Estudio proteomico diferencial entre liquidos pleurales secundarios a cáncer de pulmón versus mesotelioma / Differential proteomic study between pleural fluid secondary to lung cancer versus mesothelioma

Objetivo: Encontrar perfiles proteicos en líquido pleural que diferencien derrames pleurales secundarios a cáncer de pulmón (CP) versus mesotelioma pleural maligno (MPM).Metodología: Recogimos líquidos pleurales de 60 pacientes de tres grupos diferentes: MPM (N = 20), CP (N = 20) y derrames pleurales benignos (N = 20). Realizamos un análisis con proteómica diferencial con ITRAQ 4 plex (Applied Biosystem). Realizamos la identificación y cuantificación relativa de las proteínas con el programa Proteome Discoverer 1.4 (Termofisher Scientific). Construimos diagramas de Venn con las proteínas sobre/infra-expresadas en cada grupo. Realizamos una validación interna/externa mediante ELISA (Myobiosorce) añadiendo 25 muestras de CP y 14 de MPM.Resultados: Encontramos sobreexpresión de Pi3K en los derrames pleurales neoplásicos (16,86 +/- 25,83 ng/ml en CP; 20,66 +/- 17,26 ng/ml en MPM vs 5,92 +/- 0,99 ng/ml en controles). Hubo sobreexpresión de SPRM en MPM (30.702 +/- 30.310,53 ng/ml en el grupo MPM vs 10.404 +/- 10.157,72 ng/ml en el grupo CP vs 8.498 + /- 3.437,18 ng/ml en controles). Existió sobreexpresión de RhoB en CP (4,46 +/- 1,65 mg/ml en CP vs 1,65 +/- 2,65 mg/ml en MPM vs 0,92 +/- 1,6 mg/ml en controles). También encontramos sobreexpresión de PDGFR-alfa en derrames pleurales benignos (74,12 +/- 22,57 ng/ml en controles vs 43,05 +/- 23,96 ng/ml en CP vs 36,12 +/- 21,51 ng/ml en MPM).Conclusión: Existe un perfil diferencial proteico entre los derrames secundarios a CP (sobreexpresión de RhoB) y a MPM (sobrexpresión de SPRM). La sobrexpresión de Pi3K indica asociación a derrames pleurales malignos y la de PDGFR-alfa a derrames benignos. (AU)

Objetivo: Find protein profiles in pleural fluid that differentiate pleural effusions secondary to lung cancer (LC) versus malignant pleural mesothelioma (MPM).Metodología: We collected pleural fluids from 60 patients from three different groups: MPM (N = 20), CP (N = 20), and benign pleural effusions (N = 20). We performed differential proteomics analysis with ITRAQ 4 plex (Applied Biosystem). We performed the identification and relative quantification of the proteins with the Proteome Discoverer 1.4 program (Termofisher Scientific). We built Venn diagrams with the over/under-expressed proteins in each group. We performed an internal/external validation using ELISA (Myobiosorce) adding 25 CP and 14 MPM samples.Resultados: We found Pi3K overexpression in neoplastic pleural effusions (16.86 +/- 25.83 ng/ml in PC; 20.66 +/- 17.26 ng/ml in MPM vs 5.92 +/- 0.99 ng/ml in controls). There was overexpression of SPRM in MPM (30,702 +/- 30,310.53 ng/ml in the MPM group vs 10,404 +/- 10,157.72 ng/ml in the CP group vs 8,498 +/- 3,437.18 ng/ml in controls). There was overexpression of RhoB in CP (4.46 +/- 1.65 mg/ml in CP vs 1.65 +/- 2.65 mg/ml in MPM vs 0.92 +/- 1.6 mg/ml in controls). We also found overexpression of PDGFR-alpha in benign pleural effusions (74.12 +/- 22.57 ng/ml in controls vs 43.05 +/- 23.96 ng/ml in PC vs 36.12 +/- 21.51 ng/ml in MPM ).Conclusión: There is a differential protein profile between effusions secondary to CP (RhoB overexpression) and MPM (SPRM overexpression). Pi3K overexpression indicates association with malignant pleural effusions and PDGFR-alpha overexpression with benign effusions. (AU)

Identification of proteomic signatures associated with lung cancer and COPD.

