Autophagic Marker MAP1LC3B Expression Levels Are Associated with Carotid Atherosclerosis Symptomatology

Objectives: The mechanism by which atheroma plaque becomes unstable is not completely understood to date but analysis of differentially expressed genes in stable versus unstable plaques may provide clues. This will be crucial toward disclosing the mechanistic basis of plaque instability, and may help to identify prognostic biomarkers for ischaemic events. The objective of our study was to identify differences in expression levels of 59 selected genes between symptomatic patients (unstable plaques) and asymptomatic patients (stable plaques). Methods: 80 carotid plaques obtained by carotid endarterectomy and classified as symptomatic (>70% stenosis) or asymptomatic (>80% stenosis) were used in this study. The expression levels of 59 genes were quantified by qPCR on RNA extracted from the carotid plaques obtained by endarterectomy and analyzed by means of various bioinformatic tools. Results: Several genes associated with autophagy pathways displayed differential expression levels between asymptomatic and symptomatic (i.e. MAP1LC3B, RAB24, EVA1A). In particular, mRNA levels of MAP1LC3B, an autophagic marker, showed a 5-fold decrease in symptomatic samples, which was confirmed in protein blots. Immune system-related factors and endoplasmic reticulum-associated markers (i.e. ERP27, ITPR1, ERO1LB, TIMP1, IL12B) emerged as differently expressed genes between asymptomatic and symptomatic patients. Conclusions: Carotid atherosclerotic plaques in which MAP1LC3B is underexpressed would not be able to benefit from MAP1LC3B-associated autophagy. This may lead to accumulation of dead cells at lesion site with subsequent plaque destabilization leading to cerebrovascular events. Identified biomarkers and network interactions may represent novel targets for development of treatments against plaque destabilization and thus for the prevention of cerebrovascular events.

The E2F2 Transcription Factor Sustains Hepatic Glycerophospholipid Homeostasis in Mice

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified "lipid metabolism regulation" as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2(+/+)) and E2F2 deficient (E2F2(-/-)) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2(+/+) and E2F2(-/-) mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2(-/-) mice resembles the phenotype of proliferating E2F2(+/+) liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance.

Advantages and Versatility of Fluorescence-Based Methodology to Characterize the Functionality of LDLR and Class Mutation Assignment

Familial hypercholesterolemia (FH) is a common autosomal codominant disease with a frequency of 1:500 individuals in its heterozygous form. The genetic basis of FH is most commonly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of I-125-labeled lipoproteins (reference method) or fluorescent-labelled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations. LDLR activity of different variants has been studied by flow cytometry using FITC-labelled LDL and compared with studies performed previously with I-125-labeled lipoproteins. Flow cytometry results are in full agreement with the data obtained by the I-125 methodology. Additionally confocal microscopy allowed the assignment of different class mutation to the variants assayed. Use of fluorescence yielded similar results than I-125-labeled lipoproteins concerning LDLR activity determination, and also allows class mutation classification. The use of FITC-labelled LDL is easier in handling and disposal, cheaper than radioactivity and can be routinely performed by any group doing LDLR functional validations.

Apaf-1 Inhibitors Protect from Unwanted Cell Death in In Vivo Models of Kidney Ischemia and Chemotherapy Induced Ototoxicity

Background: Excessive apoptosis induces unwanted cell death and promotes pathological conditions. Drug discovery efforts aimed at decreasing apoptotic damage initially targeted the inhibition of effector caspases. Although such inhibitors were effective, safety problems led to slow pharmacological development. Therefore, apoptosis inhibition is still considered an unmet medical need. Methodology and Principal Findings: The interaction between Apaf-1 and the inhibitors was confirmed by NMR. Target specificity was evaluated in cellular models by siRNa based approaches. Cell recovery was confirmed by MTT, clonogenicity and flow cytometry assays. The efficiency of the compounds as antiapoptotic agents was tested in cellular and in vivo models of protection upon cisplatin induced ototoxicity in a zebrafish model and from hypoxia and reperfusion kidney damage in a rat model of hot ischemia. Conclusions: Apaf-1 inhibitors decreased Cytc release and apoptosome-mediated activation of procaspase-9 preventing cell and tissue damage in ex vivo experiments and in vivo animal models of apoptotic damage. Our results provide evidence that Apaf-1 pharmacological inhibition has therapeutic potential for the treatment of apoptosis-related diseases.

