LV revealed a global decrease in metabolites such as taurine, creatine and phosphocreatine, glutamate, glutamine and glutathione, in obese fa/fa rats, whereas lactate concentration was increased. By contrast, fatty acid concentrations in LV tissue were significantly elevated in obese fa/fa rats. CR failed to restore the LV metabolomic profile of obese fa/fa rats. In conclusion, mild and short-term CR prevented an obesity-induced cardiomyopathy phenotype in young obese fa/fa rats independently of the cardiac metabolic profile., This study was supported by grants from Spanish Ministry of Science and Innovation (BFU2011-25303), Spanish Institute of Health Carlos III (CP15/00129), UCM groups (GR-921641), SESCAMET, Fundación Mutua Madrileña, Fundación Eugenio Rodríguez Pascual and Fondos FEDER.

[Background]: Cutaneous squamous cell carcinoma (CSCC) is the second most widespread cancer in humans and its incidence is rising. These tumours can evolve as diseases of poor prognosis, and therefore it is important to identify new markers to better predict its clinical evolution. [Objectives]: We aimed to identify the expression pattern of microRNAs (miRNAs or miRs) at different stages of skin cancer progression in a panel of murine skin cancer cell lines. Owing to the increasing importance of miRNAs in the pathogenesis of cancer, we considered the possibility that miRNAs could help to define the prognosis of CSCC and aimed to evaluate the potential use of miR-203 and miR-205 as biomarkers of prognosis in human tumours. [Methods]: Seventy-nine human primary CSCCs were collected at the University Hospital of Salamanca in Spain. We identified differential miRNA expression patterns at different stages of CSCC progression in a well-established panel of murine skin cancer cell lines, and then selected miR-205 and miR-203 to evaluate their association with the clinical prognosis and evolution of human CSCC. [Results]: miR-205 was expressed in tumours with pathological features recognized as indicators of poor prognosis such as desmoplasia, perineural invasion and infiltrative growth pattern. miR-205 was mainly expressed in undifferentiated areas and in the invasion front, and was associated with both local recurrence and the development of general clinical events of poor evolution. miR-205 expression was an independent variable selected to predict events of poor clinical evolution using the multinomial logistic regression model described in this study. In contrast, miR-203 was mainly expressed in tumours exhibiting the characteristics associated with a good prognosis, was mainly present in well-differentiated zones, and rarely expressed in the invasion front. Therefore, the expression and associations of miR-205 and miR-203 were mostly mutually exclusive. Finally, using a logistic biplot we identified three clusters of patients with differential prognosis based on miR-203 and miR-205 expression, and pathological tumour features. [Conclusions]: miR-205 and miR-203 tended to exhibit mutually exclusive expression patterns in human CSCC. This work highlights the utility of miR-205 and miR-203 as prognostic markers in CSCC., J.P.-L. was partially supported by FEDER and MICINN (PLE2009-119, SAF2014-56989-R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y Leon (SA078A09, CSI034U13, CSI001U16), the ‘Eugenio Rodriguez Pascual’, the ‘Fundacion Inbiomed’ (Instituto Oncologico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa), IBSAL (IBY15/00003) and the ‘Fundacion Sandra Ibarra de Solidaridad frente al Cancer’. C.R.-C. is funded by Q3718001E (2009–2010) y GRS 612/A/11 (2011–2012) and the ‘Fundacion Eugenio Rodriguez Pascual’. A.C.-M. was supported by FIS (PI07/0057) and MICINN (PLE2009-119). J.H.M. was supported by the National Institutes of Health, a National Cancer Institute grant (R01 CA116481) and the Low-Dose Scientific Focus Area, Office of Biological & Environmental Research, U.S. Department of Energy (DE-AC02-05CH11231). J.C. was partially supported by Gerencia Regional de Salud (Junta de Castilla y Leon), GRS 1342 / A / 16.