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC. The aim of this study was to identify distinct proteomic profiles able to discriminate these two pathological entities. Protein content was assessed in the bronchoalveolar lavage (BAL) of 60 patients classified in four groups: COPD, COPD and LC, LC without COPD, and control with neither COPD nor LC. Proteins were separated into spots by bidimensional polyacrylamide gel electrophoresis (2D-PAGE) and examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF). A total of 40 proteins were differentially expressed in the LC and/or COPD groups as compared with the control group. Distinct protein profiles were identified and validated for each pathological entity (LC and COPD). The main networks involved were related to inflammatory signalling, free radical scavenging and oxidative stress response, and glycolysis and gluconeogenesis pathways. The most relevant signalling link between LC and COPD was through the NF-κB pathway. In conclusion, the protein profiles identified contribute to elucidate the underlying pathogenic pathways of both diseases, and provide new tools of potential use as biomarkers for the early diagnosis of LC. BIOLOGICAL SIGNIFICANCE: Sequence coverage. The protein sequence coverage (95%) was estimated for specific proteins by the percentage of matching amino acids from the identified peptides having confidence greater than or equal to 95% divided by the total number of amino acids in the sequence. Ingenuity Pathways Analysis. Mapping of our proteins onto biological pathways and disease networks demonstrated that 22 proteins were linked to inflammatory signalling (p-value: 1.35 10(-08)-1.42 10(-02)), 15 proteins were associated with free radical scavenging and oxidative stress response (p-value: 4.93 10(-11)-1.27 10(-02)), and 9 proteins were related with glycolysis and gluconeogenesis pathways (p-value: 7.39 10(-09)-1.58 10(-02)).

Proteomic biomarkers in lung cancer.

The correct understanding of tumour development relies on the comprehensive study of proteins. They are the main orchestrators of vital processes, such as signalling pathways, which drive the carcinogenic process. Proteomic technologies can be applied to cancer research to detect differential protein expression and to assess different responses to treatment. Lung cancer is the number one cause of cancer-related death in the world. Mostly diagnosed at late stages of the disease, lung cancer has one of the lowest 5-year survival rates at 15 %. The use of different proteomic techniques such as two-dimensional gel electrophoresis (2D-PAGE), isotope labelling (ICAT, SILAC, iTRAQ) and mass spectrometry may yield new knowledge on the underlying biology of lung cancer and also allow the development of new early detection tests and the identification of changes in the cancer protein network that are associated with prognosis and drug resistance.

PKCepsilon upregulates voltage-dependent calcium channels in cultured astrocytes.

Astrocytes express voltage-gated calcium channels (VGCCs) that are upregulated in the context of the reactive astrogliosis occurring in several CNS pathologies. Moreover, the ability of selective calcium channel blockers to inhibit reactive astrogliosis has been revealed in a variety of experimental models. However, the functions and regulation of VGCC in astrocytes are still poorly understood. Interestingly, protein kinase C epsilon (PKCepsilon), one of the known regulators of VGCC in several cell types, induces in astrocytes a stellated morphology similar to that associated to gliosis. Thereby, here we explored the possible regulation of VGCC by adenovirally expressed PKCepsilon in astrocytes. We found that PKCepsilon potently increases the mRNA levels of two different calcium channel alpha(1) subunits, Ca(V)1.2 (L-type channel) and Ca(V)2.1 (P/Q-type channel). The mRNA upregulation was followed by a robust increase in the corresponding peptides. Moreover, the new calcium channels formed as a consequence of PKCepsilon activation are functional, since overexpression of constitutively-active PKCepsilon increased significantly the calcium current density in astrocytes. PKCepsilon raised currents carried by both L- and P/Q-type channels. However, the effect on the P/Q-type channel was more prominent since an increase of the relative contribution of this channel to the whole cell calcium current was observed. Finally, we found that PKCepsilon-induced stellation was significantly reduced by the specific L-type channel blocker nifedipine, indicating that calcium influx through VGCC mediates the change in astrocyte morphology induced by PKCepsilon. Therefore, here we describe a novel regulatory pathway involving VGCC that participates in PKCepsilon-dependent astrocyte activation.

Pyruvate protects cerebellar granular cells from 6-hydroxydopamine-induced cytotoxicity by activating the Akt signaling pathway and increasing glutathione peroxidase expression.

