Lactate Oxidation in Endothelial Cells: A Feature of All Endothelial Cells?

Resumen de la comunicaciónMetabolism of endothelial cells is a topic that has gained an increasing interest in the last years. This is due to their role in the angiogenic process, which is pathologically upregulated in several diseases, such as retinopathies, diabetes and cancer. Glycolysis, among other metabolic routes, has been found to be essential for triggering the angiogenic switch. Additionally, it has been seen that endothelial cells are able to take up lactate from the extracellular media, for example in the case of the tumor microenvironment, where cancer cells would have secreted high amounts of this metabolite. Endothelial cells would oxidize this lactate for obtaining energy, but lactate can also act as a signaling molecule for the angiogenic process. However, experiments to determine the molecular fate of lactate have been performed using only macrovascular endothelial cells. The aim of the present work is to prove whether microvascular endothelial cells are also able to take up and oxidize lactate. For this purpose, fluorimetry, isotopic labeling and Seahorse experiments were used to study the metabolism of a human microvascular endothelial cell line (HMEC). The expression levels of transcripts and proteins of different enzymes and transporters related to lactate metabolism were estimated by qPCR and Western blotting. The results obtained indicate that these cells rely on glycolysis for their metabolism, while the oxidation of glucose and glutamine seems to be considerably low. On the other hand, no lactate oxidation could be detected. We then checked the mRNA expression of the two isoenzymes of lactate dehydrogenase (LDH) and the two main lactate transporters, MCT1 and MCT4, and found that levels of LDH-B and MCT1 were undetectable. We failed to measure any MCT1 mRNA or protein expression either in normoxia or hypoxia. Hence, we can conclude that at least this microvascular endothelial cell line cannot use extracellular lactate as a metabolic fuel.Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain). This communicaction has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

The coffee diterpene kahweol is an antitumoral compound for human estrogen receptor-negative breast cancer cells

Although epidemiological studies indicate that coffee consumption is associated with a low incidence of several kinds of cancer, there are still few studies on the anti-tumoral effects of pure bioactive compounds isolated from coffee. The present study aims to identify the modulatory effects of kahweol, an antioxidant diterpene contained in unfiltered coffee, on a panel of human tumor cell lines. Kahweol inhibits tumor cell proliferation and clonogenicity and induces apoptosis in several kinds of human tumor cells, with a special incidence on the estrogen receptor-negative MDA-MB231 human breast cancer cells. In these breast cancer cells, the mentioned effects are accompanied by caspases 3/7 and 9 activation and cytochrome c release, both consistent with an activation of the intrinsic pathway of apoptosis. Kahweol also increases the phosphorylation levels of protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK) observed in MDA-MB-231 cells. We also demonstrate the inhibitory effect of kahweol on the MDA-MB231 cell potential to migrate and to remodel extracellular matrix by targeting matrix metalloproteinase-9 and urokinase-type plasminogen activator, two key molecules involved in this process. Taken together, our data suggest that, indeed, kahweol behaves as an antitumor compound, especially against MDA-MB231 breast cancer cells.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The enzymatic determination of glucose in carbonated beverages: a useful tool for the undergraduate students to learn the basis of enzymatic analysis and the comparison of two analytical methods

The importance of enzymatic analysis in biochemistry, clinical chemistry and food chemistry is undoubted. The course "Applied Biochemistry" in our Faculty is aimed to undergraduate students of Chemistry and Biochemistry. In this subject, the principles and applications of enzymatic analysis are presented to the students, who receive a theoretical introductory lecture in the classroom before they carry out an experiment that should be feasible to be solved in a short laboratory period. The experimental protocol here presented, based on the enzymatic determination of glucose in carbonated beverages, has been implemented at the University of Málaga and it has been optimized according to the students’ results and commentaries along the last years. It aims to illustrate basic issues relating enzymatic analysis, including its potential application to food chemistry. Although there are several enzymatic methods that can be used for the determination of glucose, we selected the one based on the coupled reactions of glucose oxidase (GOD; EC 1.1.3.4.) and peroxidase (POD; EC 1.11.1.7.) because the kinetic constants of glucose oxidase allow the mentioned enzymatic reactions to be used in both, the end point and the kinetic enzymatic analysis methods. In this way, data for two different protocols for the determination of glucose concentration are obtained by the students from a single reaction mixture. Students construct a calibration curve for each method using a glucose standard solution, and use them to determine the glucose concentration in the problem solutions. The inclusion of replicate samples in the determination of the glucose concentration of an “ideal problem” (glucose in purified water) is used to illustrate the principles of statistics in the lab, and comparison with the “real value” allows an estimation of the accuracy of each method. The evaluation of glucose concentration in four carbonated beverages: coloured coke and uncoloured tonic sodas (regular or sugarless in both cases) makes student to recognise the appearance of interferences that should be either avoided or eliminated. Since all samples are analysed by means of end-point and kinetic methods, students can discuss the applicability of each method to these specific analytical problems. They are also encouraged to compare both analytical methods in terms of sensitivity, selectivity, accuracy, and time consumed. Chemistry and Biochemistry undergraduate students having performed this experiment in our laboratories have found it formative, interesting and challenging.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

