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Objectives: To make recommendations on the diagnosis and treatment of post-extubation laryngitis (PEL) in children with or without other comorbidities. Methods: A three-iterative modified Delphi method was applied. Specialists were recruited representing pediatric otolaryngologists, pediatric and neonatal intensivists. Questions and statements approached topics encompassing definition, diagnosis, endoscopic airway evaluation, risk factors, comorbidities, management, and follow-up. A consensus was defined as a supermajority >70%. Results: Stridor was considered the most frequent symptom and airway endoscopy was recommended for definitive diagnosis. Gastroesophageal reflux and previous history of intubation were considered risk factors. Specific length of intubation did not achieve a consensus as a risk factor. Systemic corticosteroids should be part of the medical treatment and dexamethasone was the drug of choice. No consensus was achieved regarding dosage of corticosteroids, although endoscopic findings help defining dosage and length of treatment. Non-invasive ventilation, laryngeal rest, and use of comfort sedation scales were recommended. Indications for microlaryngoscopy and bronchoscopy under anesthesia were symptoms progression or failure to improve after the first 72-h of medical treatment post-extubation, after two failed extubations, and/or suspicion of severe lesions on flexible fiberoptic laryngoscopy. Conclusions: Management of post-extubation laryngitis is challenging and can be facilitated by a multidisciplinary approach. Airway endoscopy is mandatory and impacts decision-making, although there is no consensus regarding dosage and length of treatment.

OBJETIVOS: Verificar a frequência de hipomagnesemia em pacientes pediátricos submetidos a artrodese de coluna, avaliando se há queda significativa nas dosagens de magnésio sérico nos períodos pré e pós-operatório, quais suas possíveis causas e quais as consequências clínicas para os pacientes. MÉTODOS: Estudo retrospectivo e descritivo dos pacientes admitidos em uma Unidade de Terapia Intensiva pediátrica no pós-operatório de artrodese de coluna, no período de 1º de março a 31 de agosto de 2011. Foram comparados os níveis de magnésio, fósforo, cálcio total e ionizado no pré-operatório com os valores encontrados após a admissão na Unidade de Terapia Intensiva. RESULTADOS: Foram incluídos 45 pacientes, com idade média de 13,1 anos. No pré-operatório, o valor médio do magnésio foi de 1,8±0,2 mg/dL, e no pós-operatório, de 1,4±0,2 mg/dL, o que demonstra uma queda significativa entre os dois períodos (p < 0,001). A frequência de hipomagnesemia foi de somente um paciente (2%) no pré-operatório para 31 (68%) no pós-operatório. Houve também queda significativa nos níveis de fósforo (p < 0,001) e cálcio total (p < 0,001). Houve correlação significativa entre a queda do magnésio e o volume de fluido recebido durante a cirurgia (p = 0,03), volume de transfusão sanguínea (p < 0,001) e número de vértebras fixadas (p < 0,05). Dos 31 pacientes com hipomagnesemia, sete (22%) apresentaram sintomas. CONCLUSÃO: Existe uma elevada frequência de hipomagnesemia em pacientes submetidos a artrodese de coluna. Ao realizar a dosagem do magnésio sérico no momento da admissão na Unidade de Terapia Intensiva, a reposição adequada pode ser prontamente iniciada, minimizando o risco de complicações.