Parkinson disease (PD) is the second-most common age-related neurodegenerative disease and is characterized by the selective destruction of dopaminergic neurons. Increasing evidence indicates that oxidative stress plays a crucial role in the pathogenesis of idiopathic PD. Anti-oxidant agents including catalase, manganese porphyrin and pyruvate confer cytoprotection to different cell cultures when challenged with 6-hydroxydopamine (6-OHDA). Herein we used rat cerebellar granular cell cultures to ascertain the plausible cellular pathways involved in pyruvate-induced cytoprotection against 0.1 mM 6-OHDA. Pyruvate provided cytoprotection in a concentration-dependent manner (2-10 mM). Consistent with its well-established anti-oxidant capacity, pyruvate (10 mM) prevented 6-OHDA-induced lipid peroxidation by blocking the rise in intracellular peroxides and maintaining the intracellular reduced glutathione (GSH) levels. Further experiments revealed that pyruvate increased Akt, but not extracellular signal-regulated kinase phosphorylation. Moreover, phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated pyruvate-induced cytoprotection indicating that PI3K-mediated Akt activation is necessary for pyruvate to induce cytoprotection. On the other hand, pyruvate also up-regulated glutathione peroxidase mRNA levels, but not those of the anti-oxidant enzymes superoxide dismutase-1 and -2, catalase or the anti-apoptotic oncogenes Bcl-2 or Bcl-xL. In summary, our results strongly suggest that pyruvate, besides the anti-oxidant properties related to its structure, exerts cytoprotective actions by activating different anti-apoptotic routes that include gene regulation and Akt pathway activation.

[Growth and polysaccharide formation in Sinorhizobium meliloti strains in an air-lift-type fermentor. Effect on nodulation velocity in alfalfa plants]. / Crecimiento y formación de polisacáridos de cepas de Sinorhizobium meliloti en un fermentador tipo air-lift. Influencia en la velocidad de nodulación en plantas de alfalfa.

In this paper the influence of the exopolysaccharides produced by Sinorhizobium meliloti strains on the nodulation rates in alfalfa plants has been considered. The experiments were performed in a rotary shaker and in an air-lift type fermentor. Different Sinorhizobium meliloti strains were used. Bacterial growth rates were determined by viable cell counts. Exopolysaccharide concentration was determined by precipitation with ethanol. It was observed that maximum cell concentration was in the order of 1 x 10(10) cell/ml and exopolysaccharide content was approximately 11 g/l. The experiments performed with alfalfa plants in a controlled environment chamber showed that, when inoculation was carried out with diluted suspensions (1/10), nodulation time was reduced from 10 to 4 days, while the strains retained their symbiotic properties.

Crecimiento y formación de polisacáridos de cepas de Sinorhizobium meliloti en un fermentador tipo air-lift: influencia en la velocidad de nodulación en plantas de alfalfa / Crowth of Sinorhizobium meliloti strains and exopolysaccharide formation in an air-lift fermentor: influence on the nodulation rate in alfalfa plants

En este trabajo, se estudió la influencia de los polisacáridos formados por cepas de Sinorhizobium meliloti sobre la velocidad de nodulación en plantas de alfalfa. Las experiencias fueron realizadas en agitador rotatorio y en un fermentador con circulación de líquido por inyección de aire, empleando diferentes cepas de S. meliloti, El crecimiento bacteriano fue determinado por recuento de células viables y la concentración del exopolisacárido por precipitación con etanol. Se alcanzaron concentraciones máximas del orden de 1.10 (10) células viables/ml y de 11 g/l de polisacárido. Los estudios de crecimiento de plantas de alfalfa en cámara climatizada mostraron que se producía una reducción en el tiempo de aparición de nódulos de 10 a 4 días, cuando las mismas eran inoculadas con suspensiones diluidas 1/10, manteniendo las cepas sus propiedades simbióticas.

Crecimiento y formación de polisacáridos de cepas de Sinorhizobium meliloti en un fermentador tipo air-lift: influencia en la velocidad de nodulación en plantas de alfalfa / Crowth of Sinorhizobium meliloti strains and exopolysaccharide formation in an air-lift fermentor: influence on the nodulation rate in alfalfa plants

En este trabajo, se estudió la influencia de los polisacáridos formados por cepas de Sinorhizobium meliloti sobre la velocidad de nodulación en plantas de alfalfa. Las experiencias fueron realizadas en agitador rotatorio y en un fermentador con circulación de líquido por inyección de aire, empleando diferentes cepas de S. meliloti, El crecimiento bacteriano fue determinado por recuento de células viables y la concentración del exopolisacárido por precipitación con etanol. Se alcanzaron concentraciones máximas del orden de 1.10 (10) células viables/ml y de 11 g/l de polisacárido. Los estudios de crecimiento de plantas de alfalfa en cámara climatizada mostraron que se producía una reducción en el tiempo de aparición de nódulos de 10 a 4 días, cuando las mismas eran inoculadas con suspensiones diluidas 1/10, manteniendo las cepas sus propiedades simbióticas. (AU)