Metastatic dissemination is the most frequent cause of death of sporadic colorectal cancer (sCRC) patients. Genomic abnormalities which are potentially characteristic of such advanced stages of the disease are complex and so far, they have been poorly described and only partially understood. We evaluated the molecular heterogeneity of sCRC tumors based on simultaneous assessment of the overall GEP of both coding mRNA and non-coding RNA genes in primary sCRC tumor samples from 23 consecutive patients and their paired liver metastases. Liver metastases from the sCRC patients analyzed, systematically showed deregulated transcripts of those genes identified as also deregulated in their paired primary colorectal carcinomas. However, some transcripts were found to be specifically deregulated in liver metastases (vs. non-tumoral colorectal tissues) while expressed at normal levels in their primary tumors, reflecting either an increased genomic instability of metastatic cells or theiradaption to the liver microenvironment. Newly deregulated metastatic transcripts included overexpression of APOA1, HRG, UGT2B4, RBP4 and ADH4 mRNAS and the miR-3180-3p, miR-3197, miR-3178, miR-4793 and miR-4440 miRNAs, together with decreased expression of the IGKV1-39, IGKC, IGKV1-27, FABP4 and MYLK mRNAS and the miR-363, miR-1, miR-143, miR-27b and miR-28-5p miRNAs. Canonical pathways found to be specifically deregulated in liver metastatic samples included multiple genes related with intercellular adhesion and the metastatic processes (e.g., IGF1R, PIK3CA, PTEN and EGFR), endocytosis (e.g., the PDGFRA, SMAD2, ERBB3, PML and FGFR2), and the cell cycle (e.g., SMAD2, CCND2, E2F5 and MYC). Our results also highlighted the activation of genes associated with the TGFβ signaling pathway, -e.g. RHOA, SMAD2, SMAD4, SMAD5, SMAD6, BMPR1A, SMAD7 and MYC-, which thereby emerge as candidate genes to play an important role in CRC tumor metastasis., This work has been partially supported by grants from the Instituto de Salud Carlos III (ISCIII; Ministerio de Sanidad y Consumo, Madrid, Spain) (PI12/02053-FIS), Gerencia Regional de Salud de Castilla y León, Valladolid, Spain (GRS1302/A/16), Consejería de Sanidad (Junta de Castilla y Leon, Valladolid, Spain) (BIO/SA02/13 and BIO/SA46/14), RTICC from the ISCIII (RD12/0020/0035-FEDER, RD12/0036/0048-FEDER), Fundación Memoria de Don Samuel Solórzano Barruso, (Salamanca, Spain) and Fundación Eugenio Rodríguez Pascual, (Madrid, Spain). JM Sayagués and ME Sarasquete are supported by grants (CES11/004 and CP13/00080; respectively) from the ISCIII, Ministerio de Ciencia e Innovación, Madrid, Spain.

Different data support a role for the epidermal growth factor receptor (EGFR) pathway during liver regeneration and hepatocarcinogenesis. However, important issues, such as the precise mechanisms mediating its actions and the unique versus redundant functions, have not been fully defined. Here, we present a novel transgenic mouse model expressing a hepatocyte-specific truncated form of human EGFR, which acts as negative dominant mutant (ΔEGFR) and allows definition of its tyrosine kinase-dependent functions. Results indicate a critical role for EGFR catalytic activity during the early stages of liver regeneration. Thus, after two-thirds partial hepatectomy, ΔEGFR livers displayed lower and delayed proliferation and lower activation of proliferative signals, which correlated with overactivation of the transforming growth factor-β pathway. Altered regenerative response was associated with amplification of cytostatic effects of transforming growth factor-β through induction of cell cycle negative regulators. Interestingly, lipid synthesis was severely inhibited in ΔEGFR livers after partial hepatectomy, revealing a new function for EGFR kinase activity as a lipid metabolism regulator in regenerating hepatocytes. In spite of these profound alterations, ΔEGFR livers were able to recover liver mass by overactivating compensatory signals, such as c-Met. Our results also indicate that EGFR catalytic activity is critical in the early preneoplastic stages of the liver because ΔEGFR mice showed a delay in the appearance of diethyl-nitrosamine-induced tumors, which correlated with decreased proliferation and delay in the diethyl-nitrosamine-induced inflammatory process. Conclusion: These studies demonstrate that EGFR catalytic activity is critical during the initial phases of both liver regeneration and carcinogenesis and provide key mechanistic insights into how this kinase acts to regulate liver pathophysiology. (Hepatology 2016;63:604-619), Cofunded by the Ministry of Economy and Competitiveness-MINECO and the European Regional Development Fund-FEDER, Spain (Contract grant numbers: BFU2012-35538 and ISCIII-RTICC RD12-0036-0029 to I.F.-IDIBELL; SAF2009-12477 to A.S.-UCM/IdISSC; RETICS-RD12/0019/0023 and SAF2011-30526-C02-01 to J.-C.S.-CIEMAT/CIBERER/IIS-FJD; SAF2012-33283 to A.M.V.-CSIC/CIBERDEM). People Programme (Marie Curie Actions) of the FP7-2012, under REA grant agreement #PITN-GA-2012-316549 (IT-LIVER) to I.F.-IDIBELL and to A.S.-UCM/IdISSC. AGAUR-Generalitat de Catalunya to I.F-IDIBELL (Contract grant number: 2009SGR-312). Dirección General de Investigación de la Comunidad de Madrid, Spain (Contract grant number S2010/BMD-2402 to A.S.-UCM/IdISSC). Fundación Eugenio Rodríguez Pascual to M.G.-B.-CIEMAT/CIBERER/IIS-FJD. J.L.-L. was recipient of a predoctoral fellowship from IDIBELL; D.C.-D. and A.M.-P. were recipients of predoctoral fellowships from MINECO, Spain (Contract grant number: FPI - BES-2013-064609 and BES-2007-16187, respectively).