OBJETIVO: Apresentar uma rotina de atendimento para crianças submetidas à cirurgia cardíaca. FONTES DOS DADOS: Realizada revisão bibliográfica através de bancos de dados (Medline, Mdconsult, PubMed), analisando as condutas sugeridas por diversos serviços fora do Brasil e comparando com a rotina de atendimento no Hospital Infantil Pequeno Príncipe, de Curitiba, onde foram realizadas cerca de 8.000 cirurgias cardíacas desde 1977 até abril de 2003. SÍNTESE DOS DADOS: O serviço citado é referência em cardiologia e cirurgia cardíaca no estado do Paraná e estados vizinhos. A evolução das condições de diagnóstico, preparo da equipe clínica e cirúrgica, unidade de terapia intensiva (UTI) melhor equipada com monitorização mais avançada, equipe da UTI com pessoal treinado em todas as áreas para fazer pós-operatório de cirurgia cardíaca, estrutura hospitalar adequada, oferecendo atendimento avançado em todas as especialidades pediátricas e paramédicas, fazem com que o resultado das intervenções cirúrgicas realizadas em crianças com cardiopatias congênitas ou adquiridas, principalmente nos recém-nascidos e lactentes jovens com cardiopatias complexas, apresente sensível melhora quando comparado com anos anteriores. CONCLUSÃO: As crianças com cardiopatias, principalmente as complexas, devem ser encaminhadas para um local que seja centro de referência, onde haja condições para um atendimento global no pré, per e pós-operatório.OBJECTIVE: To present a care routine for children submitted to heart surgery. SOURCE OF DATA: Literature review of Medscape, MD Consult and PubMed. Analysis of the suggested conducts adopted by various services from different countries and comparison with the care routine at the Pequeno Príncipe Children's Hospital (Curitiba, state of Paraná), where approximately 8,000 heart surgeries were performed in children from 1977 to April 2003. SUMMARY OF THE FINDINGS: Our hospital is a reference center for Cardiology and Heart Surgery in the state of Paraná and neighboring states. The improvement of conditions for diagnosis, training of the clinical and surgical teams, better equipped ICU with more modern monitoring, training of ICU personnel from all areas to handle the post-operative requirements of patients submitted to heart surgery, an adequate hospital structure with advanced care in all pediatric and paramedic specialties have resulted in marked improvement in relation to previous years in terms of the results of surgical interventions in children with congenital or acquired heart disease, especially newborns and young babies with complex heart problems. CONCLUSION: Children with heart diseases, especially complex conditions, should be receive care at reference centers that can provide global care before, during and after the surgery.

The advent of new psychoactive substances (NPS) has caused enormous difficulty for legal control since they are rapidly commercialized, and their chemical structures are routinely altered. In this aspect, derivatives phenethylamines, such as 25E-NBOH, have received great attention in the forensic scenario. Hence, we propose portable and cost-effective (U$ 5.00) 3D-printed devices for the electrochemical screening of 25E-NBOH for the first time. The cell and all electrodes were printed using acrylonitrile butadiene styrene filament (insulating material) and conductive filament (graphite embedded in a polylactic acid matrix), respectively, both by the fused deposition modeling (FDM) 3D printing technique. The electrochemical apparatus enables micro-volume analysis (50-2000 μL), especially important for low sample volumes. A mechanistic route for the electrochemical oxidation of 25E-NBOH is proposed based on cyclic voltammetric data, which showed two oxidation processes around +0.75 V and +1.00 V and a redox pair between +0.2 and -0.2 V (vs. graphite ink pseudo-reference). A fast and sensitive square-wave voltammetry method was developed, which exhibited a linear working range from 0.85 to 5.1 μmoL -1 , detection limit of 0.2 μmol L -1 , and good intra-electrode precision (n = 10, RSD <5.3 %). Inter-electrode measurements (n = 3, RSD <9.8 %) also attested that the electrode production process is reproducible. Interference tests in the presence of other drugs frequently found in blotting paper indicated high selectivity of the electrochemical method for screening of 25E-NBOH. Screening analysis of blotting paper confirmed the presence of 25E-NBOH in the seized samples. Moreover, a recovery percentage close to 100 % was found for a spiked saliva sample, suggesting the method's usefulness for quantitative purposes aimed at information on recent drug use., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fused deposition modeling (FDM) 3D printing is a promising additive manufacturing technique to produce low-cost disposable electrochemical devices. However, the print of devices like well-known screen-printed electrodes (all electrodes on the same device) is difficult using the available technology (few materials available for production of working electrodes). In this paper we present a procedure to produce disposable and robust electrochemical