Crecimiento y formación de polisacáridos de cepas de Sinorhizobium meliloti en un fermentador tipo air-lift. Influencia en la velocidad de nodulación en plantas de alfalfa. / [Growth and polysaccharide formation in Sinorhizobium meliloti strains in an air-lift-type fermentor. Effect on nodulation velocity in alfalfa plants]

In this paper the influence of the exopolysaccharides produced by Sinorhizobium meliloti strains on the nodulation rates in alfalfa plants has been considered. The experiments were performed in a rotary shaker and in an air-lift type fermentor. Different Sinorhizobium meliloti strains were used. Bacterial growth rates were determined by viable cell counts. Exopolysaccharide concentration was determined by precipitation with ethanol. It was observed that maximum cell concentration was in the order of 1 x 10(10) cell/ml and exopolysaccharide content was approximately 11 g/l. The experiments performed with alfalfa plants in a controlled environment chamber showed that, when inoculation was carried out with diluted suspensions (1/10), nodulation time was reduced from 10 to 4 days, while the strains retained their symbiotic properties.

[Production of pectinases by Penicillium simplicissimum A3263 in an amaranth-seed flour medium]. / Producción de pectinasas a partir de Penicillium simplicissimum A3263 en un medio con harina de semilla de amaranto.

The present work studies the production of pectinases using a strain of Penicillium simplicissimum A3263 and considering the influence of adding Amaranthus cruentus seed meal in a selected medium. We also considered the influence of aeration on enzyme production. Research was oriented towards the production of pectin lyase, the enzyme having the highest commercial value. This work was carried out in Erlenmeyer flasks in rotary shaker to select the medium and in a mechanically stirred fermentor to study aeration. The microorganism was developed as pellets of 1 mm diameter. Enzyme levels were of the order of 8216.21 pectin lyase units and 167.57 polygalacturonase units per gram of fungal biomass, respectively, using a medium containing 40 g/l of amaranth seed meal. As for the influence of aeration, it was determined that the higher values were obtained at 750 rpm corresponding to an oxygen absorption rate of 2691 ml O2/lh for an air flow of 1 l/l.min. The results obtained are considered very important in view of the fact that they exceeded in 550% those obtained by other authors.

Producción de pectinasas a partir de Penicillium simplicissimum A3263 en un medio con harina de semilla de amaranto / [Production of pectinases by Penicillium simplicissimum A3263 in an amaranth-seed flour medium]

The present work studies the production of pectinases using a strain of Penicillium simplicissimum A3263 and considering the influence of adding Amaranthus cruentus seed meal in a selected medium. We also considered the influence of aeration on enzyme production. Research was oriented towards the production of pectin lyase, the enzyme having the highest commercial value. This work was carried out in Erlenmeyer flasks in rotary shaker to select the medium and in a mechanically stirred fermentor to study aeration. The microorganism was developed as pellets of 1 mm diameter. Enzyme levels were of the order of 8216.21 pectin lyase units and 167.57 polygalacturonase units per gram of fungal biomass, respectively, using a medium containing 40 g/l of amaranth seed meal. As for the influence of aeration, it was determined that the higher values were obtained at 750 rpm corresponding to an oxygen absorption rate of 2691 ml O2/lh for an air flow of 1 l/l.min. The results obtained are considered very important in view of the fact that they exceeded in 550

those obtained by other authors.

Producción de pectinasas a partir de Penicillium simplicissimum A3263 en un medio con harina de semilla de amaranto. / [Production of pectinases by Penicillium simplicissimum A3263 in an amaranth-seed flour medium]

The present work studies the production of pectinases using a strain of Penicillium simplicissimum A3263 and considering the influence of adding Amaranthus cruentus seed meal in a selected medium. We also considered the influence of aeration on enzyme production. Research was oriented towards the production of pectin lyase, the enzyme having the highest commercial value. This work was carried out in Erlenmeyer flasks in rotary shaker to select the medium and in a mechanically stirred fermentor to study aeration. The microorganism was developed as pellets of 1 mm diameter. Enzyme levels were of the order of 8216.21 pectin lyase units and 167.57 polygalacturonase units per gram of fungal biomass, respectively, using a medium containing 40 g/l of amaranth seed meal. As for the influence of aeration, it was determined that the higher values were obtained at 750 rpm corresponding to an oxygen absorption rate of 2691 ml O2/lh for an air flow of 1 l/l.min. The results obtained are considered very important in view of the fact that they exceeded in 550

those obtained by other authors.