On the Dynamics of the Adenylate Energy System: Homeorhesis vs Homeostasis

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life.

Feature Selection for Speech Emotion Recognition in Spanish and Basque: On the Use of Machine Learning to Improve Human-Computer Interaction

Study of emotions in human-computer interaction is a growing research area. This paper shows an attempt to select the most significant features for emotion recognition in spoken Basque and Spanish Languages using different methods for feature selection. RekEmozio database was used as the experimental data set. Several Machine Learning paradigms were used for the emotion classification task. Experiments were executed in three phases, using different sets of features as classification variables in each phase. Moreover, feature subset selection was applied at each phase in order to seek for the most relevant feature subset. The three phases approach was selected to check the validity of the proposed approach. Achieved results show that an instance-based learning algorithm using feature subset selection techniques based on evolutionary algorithms is the best Machine Learning paradigm in automatic emotion recognition, with all different feature sets, obtaining a mean of 80,05% emotion recognition rate in Basque and a 74,82% in Spanish. In order to check the goodness of the proposed process, a greedy searching approach (FSS-Forward) has been applied and a comparison between them is provided. Based on achieved results, a set of most relevant non-speaker dependent features is proposed for both languages and new perspectives are suggested.

Novas formas da escrita de Clarice Lispector: o manuscrito Objeto gritante e a ficção tardia clariciana

Clarice Lispector foi uma das maiores escritoras brasileiras e, por essa razão, uma das mais estudadas. Muitas relações se fizeram entre sua escrita e a de alguns dos principais ícones da literatura mundial. Entretanto, o mesmo não aconteceu com seus textos produzidos aproximadamente a partir da década de 1970, a ficção derradeira da autora. As obras produzidas principalmente durante essa década, com algumas consagradas exceções, foram um tanto negligenciadas pela crítica. As pesquisadoras Marta Peixoto e Sônia Roncador procuraram provar que a pouca atenção às obras da ficção tardia da autora se deve ao fato de que as mesmas apresentam tendências que vão contra as características do que Clarice produziu anteriormente, como se a escritora tivesse desejado criar um repertório de formas que contradiziam sua produção de antes de 1970. Para demonstrar tal intenção de Lispector, este trabalho procura analisar as características do manuscrito Objeto gritante nunca publicado , o qual, após uma série de alterações significativas, acabou por se transformar na obra Água viva. Caso o projeto do manuscrito não tivesse sofrido redirecionamentos substanciais, teria sido a primeira ocorrência, em livro, das novas formas da escrita clariciana que começavam a se manifestar naquela época, mas que vieram a se consolidar nas suas obras posteriores

In Vitro Surfactant and Perfluorocarbon Aerosol Deposition in a Neonatal Physical Model of the Upper Conducting Airways

Objective: Aerosol delivery holds potential to release surfactant or perfluorocarbon (PFC) to the lungs of neonates with respiratory distress syndrome with minimal airway manipulation. Nevertheless, lung deposition in neonates tends to be very low due to extremely low lung volumes, narrow airways and high respiratory rates. In the present study, the feasibility of enhancing lung deposition by intracorporeal delivery of aerosols was investigated using a physical model of neonatal conducting airways. Methods: The main characteristics of the surfactant and PFC aerosols produced by a nebulization system, including the distal air pressure and air flow rate, liquid flow rate and mass median aerodynamic diameter (MMAD), were measured at different driving pressures (4-7 bar). Then, a three-dimensional model of the upper conducting airways of a neonate was manufactured by rapid prototyping and a deposition study was conducted. Results: The nebulization system produced relatively large amounts of aerosol ranging between 0.3 +/- 0.0 ml/min for surfactant at a driving pressure of 4 bar, and 2.0 +/- 0.1 ml/min for distilled water (H(2)Od) at 6 bar, with MMADs between 2.61 +/- 0.1 mu m for PFD at 7 bar and 10.18 +/- 0.4 mu m for FC-75 at 6 bar. The deposition study showed that for surfactant and H(2)Od aerosols, the highest percentage of the aerosolized mass (similar to 65%) was collected beyond the third generation of branching in the airway model. The use of this delivery system in combination with continuous positive airway pressure set at 5 cmH(2)O only increased total airway pressure by 1.59 cmH(2)O at the highest driving pressure (7 bar). Conclusion: This aerosol generating system has the potential to deliver relatively large amounts of surfactant and PFC beyond the third generation of branching in a neonatal airway model with minimal alteration of pre-set respiratory support.