devices by FDM 3D printing that allows reproducible analysis of small volumes (50-2000 μL). The device consists of just two printed parts that allow easy coupling of different conductive materials for using as disposable or non-disposable working electrodes with reproducible geometric area. Printed counter and pseudo-reference electrodes can also be easily fitted into the microcell. Moreover, conventional counter (platinum wire) and mini reference electrodes can also be used. As a proof of concept, paracetamol, cocaine and uric acid were used as model analytes using different materials as working electrodes. Linear calibration curves (r > 0.99) with similar slopes (0.29 ± 0.01 μA μmol L -1 ; RSD = 3.4%) were obtained by square wave voltammetry (SWV) using a complete printed system and different volumes of standard solutions of paracetamol (50, 100, and 200 μL). For uric acid, a linear range of 10-125 μmol L -1 (r > 0.99), was obtained using differential pulse voltammetry as the electrochemical technique and a disposable laser-induced graphene base as the working electrode. With the coupling of boron-doped diamond working electrode, screening tests were successfully performed in seized cocaine samples with selective detection of cocaine in the presence of its most common adulterants. The production cost per unit of a complete electrochemical system is around US 5.00. In large-scale production, only the working electrode needs to be replaced while the microcell and counter/pseudo reference electrodes do not need to be discarded., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In recent times, bladder cancer has increased significantly in terms of incidence and mortality. Currently, two subtypes are known based on tumour growth: non-muscle invasive (NMIBC) and muscle-invasive bladder cancer (MIBC). In this work, we focus on the MIBC subtype because it has the worst prognosis and can spread to adjacent organs. We present a self-learning framework to grade bladder cancer from histological images stained by immunohistochemical techniques. Specifically, we propose a novel Deep Convolutional Embedded Attention Clustering (DCEAC) which allows for the classification of histological patches into different levels of disease severity, according to established patterns in the literature. The proposed DCEAC model follows a fully unsupervised two-step learning methodology to discern between non-tumour, mild and infiltrative patterns from high-resolution 512 × 512 pixel samples. Our system outperforms previous clustering-based methods by including a convolutional attention module, which enables the refinement of the features of the latent space prior to the classification stage. The proposed network surpasses state-of-the-art approaches by 2-3% across different metrics, reaching a final average accuracy of 0.9034 in a multi-class scenario. Furthermore, the reported class activation maps evidence that our model is able to learn by itself the same patterns that clinicians consider relevant, without requiring previous annotation steps. This represents a breakthrough in MIBC grading that bridges the gap with respect to training the model on labelled data., (Copyright © 2021. Published by Elsevier Ltd.)

Background & Aims: Perturbations of intracellular magnesium (Mg 2+ ) homeostasis have implications for cell physiology. The cyclin M family, CNNM, perform key functions in the transport of Mg 2+ across cell membranes. Herein, we aimed to elucidate the role of CNNM4 in the development of non-alcoholic steatohepatitis (NASH)., Methods: Serum Mg 2+ levels and hepatic CNNM4 expression were characterised in clinical samples. Primary hepatocytes were cultured under methionine and choline deprivation. A 0.1% methionine and choline-deficient diet, or a choline-deficient high-fat diet were used to induce NASH in our in vivo rodent models. Cnnm4 was silenced using siRNA, in vitro with DharmaFECT and in vivo with Invivofectamine® or conjugated to N-acetylgalactosamine., Results: Patients with NASH showed hepatic CNNM4 overexpression and dysregulated Mg 2+ levels in the serum. Cnnm4 silencing ameliorated hepatic lipid accumulation, inflammation and fibrosis in the rodent NASH models. Mechanistically, CNNM4 knockdown in hepatocytes induced cellular Mg 2+ accumulation, reduced endoplasmic reticulum stress, and increased microsomal triglyceride transfer activity, which promoted hepatic lipid clearance by increasing the secretion of VLDLs., Conclusions: CNNM4 is overexpressed in patients with NASH and is responsible for dysregulated Mg 2+ transport. Hepatic CNNM4 is a promising therapeutic target for the treatment of NASH., Lay Summary: Cyclin M4 (CNNM4) is overexpressed in non-alcoholic steatohepatitis (NASH) and promotes the export of magnesium from the liver. The liver-specific silencing of Cnnm4 ameliorates NASH by reducing endoplasmic reticulum stress and promoting the activity of microsomal triglyceride transfer protein., Competing Interests: Conflicts of interest The authors declare no conflicts of interest related to this submitted work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. All rights reserved.)