et al., Aging remains the main risk factor to suffer Alzheimer's disease (AD), though epidemiological studies also support that type 2 diabetes (T2D) is a major contributor. In order to explore the close relationship between both pathologies we have developed an animal model presenting both AD and T2D, by crossing APP/PS1 mice (AD model) with db/db mice (T2D model). We traced metabolic and cognitive evolution before T2D or AD pathology is present (4 weeks of age), when T2D has debuted but no senile plaques are present (14 weeks of age) and when both pathologies are well established (26 weeks of age). APP/PS1xdb/db mice showed an age-dependent synergistic effect between T2D and AD. Significant brain atrophy and tau pathology were detected in the cortex by 14 weeks, that spread to the hippocampus by 26 weeks of age. Severe cognitive impairment was also detected as soon as at 14 weeks of age. Interestingly, in APP/PS1xdb/db mice we observed a shift in Aβ soluble/insoluble levels, and whereas more toxic soluble species were favoured, senile plaques (SP) were reduced. An overall increase of microglia activation was observed in APP/PS1xdb/db mice. We also found exacerbated hemorrhagic burden in APP/PS1xdbd/db mice, suggesting that blood brain barrier alterations may be responsible for the early pathological features observed. Moreover, metabolic parameters can predict many of these alterations, supporting a role for T2D in AD pathology. This new model provides a relevant tool to further explore the relationship between T2D, AD and vascular implications, offering the possibility to assess therapeutic approaches, that by improving T2D metabolic control could delay or prevent AD pathology., Junta de Andalucia, Proyectos de Excelencia, Consejería de Economía, Innovación, Ciencia y Empleo (P11-CTS-7847), Fundación Eugenio Rodríguez Pascual 2015, ISCIII–Subdirección General de Evaluación y Fomento de la Investigación and cofinanced by the European Union (Fondo Europeo de Desarrollo Regional, FEDER) “Una manera de hacer Europa” PI12/00675 (Monica Garcia-Alloza).

Myocardial fibrosis detected via delayed-enhanced magnetic resonance imaging (MRI) has been shown to be a strong indicator for ventricular tachycardia (VT) inducibility. However, little is known regarding how inducibility is affected by the details of the fibrosis extent, morphology, and border zone configuration. The objective of this article is to systematically study the arrhythmogenic effects of fibrosis geometry and extent, specifically on VT inducibility and maintenance. We present a set of methods for constructing patient-specific computational models of human ventricles using in vivo MRI data for patients suffering from hypertension, hypercholesterolemia, and chronic myocardial infarction. Additional synthesized models with morphologically varied extents of fibrosis and gray zone (GZ) distribution were derived to study the alterations in the arrhythmia induction and reentry patterns. Detailed electrophysiological simulations demonstrated that (1) VT morphology was highly dependent on the extent of fibrosis, which acts as a structural substrate, (2) reentry tended to be anchored to the fibrosis edges and showed transmural conduction of activations through narrow channels formed within fibrosis, and (3) increasing the extent of GZ within fibrosis tended to destabilize the structural reentry sites and aggravate the VT as compared to fibrotic regions of the same size and shape but with lower or no GZ. The approach and findings represent a significant step toward patient-specific cardiac modeling as a reliable tool for VT prediction and management of the patient. Sensitivities to approximation nuances in the modeling of structural pathology by image-based reconstruction techniques are also implicated. Makarand Deo is supported by American heart association scientist development grant no. 12sdg11480010. the work is also supported by Pro CniC Foundation, Eugenio Rodríguez Pascual Foundation, Fondo Europeo de Desarrollo Regional (Feder), European Union, and Instituto de Salud Carlos iii, Spain [rd12/0042/0036 (RIC)] (David Filgueiras-Rama, Gonzalo Pizarro, and Borja ibañez). Sí