A practice project to prevent the cookbook model as modus operandi for biochemistry laboratory learning

Laboratory learning is a crucial component of chemistry and biochemistry instruction and should be conceived as a way to develop students’ reasoning, technical or practical skills, introducing them into the scientific method principles. Nevertheless, the heavily criticized “expository instruction style”, characterized by a cookbook nature, is still the most widespread style of laboratory instruction in our universities. Alternative learning styles based in the inquiry, discovery and problem-based pedagogical approaches, have been reported to promote students’ problem solving skills, critical thought and self-confidence development. We are currently involved in the Educative Innovation Project PIE17-065, funded by University of Malaga, aimed to improve the teaching practice of Biochemistry laboratory to undergraduate students. Based on an enzymatic analysis of glucose in soft-drinks we have developed a laboratory protocol as a part of a full practice project where students must work before and after the lab session, in order to prevent the cookbook model as modus operandi, therefore preventing the situation where the students get a first glimpse of the experiment protocol whereas they put on their lab coat. The learning activities have been designed to move our students from the passive role that characterizes the step-by-step procedures, to an active and critical attitude that starts before and remains after their laboratory session, also minimizing time, space, and equipment resources. Our results have shown that this experiment has improved the learning of both, future biochemists and chemists, which showed a very positive perception of the whole practical project.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. PIE 17-06

Evaluation of the anti-angiogenic potential of hydroxytyrosol derivatives

Angiogenesis, a process which allows the formation of new vessels from pre-existing ones, is an essential phenomenon for tumor survival since it allows cancer cells to obtain nutrients and oxygen. This explains the increasing interest showed by many groups of research and pharmaceutical companies to find compounds with potential to disrupt at least one of the steps within the angiogenic process. Hydroxytyrosol (3,4-dihydroxyphenyl ethanol) has been identified as the most important health-related phenolic compound of virgin olive oil because of its pleiotropic effects on multiple targets. In 2012, our group identified hydroxytyrosol as an anti-angiogenic compound able to inhibit several key steps in the angiogenic process. In the present study, the potential effects of six hydroxytyrosol derivatives are tested and compared with those exhibited by hydroxytyrosol by making use of several in vitro and in vivo assays. Results indicate that these are candidate new anti-angiogenic compounds with potential utility in anti-tumor and anti-angiogenic therapies.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech [Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain)]. This communication has the support of a travel grant

Transport in the blood of an anti-tumor water-soluble rutheniun cyclopentadienyl complex: a fluorescence study on albumin binding