Constructed wetlands are an alternative biotechnology for wastewater treatment that have several advantages over conventional systems. In this work, a biokinetic model for surface flow constructed wetlands is presented (SURFWET). SURFWET belongs to a class of models that are not only interesting from a theoretical viewpoint, as they allow to improve the understanding of the underlying processes; but also from a practical viewpoint, because they can be useful for optimal designs of constructed wetlands, complementing current empirical methods. The proposed model is centered on the intervening physical and biochemical processes involved in pollutant removal in wastewater (organic matter, nitrogen, phosphorus, suspended solids), capturing the interplay of the main agents on contaminant removal (bacteria, macrophytes and phytoplankton). Furthermore, the hydraulic model considers water volume as a variable depending on the outlet hydraulic capacity, and dissolved oxygen has also been introduced as a key driver of reaction kinetics of wetlands. Beyond putting forward a theoretical framework, SURFWET has been applied to simulate a specific case to demonstrate its robustness, in a 12-year-interval simulation. The results show the typical seasonality of this biotechnology, highlighting the importance of dissolved oxygen, which is a key limiting factor on a large number of biochemical processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The modeling of free-water surface constructed wetlands (FWS-CWs) provides an improved understanding of their processes and constitutes a useful tool for the design and management of these systems. In this work, a dynamic simulation model for FWS-CWs was developed and used to simulate the operation of a FWS-CW proposed for improving the treatment of sewage effluents entering the Tablas de Daimiel National Park in central Spain. The process-based model simulates carbon, nitrogen and phosphorus dynamics, including key hydrological processes for wetlands under a fluctuating Mediterranean semiarid climate. The model allows for the simulation of the operation of FWS-CWs with variable flooding regimes, relating the surface water level to the flooded area and the water outflow. Simulations of the proposed FWS-CW under different water management schemes and scenarios were run, and the consequences of those management strategies on the treatment efficiency were analyzed. Under the Mediterranean climate and geology of the study area, namely, high water losses through evapotranspiration and infiltration, the decrease in nutrient concentrations was higher when the flooded area was reduced in summer than when a constant flooded area was maintained. Moreover, the meteorological variability introduced in different scenarios produced different results in terms of water outflow, but differences in terms of nutrient concentrations were not significant. The ability of the model to simulate different hydrological scenarios and their consequences on water quality makes it a useful decision-support tool., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chronic exposure to heavy metals such as Pb, As, and MeHg can be associated with an increased risk of developing neurodegenerative diseases. Our in vitro bioassays results showed the potency of heavy metals in the order of Pb < As < MeHg on hippocampal cells. The main objective of this study was combining in vitro label free proteomics and systems biology approach for elucidating patterns of biological response, discovering underlying mechanisms of Pb, As, and MeHg toxicity in hippocampal cells. The omics data was refined by using different filters and normalization and multilevel analysis tools were employed to explore the data visualization. The functional and pathway visualization was performed by using Gene ontology and PathVisio tools. Using these all integrated approaches, we identified significant proteins across treatments within the mitochondrial dysfunction, oxidative stress, ubiquitin proteome dysfunction, and mRNA splicing related to neurodegenerative diseases. The systems biology analysis revealed significant alterations in proteins implicated in Parkinson's disease (PD) and Alzheimer's disease (AD). The current proteomics analysis of three metals support the insight into the proteins involved in neurodegeneration and the altered proteins can be useful for metal-specific biomarkers of exposure and its adverse effects., Significance: The proteomics techniques have been claimed to be more sensitive than the conventional toxicological assays, facilitating the measurement of responses to heavy metals (Pb, As, and MeHg) exposure before obvious harm has occurred demonstrating their predictive value. Also, proteomics allows for the comparison of responses between Pb, As, and MeHg metals, permitting the evaluation of potency differences hippocampal cells of the brain. Hereby, the molecular information provided by pathway and gene functional analysis can be used to develop a more thorough understanding of each metal mechanism at the protein level for different neurological adverse outcomes (e.g. Parkinson's disease, Alzheimer's diseases). Efforts are put into developing proteomics