The 8–32 fragment of salmon calcitonin ((8–32) sCT) has been proposed as a highly selective amylin receptor antagonist. In the present study, we have studied the influence of (8–32) sCT on the inhibitory effect of both amylin and its structural congener, calcitonin gene-related peptide (CGRP), on insulin secretion in the rat perfused pancreas. Both amylin and CGRP, at 75 pM, clearly inhibited glucose-induced insulin release (by 80% and by 70%, respectively). Simultaneous infusion of 10 μm (8–32) sCT reversed the inhibitory effect of amylin (by 80%; P, This work was supported by grants from the Fondo de Investigacion Sanitaria de la Seguridad Social (FIS no. 94/0282 and 93/0184) and the Fundaci6n Eugenio Rodriguez-Pascual, Spain.

Ghrelin is an endogenous regulator of energy homeostasis synthesized by the stomach to stimulate appetite and positive energy balance. Similarly, the endocannabinoid system is part of our internal machinery controlling food intake and energy expenditure. Both peripheral and central mechanisms regulate CB1-mediated control of food intake and a functional relationship between hypothalamic ghrelin and cannabinoid CB1 receptor has been proposed. First of all, we investigated brain ghrelin actions on food intake in rats with different metabolic status (negative or equilibrate energy balance). Secondly, we tested a sub-anxiogenic ultra-low dose of the CB1 antagonist SR141716A (Rimonabant) and the peripheral-acting CB1 antagonist LH-21 on ghrelin orexigenic actions. We found that: 1) central administration of ghrelin promotes food intake in free feeding animals but not in 24 h food-deprived or chronically food-restricted animals; 2) an ultra-low dose of SR141716A (a subthreshold dose 75 folds lower than the EC50 for induction of anxiety) completely counteracts the orexigenic actions of central ghrelin in free feeding animals; 3) the peripheral-restricted CB1 antagonist LH-21 blocks ghrelin-induced hyperphagia in free feeding animals. Our study highlights the importance of the animaĺs metabolic status for the effectiveness of ghrelin in promoting feeding, and suggests that the peripheral endocannabinoid system may interact with ghrelińs signal in the control of food intake under equilibrate energy balance conditions. © 2013 Alen et al.

Type 2 diabetes (T2D) mellitus and Alzheimer's disease (AD) are two prevalent diseases with comparable pathophysiological features and genetic predisposition. Patients with AD are more susceptible to develop T2D. However, the molecular mechanism linking AD and T2D remains elusive. In this study, we have generated a new mouse model to test the hypothesis that AD would prompt the onset of T2D in mice. To test our hypothesis, we crossed Alzheimer APPswe/PS1dE9 (APP/PS1) transgenic mice with mice partially deficient in leptin signaling (db/+). Body weight, plasma glucose, and insulin levels were monitored. Phenotypic characterization of glucose metabolism was performed using glucose and insulin tolerance tests. β-Cell mass, islet volume, and islet number were analyzed by histomorphometry. APP/PS1 coexpression in mice with intact leptin receptor signaling did not show any metabolic perturbations in glucose metabolism or insulin sensitivity. In contrast, APP/PS1 coexpression in db/+ mice resulted in nonfasting hyperglycemia, hyperinsulinemia, and hypercholesterolemia without changes in body weight. Conversely, fasting blood glucose and cholesterol levels remained unchanged. Coinciding with altered glucose metabolism, APP/PS1 coexpression in db/+ mice resulted in glucose intolerance, insulin resistance, and impaired insulin signaling. In addition, histomorphometric analysis of pancreata revealed augmented β-cell mass. Taken together, these findings provide experimental evidence to support the notion that aberrant Aβ production might be a mechanistic link underlying the pathology of insulin resistance and T2D in AD., This work was supported by grants from Instituto de Salud Carlos III (ISCIII)-Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00671 and CP08/00094), and a Europe-FP7 Marie Curie grant (IRG-247835) to I. Cózar-Castellano and by ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, Spain (PS09/00969), RYC-2008- 02333, Proyecto de Excelencia Junta de Andalucía (P11-CTS-7847), and Fundación Dr. Eugenio Rodríguez Pascual to M. García-Alloza.