Obtención de xantano a partir de Xanthomonas campestris B-1459 en fermentadores con agitación mecánica / [Isolation of xanthan from Xanthomonas campesteris B-1459 in mechanically agitated fermentors]

Xanthan production from Xanthomonas campestris was studied by a mechanically shaken fermentor. Influence of glucose concentration, aeration of culture media, rheology of broths and pH control was evaluated. Different aeration conditions based on variation of stirring rates were assayed. Substrate concentration was determined according to the Miller method, and polymer production was performed by the Cadmus method. The higher xanthan levels (i.e. 2.3

) were obtained at 750 rpm, with 1 v/v. min. In such conditions, viscosity ranges about 7000 cPoise and a low level of dissolved oxygen were detected in the culture medium. Xanthan production was influenced by the glucose concentration and the presence of amaranth within the culture medium. In the processes wherein an automatic control of pH was performed, the polymer concentration did not increase regarding to processes involving regular pH evolution.

[Isolation of xanthan from Xanthomonas campesteris B-1459 in mechanically agitated fermentors]. / Obtención de xantano a partir de Xanthomonas campestris B-1459 en fermentadores con agitación mecánica.

Xanthan production from Xanthomonas campestris was studied by a mechanically shaken fermentor. Influence of glucose concentration, aeration of culture media, rheology of broths and pH control was evaluated. Different aeration conditions based on variation of stirring rates were assayed. Substrate concentration was determined according to the Miller method, and polymer production was performed by the Cadmus method. The higher xanthan levels (i.e. 2.3%) were obtained at 750 rpm, with 1 v/v. min. In such conditions, viscosity ranges about 7000 cPoise and a low level of dissolved oxygen were detected in the culture medium. Xanthan production was influenced by the glucose concentration and the presence of amaranth within the culture medium. In the processes wherein an automatic control of pH was performed, the polymer concentration did not increase regarding to processes involving regular pH evolution.

Obtención de xantano a partir de Xanthomonas campestris B-1459 en fermentadores con agitación mecánica. / [Isolation of xanthan from Xanthomonas campesteris B-1459 in mechanically agitated fermentors]

Xanthan production from Xanthomonas campestris was studied by a mechanically shaken fermentor. Influence of glucose concentration, aeration of culture media, rheology of broths and pH control was evaluated. Different aeration conditions based on variation of stirring rates were assayed. Substrate concentration was determined according to the Miller method, and polymer production was performed by the Cadmus method. The higher xanthan levels (i.e. 2.3

) were obtained at 750 rpm, with 1 v/v. min. In such conditions, viscosity ranges about 7000 cPoise and a low level of dissolved oxygen were detected in the culture medium. Xanthan production was influenced by the glucose concentration and the presence of amaranth within the culture medium. In the processes wherein an automatic control of pH was performed, the polymer concentration did not increase regarding to processes involving regular pH evolution.

[Xanthan production by Xanthomonas campestris B-1459]. / Producción de xantano a partir de Xanthomonas campestris B-1459.

The production of xantano from Xanthomonas campestris B-1459 was analyzed. The experiments were performed in shaked flasks at 250 rpm and 2.5 cm eccentricity. Using a base modified medium it was possible to achieve xantano concentration of 35 g/l in 72 h of process. The modified medium contained glucose as carbon source, and yeast extract, meat peptone, malt extract and amaranth meal as growth factors and nitrogen sources, in a KH2PO4/K2HPO4 buffer.

Producción de xantano a partir de Xanthomonas campestris B-1459 / [Xanthan production by Xanthomonas campestris B-1459]

The production of xantano from Xanthomonas campestris B-1459 was analyzed. The experiments were performed in shaked flasks at 250 rpm and 2.5 cm eccentricity. Using a base modified medium it was possible to achieve xantano concentration of 35 g/l in 72 h of process. The modified medium contained glucose as carbon source, and yeast extract, meat peptone, malt extract and amaranth meal as growth factors and nitrogen sources, in a KH2PO4/K2HPO4 buffer.

Producción de xantano a partir de Xanthomonas campestris B-1459. / [Xanthan production by Xanthomonas campestris B-1459]

The production of xantano from Xanthomonas campestris B-1459 was analyzed. The experiments were performed in shaked flasks at 250 rpm and 2.5 cm eccentricity. Using a base modified medium it was possible to achieve xantano concentration of 35 g/l in 72 h of process. The modified medium contained glucose as carbon source, and yeast extract, meat peptone, malt extract and amaranth meal as growth factors and nitrogen sources, in a KH2PO4/K2HPO4 buffer.