based toxicity testing methods using in vitro models for improving human risk assessment. Some of the key proteins identified can also potentially be used as biomarkers in epidemiologic studies. These heavy metal response patterns shed new light on the mechanisms of mRNA splicing, ubiquitin pathway role in neurodegeneration, and can be useful for the development of molecular biomarkers of heavy metals exposure., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The number of old people with breast cancer is estimated to increase during the next years in developed countries. However, management of breast cancer in octogenarians is not well established. The main objective was to evaluate if patients older than 80 years with breast cancer are receiving the most convenient treatment by using a population registry cancer analysis., Patients and Methods: A retrospective analysis of a population cancer registry was designed. Data were retrieved from the Castellon Cancer Registry (Valencian Community, Spain). Patient records were analyzed from January 1, 1995 to December 31, 2013. Two groups were defined: group A, <80 years; and group B, >= 80 years. Survival analyses were sequentially performed into three phases. First, a non-adjusted Kaplan Meier analysis was conducted. For the second survival analyses, Cox's proportional hazards model of Overall survival was used adjusting for condensed-TNM stage and adjuvant treatments. Finally, the third specific adjustment was carried out adding information of life expectancy by age for Spanish women, corresponding to year 2008 with condensed-TNM stage and Propensity Score variable, as an approximation to relative or disease-specific survival., Results: The total number of included patients was 1304. Women ≥80 years presented a more extended disease, larger tumors and less in situ carcinomas. A lower proportion of women ≥80 years received adjuvant therapies. In the absence of any adjustment of results, the overall survival in women ≥80 years was poorer than in younger patients (median of 14.1 years for <80y vs. 5.7 years for ≥80y), the crude HR was 4.6 (95% CI: 2.9-7.5) p < 0.001. For second survival analysis, the HR was 2.5 (95% CI: 1.8-3.5) p < 0.001. After the third adjustment the HR was 1.7 (95% CI: 1.2-2.4) p = 0.004., Conclusions: Octogenarians with operable breast cancer are receiving suboptimal treatments, which can have repercussions on survival. New studies are required to identify a subgroup of women age ≥80 years who may benefit from more aggressive treatment and a population of older women on the basis of tumor characteristics, comorbidities and life expectancy who may not need as aggressive treatment., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Humans are exposed to a cocktail of heavy metal toxicants in the environment. Though heavy metals are deleterious, there is a paucity of information on the toxicity of mixtures. In this study, four common neurotoxicity heavy metals lead (Pb) cadmium (Cd), arsenic (As), and methylmercury (MeHg) were exposed individually and as mixtures to HT-22 cell line for 8days. The study established that low dose exposures induced toxicity to the HT-22 cell line during 8days. The results indicates potency dependent response, the toxicity of single metals on the HT-22 cells; MeHg > As > Cd > Pb. The cytotoxicity data of single metals were used to determine the mixtures interaction profile by using the dose additivity and effect additivity method. Metal mixtures showed higher toxicities compared to individual metals. Synergistic, antagonistic or additive effects of the toxicity were observed in different mixtures in low dose exposure. The interactive responses of mixtures depend on the co-exposure metal and their respective concentration. We concluded that the combined effects should be considered in the risk assessment of heavy metal co-exposure and potency. In future, comprehensive mechanistic based investigations needed for understanding the real interactive mixtures effects at molecular level., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Constructed wetlands are currently recognized as an effective environmental biotechnology for wastewater treatment, but the influence of their design parameters on internal functioning and contaminant removal efficiency is still under discussion. In this work, the effect of aspect ratio and water depth on bacteria communities as well as treatment efficiency of horizontal subsurface flow constructed wetlands (HSSF) under the Mediterranean climate was evaluated, using a mathematical model. For this purpose, experimental results from four pilot-scale wetlands of equal surface area but different aspect ratios and water depth were used. The HSSF system was fed with municipal wastewater. The experimental data were simulated using the BIO&#95;PORE model, developed in the COMSOL Multiphysics™ platform. Simulations with the BIO&#95;PORE model fitted well to the experimental results, showing a higher removal efficiency for the shallower HSSF for COD (93.7% removal efficiency) and ammonia nitrogen (73.8%). The aspect ratio had a weak relationship with the bacteria distribution and the removal efficiency. In contrast, the