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014O Cancro é a segunda maior causa de morte em países desenvolvidos. Estima-se que o número global de mortes por cancro aumente em 50% até 2030, criando a necessidade urgente de encontrar soluções para este problema de saúde global. A cisplatina, carboplatina e oxaliplatina são os únicos metalofármacos aprovados para uso clínico em todo o mundo mas, apesar da sua eficácia, apresentam efeitos secundários que limitam muito a sua utilização. Os compostos de ruténio são já reconhecidos como uma alternativa promissora aos fármacos de platina usados em quimioterapia, dada a sua menor citotoxidade e maior selectividade. Além disso, oferecem diferentes mecanismos de acção e um espectro de atividade diferente, mais alargado, que os metalofármacos de Pt. O seu mecanismo de acção é intensamente estudado actualmente e a ligação a proteínas séricas parece ter um papel importante na sua resposta biológica. O estudo da interacção com proteínas séricas e com a albumina do plasma humano (HSA) em particular reveste-se de uma grande importância no contexto do desenvolvimento de metalofármacos. Por um lado, a interacção com a albumina tem um impacto importante na biodisponibilidade do complexo em estudo. Por outro lado, a HSA desempenha um papel fulcral no transporte de compostos endógenos e exógenos, sendo também crucial como veiculo transportador de compostos terapêuticos para lhe permitir atingir as células e tecidos-alvo onde exercem a sua acção. Além disso, é um requisito da FDA (Food and Drug Administration) que resultados da interacção com proteínas séricas sejam incluídos no processo de pre-selecção de potenciais fármacos e metalofármacos. Neste trabalho estudou-se a interacção de um complexo organometálico de ruténio(II) de estrutura em banco de piano com a albumina de plasma humano (sem ácidos gordos, fafHSA). O complexo [RuII(Cp)(bipy)(CO)][PF6] (designado neste trabalho por RuC), com uma actividade moderada para o adenocarcinoma de ovário humano, é solúvel em água o que o torna muito interessante no contexto de desenvolvimento de novos agentes terapêuticos. A sua ligação à HSA foi estudada em meio aquoso a pH 7.4 (tamponizado com HEPES) e à temperatura de 37ºC por espectroscopia de UV-Vis e espectroscopia de Fluorescência (em estado estacionário e resolvida no tempo) usando a emissão do resíduo Trp214 da proteína como sonda de fluorescência intrínseca. A interacção do complexo com a HSA ocorre rapidamente e resulta na extinção da fluorescência do Trp214 por um processo misto estático e dinâmico, para o qual se obtiveram as constantes de Stern-Volmer KD = (4,8 ± 0,3)x104 M-1 e KS =(2,3 ± 0,7)x104 M-1 (calculadas a em=338 nm pelo método dos mínimos quadráticos para um nível de confiança de 95%). O modelo proposto para a interacção RuC–HSA engloba a formação de dois aductos complexo-proteína de estequiometrias 1:1 e 1:2 para os quais são calculadas constantes de formação condicionais log β’1= (4,78 ± 0,01) (para o aducto 1:1 {HSA-RuC}) e log β’2= (9,52 ± 0,01) (para o aducto 1:2 {HSA-(RuC)2}. Reacções de competição com “marcadores” conhecidos como a varfarina e a dansilglicina, para os quais a ligação à HSA é específica e está bem estudada, são utilizadas para obter informação sobre os locais de ligação do RuC à proteína. Os resultados obtidos neste trabalho permitem concluir que o complexo organometálico estudado interactua com a HSA de forma moderada, podendo ser eficientemente transportado pelo sangue através da ligação a esta proteína.Ruthenium (Ru) complexes have captivated an increasing interest in recent years; they are a promising alternative to platinum-based complexes due to the fact that some of them have been repeatedly described for showing higher selectivity and lower cytotoxicity regarding their effects on cancer cells. The interactions studied between the Ru-complexes and serum proteins such as fatty acid free Human Serum Albumin (fafHSA) are of very high importance considering that their bioavailability and effects depend on their binding to albumin to reach the targeted cells. In addition, the Food and Drug Administration (FDA) has set the report on plasma protein binding for prodrugs as a requirement in order to do the screening for potential therapeutic agents. The interactions and several aspects related to the binding between the ruthenium (II) complex [RuII(Cp)(bipy)(CO)][PF6] (or RuC, also named pmc44) and HSA are addressed using fluorescence spectroscopy, steady-state and time-resolved measurements, and UV-Visible absorption in 10 mM HEPES buffered medium pH 7,4. Additionally, in order to obtain a deeper insight into the HSA binding properties of the complex at the specific drug binding sites, displacement experiments with the site markers Warfarin and Dansylglycine (for which the binding site is well known) were performed. The results show that the complex strongly quenches the intrinsic fluorescence of albumin and that the interaction is quite fast, indicating that the complex successfully binds the protein. The outcome from the Stern-volmer linearizations (both time-resolved and steady-state measurements) displayed more than one type of quenching mechanism; albumin fluorescence is quenched by a combination of dynamic and static quenching mechanisms. The most representative values obtained for KD and KS from the averages were (4,8 ± 0,3)x104 M-1 and (2,3 ± 0,7)x104 M-1 respectively, calculated with the Stern-Volmer linearization at em=338 nm by a Minimum Square Fit procedure with variance analysis (ANOVA) with a 95% confidence level. A speciation model with two protein-RuC adducts is proposed with global conditional binding constants of log β’1= (4,78 ± 0,01) (for the 1:1 adduct {HSA-RuC}) and log β’2= (9,52 ± 0,01) (for the 1:2 adduct {HSA-(RuC)2} with a fitting parameter of 1,16x105 using the computer program PSEQUAD. The conclusions achieved with the site markers´ experiments revealed that the complex binds to both sites I and II of the protein. Altogether, the results from this work indicated that the complex studied can bind human serum albumin in a moderate way and thus it be efficiently transported in the blood by HSA