17 páginas, 8 figuras, 2 tablas., Murine models of Alzheimer’s disease (AD) provide means to detect and follow biomarker changes similar to thoseobserved in humans. Non-invasive biomarkers, such as those provided by magnetic resonance imaging (MRI) and spectroscopy(MRS) methods are highly desirable, however, systematic studies ofin vivoMRI/MRS methods to characterize the cerebralmorphology and metabolic pattern of these mice remain scarce. We investigated sixteen consecutive slices from the brain ofwild-type and A PP/PS1 mice, obtaining a collection of T2-weighted, diffusion weighted and magnetization transfer weightedimages as well as1H PRESS spectra from the cortical and subcortical areas. Compared to controls, A PP/PS1 mice showsignificant regional hyperintensities in T2-weighted images of the cerebral cortex, significant ventricular enlargement, anddecreased hippocampal area and fractional magnetization transfer. MRS demonstrated an increase in the ratio of choline (Cho)to creatine (Cr) in the cortical and subcortical areas of the transgenic animals. A logistic regression classifier was implementedconsidering all parameters investigated, and revealed the most characteristic changes and allowed for the correct classification ofcontrol and A PP/PS1 mice. In summary, the present results provide a useful frame to evaluate optimal MRI/MRS biomarkersfor the characterization of AD models, potentially applicable in drug discovery processes, because of their non-invasive andrepeatable nature in longitudinal studies., This work has been supported by grants from Ministerio de Ciencia e Innovación (SAF2007-624505 andoSAF2010-1700) and Fundación Eugenio Rodríguez Pascual to A.M.R., grants from Instituto de SaludCarlos III (PI060155), Fundación Investigación Médica Mutua Madrileña (2006.125, 2008.93), and CIBERNED to E.C., grants SAF 2008-01327 andBIO2006-170 from Ministerio de Ciencia e Innovación and the Comunidad Autónoma de Madrid to S.C. N.E. holds a fellowship of the JAE predoctoral program of the CSIC

Glioblastoma multiforme (GBM) is one of the deadliest forms of cancer, with an average survival time of approximately 1 year despite aggressive surgery, radiotherapy and chemotherapy. Here, we report a preclinical study by which the two main energy pathways of the tumor cells, oxidative phosphorylation and aerobic glycolysis, are simultaneously disrupted. The therapy is based on a plant gene encoding a β-glucosidase, linamarase (lis), which react with the substrate linamarin (lin) producing cyanide. We also use glucose oxidase (GO) to enhance oxidative stress and to induce cell death in the tumor. To test in vivo this suicide gene therapy system (lis/lin/GO), we used an orthotopic model of the human U87MG glioma cells, genetically modified to express the lis gene, and stereotactically implanted into the brains of nude rats (rnu/rnu). Despite its genetic condition, 6% of the animals immunorejected the xenotransplanted cells giving false curative results. We tried several delivery methods with limited success. The therapeutic cocktail, at dosages that perhaps eliminated the brain tumors, is too toxic for the animal causing its premature death., This work was supported by funds from: FINA Biotech; Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo (PI06/0554) Spain; Ministerio de Ciencia e Innovación (SAF 2009-07259) Spain, and Fundación Eugenio Rodriguez Pascual (Madrid). The Centro de Biología Molecular S.O. is also the recipient of an institutional grant from the Ramón Areces Foundation. A.C. was the recipient of a grant MAEC-AECID, Spain.