water depth was a factor. The results of the present study confirm a previous hypothesis in which depth has an important impact on the biochemical reactions causing contaminants transformation and degradation., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aims: The reduction of delay times as well as the rate of false alarms (FA) have become some of the main points of the different infarction networks. We propose a simple way of classifying patients derived for primary PCI (pPCI) into well-defined simple groups by colors, where we can assess real delays of each clinical presentation, define the FA and, furthermore, establish their immediate and short term prognosis., Methods and Results: Prospective study of STEMI consecutive patients derived for pPCI during 2014. Patients were categorized into one of the 3 predesigned groups [(i) Green: diagnostic-ECG with compatible clinical presentation for pPCI; (ii) Yellow: LBBB, pacemaker rate or non-diagnostic ECG; and (iii) Red: very complex patients], always before performing the angiography in 518 patients. Delay times were highest in the Yellow group, with much longer first medical contact (FMC) to balloon time (median Green 118'; Yellow 163'; Red 130'; p<0.001) mainly due to higher times from the first medical contact to the diagnosis and team activation (median Green 30'; Yellow 70'; Red 39'; p<0.001). In the whole cohort, pPCI was performed in 80.2% of patients, with 11.9% of FA. The Green group had only a 2.5% FA rate, in contrast to the Yellow group where FA were 43.2%., Conclusions: This simple classification differentiates the 3 very clear groups in which delay times and prognosis are very different. This classification allows us to measure, evaluate and compare the performance of each of our pPCI networks with others and within different periods of times., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background and Objectives: The relationship between mineral metabolism disorders, bone fractures and vascular calcifications in kidney transplant recipients has not been established., Method: We performed a cross-sectional study in 727 stable recipients from 28 Spanish transplant clinics. Mineral metabolism parameters, the semi-quantification of vertebral fractures and abdominal aortic calcifications were determined centrally., Results: Vitamin D deficiency (25OHD3<15ng/ml) was more common in female recipients at CKD-T stages I-III (29.6% vs 44.4%; p=0.003). The inverse and significant correlation between 25OHD3 and PTH was gender-specific and women exhibited a steeper slope than men (p=0.01). Vertebral fractures (VFx) with deformity grade ≥2 were observed in 15% of recipients. Factors related to VFx differed by gender; in males, age (OR 1.04; 95% CI 1.01-1.06) and CsA treatment (OR: 3.2; 95% CI: 1.6-6.3); in females, age (OR 1.07; 95% CI: 1.03-1.12) and PTH levels (OR per 100pg/ml increase: 1.27; 95% CI: 1.043-1.542). Abdominal aortic calcifications were common (67.2%) and related to classical risk factors but not to mineral metabolism parameters., Conclusions: Vitamin D deficiency is more common among female kidney transplant recipients at earlier CKD-T stages, and it contributes to secondary hyperparathyroidism. Prevalent vertebral fractures are only related to high serum PTH levels in female recipients., (Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.)

Background & Aims: Glycine N-methyltransferase (GNMT) expression is decreased in some patients with severe non-alcoholic fatty liver disease. Gnmt deficiency in mice (Gnmt-KO) results in abnormally elevated serum levels of methionine and its metabolite S-adenosylmethionine (SAMe), and this leads to rapid liver steatosis development. Autophagy plays a critical role in lipid catabolism (lipophagy), and defects in autophagy have been related to liver steatosis development. Since methionine and its metabolite SAMe are well known inactivators of autophagy, we aimed to examine whether high levels of both metabolites could block autophagy-mediated lipid catabolism., Methods: We examined methionine levels in a cohort of 358 serum samples from steatotic patients. We used hepatocytes cultured with methionine and SAMe, and hepatocytes and livers from Gnmt-KO mice., Results: We detected a significant increase in serum methionine levels in steatotic patients. We observed that autophagy and lipophagy were impaired in hepatocytes cultured with high methionine and SAMe, and that Gnmt-KO livers were characterized by an impairment in autophagy functionality, likely caused by defects at the lysosomal level. Elevated levels of methionine and SAMe activated PP2A by methylation, while blocking PP2A activity restored autophagy flux in Gnmt-KO hepatocytes, and in hepatocytes treated with SAMe and methionine. Finally, normalization of methionine and SAMe levels in Gnmt-KO mice using a methionine deficient diet normalized the methylation capacity, PP2A methylation, autophagy, and ameliorated liver steatosis., Conclusions: These data suggest that elevated levels of methionine and SAMe can inhibit autophagic catabolism of lipids contributing to liver steatosis., (Copyright © 2015 European Association for the Study of the Liver. All rights reserved.)