A Delphi Process to Optimize Quality and Performance of Drug Evaluation in Neonates

Background: Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. The objective of this work was to identify the major criteria considered necessary for selecting neonatal intensive care units that are able to perform drug evaluations competently. Methodology and Main Findings: This Delphi process was conducted with an international multidisciplinary panel of 25 experts from 13 countries, selected to be part of two committees (a scientific committee and an expert committee), in order to validate criteria required to perform drug evaluation in neonates. Eighty six items were initially selected and classified under 7 headings: "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6). Sixty-one items were retained and headings were rearranged after the first round, 34 were selected after the second round. A third round was required to validate 13 additional items. The final set includes 47 items divided under 5 headings. Conclusion: A set of 47 relevant criteria will help to NICUs that want to implement, conduct or participate in drug trials within a neonatal network identify important issues to be aware of. Summary Points: 1) Neonatal trials remain difficult to conduct for several reasons: in particular the need for study sites to have an existing infrastructure in place, with trained investigators and validated quality procedures to ensure good clinical, laboratory practices and a respect for high ethical standards. 2) The present Delphi study was conducted with an international multidisciplinary panel of 25 experts from 13 countries and aims to identify the major criteria considered necessary for selecting neonatal intensive care units (NICUs) that are able to perform drug evaluations competently. 3) Of the 86 items initially selected and classified under 7 headings - "NICUs description - Level of care'' (21), "Ability to perform drug trials: NICU organization and processes (15), "Research Experience'' (12), "Scientific competencies and area of expertise'' (8), "Quality Management'' (16), "Training and educational capacity'' (8) and "Public involvement'' (6) - 47 items were selected following a three rounds Delphi process. 4) The present consensus will help NICUs to implement, conduct or participate in drug trials within a neonatal network.

Self-Organized Criticality, Plasticity and Sensorimotor Coupling. Explorations with a Neurorobotic Model in a Behavioural Preference Task

During the last two decades, analysis of 1/f noise in cognitive science has led to a considerable progress in the way we understand the organization of our mental life. However, there is still a lack of specific models providing explanations of how 1/f noise is generated in coupled brain-body-environment systems, since existing models and experiments typically target either externally observable behaviour or isolated neuronal systems but do not address the interplay between neuronal mechanisms and sensorimotor dynamics. We present a conceptual model of a minimal neurorobotic agent solving a behavioural task that makes it possible to relate mechanistic (neurodynamic) and behavioural levels of description. The model consists of a simulated robot controlled by a network of Kuramoto oscillators with homeostatic plasticity and the ability to develop behavioural preferences mediated by sensorimotor patterns. With only three oscillators, this simple model displays self-organized criticality in the form of robust 1/f noise and a wide multifractal spectrum. We show that the emergence of self-organized criticality and 1/f noise in our model is the result of three simultaneous conditions: a) non-linear interaction dynamics capable of generating stable collective patterns, b) internal plastic mechanisms modulating the sensorimotor flows, and c) strong sensorimotor coupling with the environment that induces transient metastable neurodynamic regimes. We carry out a number of experiments to show that both synaptic plasticity and strong sensorimotor coupling play a necessary role, as constituents of self-organized criticality, in the generation of 1/f noise. The experiments also shown to be useful to test the robustness of 1/f scaling comparing the results of different techniques. We finally discuss the role of conceptual models as mediators between nomothetic and mechanistic models and how they can inform future experimental research where self-organized critically includes sensorimotor coupling among the essential interaction-dominant process giving rise to 1/f noise.

Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding beta-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic beta-amylase encoding genes in pgi1 leaves, which was accompanied by increased beta-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P) H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Design and implementation of a cloud computing adoption decision tool: Generating a cloud road

Migrating to cloud computing is one of the current enterprise challenges. This technology provides a new paradigm based on "on-demand payment" for information and communication technologies. In this sense, the small and medium enterprise is supposed to be the most interested, since initial investments are avoided and the technology allows gradual implementation. However, even if the characteristics and capacities have been widely discussed, entry into the cloud is still lacking in terms of practical, real frameworks. This paper aims at filling this gap, presenting a real tool already implemented and tested, which can be used as a cloud computing adoption decision tool. This tool uses diagnosis based on specific questions to gather the required information and subsequently provide the user with valuable information to deploy the business within the cloud, specifically in the form of Software as a Service (SaaS) solutions. This information allows the decision makers to generate their particular Cloud Road. A pilot study has been carried out with enterprises at a local level with a two-fold objective: To ascertain the degree of knowledge on cloud computing and to identify the most interesting business areas and their related tools for this technology. As expected, the results show high interest and low knowledge on this subject and the tool presented aims to readdress this mismatch, insofar as possible. Copyright: © 2015 Bildosola 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.

The Interplay between Natural Selection and Susceptibility to Melanoma on Allele 374F of SLC45A2 Gene in a South European Population

We aimed to study the selective pressures interacting on SLC45A2 to investigate the interplay between selection and susceptibility to disease. Thus, we enrolled 500 volunteers from a geographically limited population (Basques from the North of Spain) and by resequencing the whole coding region and intron 5 of the 34 most and the 34 least pigmented individuals according to the reflectance distribution, we observed that the polymorphism Leu374Phe (L374F, rs16891982) was statistically associated with skin color variability within this sample. In particular, allele 374F was significantly more frequent among the individuals with lighter skin. Further genotyping an independent set of 558 individuals of a geographically wider population with known ancestry in the Spanish population also revealed that the frequency of L374F was significantly correlated with the incident UV radiation intensity. Selection tests suggest that allele 374F is being positively selected in South Europeans, thus indicating that depigmentation is an adaptive process. Interestingly, by genotyping 119 melanoma samples, we show that this variant is also associated with an increased susceptibility to melanoma in our populations. The ultimate driving force for this adaptation is unknown, but it is compatible with the vitamin D hypothesis. This shows that molecular evolution analysis can be used as a useful technology to predict phenotypic and biomedical consequences in humans.

Machado de Assis e o exercício dos gêneros da intimidade

Observando o crescente interesse pela escrita do eu e levando em conta a afirmação de Jorge de Sena no ensaio Machado de Assis e o seu quinteto carioca, que declara haver como unidade entre as cinco obras finais de Machado de Assis o questionamento dos gêneros que expressam a subjetividade moderna, buscamos investigar, nesta dissertação, a maneira como Machado de Assis lida com alguns desses gêneros da intimidade, dentro e fora do espaço ficcional. Para tanto, selecionamos duas das obras finais que empregam mais diretamente a escrita do eu e que, do mesmo modo, mais transgridem essa escrita: Memórias Póstumas de Brás Cubas, uma autobiografia escrita por um defunto autor; e Memorial de Aires, um diário que registra mais frequentemente a vida dos outros do que a do próprio diarista Aires. Além das obras ficcionais, trazemos para a análise as missivas machadianas, observando como o escritor constrói a imagem de si na troca de cartas com intelectuais de seu convívio. Refletindo sobre o exercício desses gêneros da intimidade, por parte de Machado de Assis, observamos que este, na sua correspondência, não ultrapassou os limites que, à época, regiam a troca de confidências entre pares e amigos. Comedido e discreto, assume, de modo geral, o papel de mestre e aconselhador, apresentando em suas cartas um projeto literário de cunho pedagógico, conduzindo os mais jovens na direção da literatura. Já na ficção, ao fazer um uso engenhoso e criativo das retóricas da autobiografia e do diário, o escritor utilizou tais gêneros para, ao mesmo tempo, recobrir e agudizar a crítica corrosiva à sociedade de seu tempo