Livers isolated from fasted rats were perfused in a blood-free recirculating system using alanine (10 mm) as the carbon source. Glucagon at a concentration of 2.1 × 10−9m enhanced gluconeogenesis, ureogenesis, and ketogenesis. The proportion of alanine utilized to glucose formed remained rather constant in all the situations studied, suggesting that the contribution of glycogen breakdown to the total glucose output was negligible. The glucagon stimulation of gluconeogenesis was accompanied by a decrease in the [ATP]/[ADP]ratio and a rise in the reduction state of the cytosolic and mitochondrial NAD systems. The calculation of the intracellular distribution of metabolites indicates that glucagon increases the intramitochondrial oxaloacetate concentration. This finding seems to support the hypothesis of pyruvate carboxylation, the first nonequilibrium enzymic step in the gluconeogenic sequence, as one of the main sites of glucagon action. The rise in the mitochondrial:cytosolic concentration gradient of malate suggests that glucagon may also act by facilitating the transfer of three-carbon units from the mitochondria to the cytosol. The fact that insulin reversed virtually all the glucagon-induced changes strongly suggests that both hormones act on common steps. It is remarkable that these insulin effects occur at glucagon/insulin ratios similar to those normally found in the portal vein of the intact animal., This work has been supported by grants from Lilly Indiana S.A., Fundación Rodríguez Pascual and Comisión Asesora para el Desarrollo de la Investigación

The metabolic effects of glucagon and glucagon plus insulin on the isolated rat livers perfused with 10 mM sodium L‐lactate as substrate were studied. Glucagon stimulated gluconeogenesis, ketogenesis and ureogenesis at the concentration used of 2.1 nM. The addition of insulin to give a glucagon‐to‐insulin ratio of 0.2 reversed all the glucagon effects. The glucagon enhancement of gluconeogenesis was accompanied by a rise in the cytosolic and mitochondrial state of reduction of the NAD system and a fall in the [ATP]/[ADP] ratio. The analysis of the intermediary metabolite concentrations suggested, as possible sites of glucagon action, the steps between pyruvate and phosphoenolpyruvate as well as the reactions catalyzed by phosphofructokinase and/or fructose bisphosphatase. All the changes in metabolite contents were abolished when insulin was present. Glucagon increased the intramitochondrial concentration of all the metabolites, whose intracellular distribution was calculated. The finding of a significant rise in the calculated intramitochondrial concentration of oxaloacetate points to pyruvate carboxylation as an important site of glucagon interaction with the gluconeogenic pathway. A primary event in the glucagon action redistributing intracellular metabolites seems to be the mitochondrial entry of malate. The possibility is discussed that the changes in metabolite cellular distribution were brought about by the increased cellular state of reduction caused by the hormone., This work has been supported by grants from Lilly Indiana S. A.. Fundación Rodríguez Pascual and Comisión Asesora para el Desarrollo de la Investigación.

The relationship between the cellular energetic state and glucose uptake by isolated lung cells has been studied by perturbing the oxidative metabolism by anoxia, treatment with uncouplers of the oxidative phosphorylation, inhibitors of the respiratory chain or inhibitors of the adenine nucleotide translocation across the mitochondrial membrane. Anoxia did not produce a significant decrease in the cellular ATP content or total adenine nucleotides. Inhibitors of the respiratory chain produced a fall of 60% in ATP content and a significant decrease in the [ATP]/[ADP] ratio. Dinitrophenol or atractyloside decreased ATP by 30% and produced also a milder decrease in the [ATP]/[ADP] ratio. Under none of these situations the glucose uptake was significantly enhanced, however, the percentage of glucose accounted for as lactate was increased. These observations, together with the fact that the intracellular glucose concentration was undetectable, seem to indicate that the glucose transport into cells, not the glycolytic flux as previously suggested, is the rate limiting factor for its utilization, and that both events, glucose transport into the cells and glucose breakdown, are not under the same control mechanism. Anoxia, although it does not accelerate significantly the glucose uptake when a saturating dose of glucose was used, acts on lung cells lowering the apparent 'K(m)' for glucose utilization. This effect, nevertheless, may not be very important from a physiological point of view since the 'K(m)' for glucose uptake is well below the plasma glucose concentration., This work was supported by grants from Fundación Rodríguez Pascual, Comisión Asesora para el desarrollo de la Investigación and Essex España, S.A.J.P.D. and A.M.R. are recipients of research, fellowship from Spanish Secretary of Education and Science.