Background & Aims: Very-low-density lipoproteins (VLDLs) export lipids from the liver to peripheral tissues and are the precursors of low-density-lipoproteins. Low levels of hepatic S-adenosylmethionine (SAMe) decrease triglyceride (TG) secretion in VLDLs, contributing to hepatosteatosis in methionine adenosyltransferase 1A knockout mice but nothing is known about the effect of SAMe on the circulating VLDL metabolism. We wanted to investigate whether excess SAMe could disrupt VLDL plasma metabolism and unravel the mechanisms involved., Methods: Glycine N-methyltransferase (GNMT) knockout (KO) mice, GNMT and perilipin-2 (PLIN2) double KO (GNMT-PLIN2-KO) and their respective wild type (WT) controls were used. A high fat diet (HFD) or a methionine deficient diet (MDD) was administrated to exacerbate or recover VLDL metabolism, respectively. Finally, 33 patients with non-alcoholic fatty-liver disease (NAFLD); 11 with hypertriglyceridemia and 22 with normal lipidemia were used in this study., Results: We found that excess SAMe increases the turnover of hepatic TG stores for secretion in VLDL in GNMT-KO mice, a model of NAFLD with high SAMe levels. The disrupted VLDL assembly resulted in the secretion of enlarged, phosphatidylethanolamine-poor, TG- and apoE-enriched VLDL-particles; special features that lead to increased VLDL clearance and decreased serum TG levels. Re-establishing normal SAMe levels restored VLDL secretion, features and metabolism. In NAFLD patients, serum TG levels were lower when hepatic GNMT-protein expression was decreased., Conclusions: Excess hepatic SAMe levels disrupt VLDL assembly and features and increase circulating VLDL clearance, which will cause increased VLDL-lipid supply to tissues and might contribute to the extrahepatic complications of NAFLD., (Copyright © 2014 European Association for the Study of the Liver. All rights reserved.)

Background: Triple negative breast cancers (negative estrogen receptor, progestagen receptor and no overexpression of HER2) seems to be more aggressive than other breast carcinoma subtypes. Therefore, it is necessary to analyze if a more aggressive surgical treatment should be offered to this subgroup of patients., Patients and Methods: The Castellon Cancer Registry Database (C.Valenciana, Spain) was used to identify eligible breast cancer patients. Female patients who were diagnosed and/or treated from January 2000 to December 2008 with primary, invasive, unilateral breast cancer at our hospital were included. A total of 410 patients make up the study population. Kaplan-Meier curves and log-rank tests were used to estimate 5-year local recurrence functions and to compare them across strata. Cox proportional hazards models were used to calculate hazard ratios and 95% confidence intervals to fit the effect of conservative surgery and other independent variables on local recurrence., Results: Median follow-up time was 60 months (1-127 months). A total of 21 patients (5%) presented a local recurrence during follow-up. There was a 9% difference in terms of local recurrence between conservative and non-conservative techniques for triple negative patients, whereas such difference was only 1% for no triple negative patients. On univariate analysis for local recurrence, only tumor size and lymph node metastasis were statistically associated with local recurrence. All other variables (type of surgery, triple negative status, molecular classification, tumor grade, age, adjuvant treatments, and total number of analyzed lymph nodes) were not statistically significant. On multivariate analysis, independent prognostic factors were breast conservative surgery (HR 4.62 95%CI 1.12-16.82), number of positive lymph nodes (HR 1.07 95%CI 1.01-1.17) and millimetre tumor size (HR 1.02 95%CI 1.01-1.06). In contrast, triple negative status trended to be a risk factor but without statistical significance (HR 1.59 95%CI 0.42-8.04)., Conclusions: It was not possible to find statistically significant differences between conservative and non-conservative surgery for triple negative breast cancer. However, a trend was observed for higher recurrence rates after breast conservative surgery for this group of patients. Prospective clinical trials are needed to confirm this observation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Sentinel node biopsy (SNB) is an effective alternative to axillary lymph node dissection (ALD) for axillary staging. SNB (test) needs a validation period in which ALD (the gold standard) is always performed. Sensitivity, specificity and predictive values (PV) are used to define the accuracy of the procedure. We hypothesise that, during the period of validation, a bias is produced if the result of SNB is included as a part of the ALD., Patients and Methods: A hypothetical population of 350 patients was analysed. First analyses were performed by including the sentinel lymph node as a part of 'the rest of the axilla'. Second analyses were re-done according to our theory, and sentinel lymph node was considered outside 'the rest of the axilla'. Sensitivity, specificity and PV were compared for both models., Results: First group (classic) - sensitivity: 94%; specificity: 100%; positive PV: 100%; negative PV: 97%. Second group (new proposed model) - sensitivity: 87%; specificity: 81%; positive PV: 44%; negative PV: 97%., Conclusion: The classic concept of sentinel lymph node to calculate sensitivity, specificity and positive PV can result in a bias. The magnitude of this bias will vary in terms of the obtained values, but its direction is always optimistic.