PRODUCTION OF HIGHLY POROUS AL-NI FOAMS BY POWDER METALLURGY USING DOLOMITE AS A FOAMING AGENT

In this study, metal foams were fabricated using Al-Ni combined with dolomite(CaMg(CO_3 )_2) as an alternative foaming agent via the powder metallurgy (PM) route. For first-time dolomite, a dual carbonate (magnesium-calcium) was found to be a highly effective foaming and an excellent stabilizing agent. It has a gradual decomposition and at different temperatures produces oxides, improving foaming conditions and expansion performance. The experimental findings showed that the addition of 7wt% of dolomite is the optimal amount for the development of well-formed pores with uniform cell distribution, spherical shape, and thick cell walls. The addition of Ni to the mixture increased the melting temperature and decreased the difference between the alloying melting point and dolomite decomposition temperature. Specific Al-Ni compositions showed appropriate viscosities for enclosing the gas as it was released. Al-10Ni and Al-15Ni were found the optimum combinations to form intermetallics which further helped stabilize the foams. Moreover, as a result of this investigation, a modification of the powder metallurgy technique was proposed, by partial-sintering the precursors before the foaming process (i.e. raising the temperature of the precursor for a period of time prior to foaming). This step allowed the formation of particle sintering, enhancing foam expansion, leading to more homogeneous porosity, cell morphology and microstructure. The following thesis is based in one paper, which has been submitted to the journal, Advanced Engineering Materials

Learning contract, co-operative and flipped learning as useful tools for studying metabolism

Es el Abstract de una comunicación a un congreso internacional sobre educaciónUndergraduate students in Biology identify Metabolic Biochemistry as a particularly difficult subject. This is due to the fact that students need to interconnect properly all the contents of its syllabus throughout their study of the subject in order to get a global insight of the complex regulatory features controlling metabolic pathways within the metabolic network under different physiologic and pathologic conditions, as well as metabolism as a whole. Due to these objective difficulties, a high percentage of our students face the study of this subject as a very hard task beyond their forces and capacities. This perception leads to high rates of premature dropout. In previous years, less than 40% of all the registered students attended the examinations of Metabolic Biochemistry (a subject in the second year of the Degree of Biology at our University). Even worse, less than 25% of our students passed the exams. From the academic year 2015/16 on, we are developing innovative teaching projects (PIE15-163 and PIE17-145, funded by University of Malaga) aimed to increase our student loyalty to the subject (and hence to increase their attendance to exams) and to help them to learn more effectively metabolism and its regulation. These innovative teaching projects are based on the use of several powerful tools: a learning contract and problem-based learning within the framework of group tasks promoting an actual collaborative learning in a flipped classroom. The present communication will show the implementation of the PIE15-163 and PIE17-145 projects and some results obtained from them.This work was supported by Malaga University funds granted to the educational innovation project PIE17-145. The attendance to the END2018 International Conference on Education and New Developments (June 2018, Budapest, Hungary) has received a grant from "I Plan Propio Integral de Docencia. Universidad de Málaga"]

Evaluation of metabolism and biosignaling in the angiogenic microenvironment as potential targets for therapeutic intervention

The "re-discovery" of Warburg effect at the turn of the present millennium has been a key determinant of the current renewed interest on cancer metabolism. In fact, metabolic reprogramming has been identified as one of the hallmarks of cancer. However, cancers grow in tight contact with non-tumoral accompanying cells and the surrounding extracellular matrix, as underlined by the concept of tumor microenvironment. Endothelial cells are key components of this tumor microenvironment, since they are requested for angiogenesis, another hallmark of cancer. In this complex system, rewiring of metabolism and signaling pathway in cancer, endothelial and other accompanying cell emerges as new potential targets for therapeutic intervention. In this communication, we will present the drug discovery and characterization approach of our group and our more recent results in this field, including new modeling with an evolutionary and ecological point of view.[Our experimental work is supported by grants BIO2014-56092-R (MINECO and FEDER) and P12-CTS-1507 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian Government). The "CIBER de Enfermedades Raras" is an initiative from the ISCIII (Spain)]. This communication has the support of a travel grant "Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech"

Influence of a rocky platform in the profile morphology: Victoria Beach, Cadiz (Spain)

The equilibrium profile concept is a useful tool for the knowledge and management of the coastline. This concept is based on several assumptions that rarely are satisfied in nature. One of these considers that the geologic setting has no influence in the profile morphology. The Victoria beach is a good example that questions this hypothesis. Muñoz-Pérez (1996) and Muñoz-Pérez et al. (1999) proposed an analytical expression of equilibrium beach profile for reefprotected beaches. In this work, data measured at Victoria beach are used to confirm the goodness of this model. The results obtained show that the occurrence of a horizontal rocky platform in subtidal zone modifies the beach slope and the seasonal evolution of the profile.Xunta de Galici