Collagenase digestion of minced lung tissue yielded isolated cells, functionally viable as judged by several metabolic and morphological criteria, representative of all the cell species normally present in the tissue. The efficiency of the isolation procedure was about 25 per cent. Aerobic metabolism was not affected by most of the substrates tested except by succinate which increased oxygen utilization, and glucose, fructose and octanoate which significantly decreased oxygen uptake. Since no significant changes have been observed in the cellular adenine nucleotide content during glucose depression of aerobic metabolism it is concluded that the glycolytic flux had to be sufficient as to account for the decrease in the mitochondrial energy production. The mechanism responsible for these effects as well as their physiological significance are discussed herewith., This work has been supported in part by grants from Fundación Rodríguez Pascual and Comisión Asesora para el Desarrollo de la Investigación. J. P. D. is recipient of a research fellowship from the Spanish Secretary of Education and Science.

Glucose decreases the oxygen utilization by isolated rat lung cells. Its effect displays saturation type kinetics with a “Ki” of 2.2. mM. The similarity of this value with the reported “Km” of 2.4 mM described for glucose uptake by these cells, suggests that both processes may be intimately related and both of them are under the control of the same rate limiting step. Several arguments point to glucose transport into these cells as the most important rate limiting step for its utilization: 1) Phloridzin prevented glucose inhibition of oxygen uptake while mannoheptulose did not; 2) The activity of hexokinase which is the least active glycolytic enzyme in these cells far exceeded the observed rates of glucose utilization and a decrease of 45 per cent in its activity in starved animals did not affect the rate of glucose uptake; 3) The “Km” of hexokinase for glucose is two orders of magnitude below the observed “Km” for glucose uptake and the “Ki” for glucose inhibition of respiration., This work was supported by grants from Fundación Rodríguez Pascual, Essex (Espana) S.A, and Comisión Asesora para el Desarrollo de la Investigación, J,P .D . and A,M.R . are recipients of research fellowships from the Spanish Secretary of Education and Science.

[Background]: Hyperglycemia is acknowledged as a pro-inflammatory condition and a major cause of vascular damage. Nevertheless, we have previously described that high glucose only promotes inflammation in human vascular cells previously primed with pro-inflammatory stimuli, such as the cytokine interleukin (IL)1β. Here, we aimed to identify the cellular mechanisms by which high glucose exacerbates the vascular inflammation induced by IL1β. [Methods]: Cultured human aortic smooth muscle cells (HASMC) and isolated rat mesenteric microvessels were treated with IL1β in medium containing 5.5-22 mmol/L glucose. Glucose uptake and consumption, lactate production, GLUT1 levels, NADPH oxidase activity and inflammatory signalling (nuclear factor-ΚB activation and inducible nitric oxide synthase expression) were measured in HASMC, while endothelium-dependent relaxations to acetylcholine were determined in rat microvessels. Pharmacological inhibition of IL1 receptors, NADPH oxidase and glucose-6-phosphate dehydrogenase (G6PD), as well as silencing of G6PD, were also performed. Moreover, the pentose phosphate pathway (PPP) activity and the levels of reduced glutathione were determined. [Results]: We found that excess glucose uptake in HASMC cultured in 22 mM glucose only occurred following activation with IL1β. However, the simple entry of glucose was not enough to be deleterious since over-expression of the glucose transporter GLUT1 or increased glucose uptake following inhibition of mitochondrial respiration by sodium azide was not sufficient to trigger inflammatory mechanisms. In fact, besides allowing glucose entry, IL1β activated the PPP, thus permitting some of the excess glucose to be metabolized via this route. This in turn led to an over-activation NADPH oxidase, resulting in increased generation of free radicals and the subsequent downstream pro-inflammatory signalling. Moreover, in rat mesenteric microvessels high glucose incubation enhanced the endothelial dysfunction induced by IL1β by a mechanism which was abrogated by the inhibition of the PPP. [Conclusions]: A pro-inflammatory stimulus like IL1β transforms excess glucose into a vascular deleterious agent by causing an increase in glucose uptake and its subsequent diversion into the PPP, promoting the pro-oxidant conditions required for the exacerbation of pro-oxidant and pro-inflammatory pathways. We propose that over-activation of the PPP is a crucial mechanism for the vascular damage associated to hyperglycemia., This study was supported with grants from Plan Nacional de I+ D (SAF2014-52762-R), Sociedad Española de Farmacología-Almirall, Fundación Eugenio Rodríguez Pascual, and ISCIII (RETICEF-R12/0043/0021) and Junta de Castilla y León (SA003U13). LV was supported by a fellowship from CONACYT (Mexico). TR is the recipient of a Marie Curie Intra-European Fellowship (2012-IEF-328793 ADDIO).