Molecular characterization of autophagic and apoptotic signaling induced by sorafenib in liver cancer cells

Sorafenib is the unique accepted molecular targeted drug for the treatment of patients in advanced stage of hepatocellular carcinoma. The current study evaluated cell signaling regulation of endoplasmic reticulum (ER) stress, c-Jun-N-terminal kinase (JNK), Akt, and 5′AMP-activated protein kinase (AMPK) leading to autophagy and apoptosis induced by sorafenib. Sorafenib induced early (3–12 hr) ER stress characterized by an increase of Ser51P-eIF2α/eIF2α, C/EBP homologous protein (CHOP), IRE1α, and sXBP1, but a decrease of activating transcription factor 6 expression, overall temporally associated with the increase of Thr183,Tyr185P-JNK1/2/JNK1/2, Thr172P-AMPKα, Ser413P-Foxo3a, Thr308P-AKt/AKt and Thr32P-Foxo3a/Foxo3a ratios, and reduction of Ser2481P-mammalian target of rapamycin (mTOR)/mTOR and protein translation. This pattern was related to a transient increase of tBid, Bim EL, Beclin-1, Bcl-xL, Bcl-2, autophagy markers, and reduction of myeloid cell leukemia-1 (Mcl-1) expression. The progressive increase of CHOP expression, and reduction of Thr308P-AKt/AKt and Ser473P-AKt/AKt ratios were associated with the reduction of autophagic flux and an additional upregulation of Bim EL expression and caspase-3 activity (24 hr). Small interfering-RNA (si-RNA) assays showed that Bim, but not Bak and Bax, was involved in the induction of caspase-3 in sorafenib-treated HepG2 cells. Sorafenib increased autophagic and apoptotic markers in tumor-derived xenograft model. In conclusion, the early sorafenib-induced ER stress and regulation of JNK and AMPK-dependent signaling were related to the induction of survival autophagic process. The sustained drug treatment induced a progressive increase of ER stress and PERK-CHOP-dependent rise of Bim EL, which was associated with the shift from autophagy to apoptosis. The kinetic of Bim EL expression profile might also be related to the tight balance between AKt- and AMPK-related signaling leading to Foxo3a-dependent BIM EL upregulation.Ministerio de Economía y Competitividad BFU2016‐75352‐PInstituto de Salud Carlos III PI15/00034, PI13/ 00021, PI16/00090, PI14/01349Ministerio de Educación FPU16/05127, FPU12/01433, FPU13/01237Junta de Andalucía CTS-6264, PI-00025-2013, PI-0127-2013, PI-0198-201

Petrographical and geochemical characterization and deformation conditions of the San Cristóbal pluton, Sierra de Velasco, La Rioja, Argentina

El plutón San Cristóbal constituye un cuerpo granítico de 35 km2 que aflora en el extremo sudeste de la Sierra de Velasco, situada al oeste de la ciudad de La Rioja, Argentina. Está formado por monzogranitos y sienogranitos con escasas granodioritas, de texturas equigranulares de grano medio a fino a ligeramente porfíricas. La asociación mineral es cuarzo + microclino + plagioclasa + biotita ± moscovita + circón + apatito + magnetita. Contiene enclaves magmáticos máficos de composición diorítica a tonalítica. El granito fue afectado en su parte media y este por una zona de cizalla, formada por milonitas que integran la Faja Milonítica Sur, de rumbo NNO-SSE. La roca de caja se reconoce por xenolitos de esquistos y cuarcitas en facies esquistos verdes, y septos de corneanas con la paragénesis feldespato potásico - cordierita - biotita ± sillimanita. Los granitos son calcoalcalinos, débil a moderadamente peraluminosos, emplazados en un ambiente de arco magmático continental desarrollado en el borde occidental de Gondwana durante el Paleozoico Inferior. La profundidad de emplazamiento del plutón estaría en el orden de los 12 km.The San Cristóbal pluton is a 35 km2 granitic body that outcrops at the southestern tip of the Sierra de Velasco, located west of La Rioja city, Argentina. It is formed by monzogranites and syenogranites, together with scarce granodiorites, with medium to fine-grained, equigranular to slightly porphyritic textu- res. Their mineral assemblage consists of quartz + microcline + plagioclase + biotite ± muscovite + zir- con + apatite + magnetite. The granite contains dioritic to tonalitic mafic enclaves. The central and eas- tern parts of the granite have been deformed by the NNW-SSE trending South Mylonitic shear zone for- med by mylonitic rocks. The metamorphic host-rock is represented by scarce greenschist facies xeno- liths and hornfels with the high T/P assemblage K-feldspar – cordierite – biotite ± sillimanite. The grani- tes are calc-alkaline, weak- to moderately peraluminous, and formed as part of a continental magmatic arc developed along the active margin of western Gondwana during the Early Paleozoic. The depth of emplacement of the San Cristóbal pluton is estimated at ~12 km.Fil: Bellos, Laura Iudith. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Correlación Geológica. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Departamento de Geología. Cátedra Geología Estructural. Instituto Superior de Correlación Geológica; ArgentinaFil: Toselli, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Correlación Geológica. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Departamento de Geología. Cátedra Geología Estructural. Instituto Superior de Correlación Geológica; ArgentinaFil: Rossi, J. N.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Correlación Geológica. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo. Departamento de Geología. Cátedra Geología Estructural. Instituto Superior de Correlación Geológica; ArgentinaFil: Grosse, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán; ArgentinaFil: de la Rosa, J. D.. Universidad de Huelva; EspañaFil: Castro, A.. Universidad de Huelva; Españ

Infrared Spectroscopy as a Tool to Study the Antioxidant Activity of Polyphenolic Compounds in Isolated Rat Enterocytes

The protective effect of different polyphenols, catechin (Cat), quercetin (Qc) (flavonoids), gallic acid (GA), caffeic acid (CfA), chlorogenic acid (ChA) (phenolic acids), and capsaicin (Cap), against H2O2-induced oxidative stress was evaluated in rat enterocytes using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy and Fourier Transform Infrared Microspectroscopy (FTIRM), and results were compared to standard lipid peroxidation techniques: conjugated dienes (CD) and Thiobarbituric Acid Reactive Substances (TBARS). Analysis of ATR-FTIR and FTIRM spectral data allowed the simultaneous evaluation of the effects of H2O2 and polyphenols on lipid and protein oxidation. All polyphenols showed a protective effect against H2O2-induced oxidative stress in enterocytes, when administered before or after H2O2. Cat and capsaicin showed the highest protective effect, while phenolic acids had weaker effects and Qc presented a mild prooxidative effect (IR spectral profile of biomolecules between control and H2O2-treated cells) according to FTIR analyses. These results demonstrated the viability to use infrared spectroscopy to evaluate the oxidant and antioxidant effect of molecules in cell systems assays

Inhibición de lipasa pancreática por flavonoides: importancia del doble enlace C2=C3 y la estructura plana del anillo C//Inhibition of pancreatic lipase by flavonoids: relevance of the C2=C3 double bond and C-ring planarity

Lipasa pancreática es una enzima clave en el metabolismo de lípidos. Los flavonoides son compuestos bioactivos de gran relevancia debido a sus interacciones con enzimas digestivas. Se evaluó la actividad de lipasa pancreática en presencia de flavonoides. Mediante espectroscopía UVVisible se determinó que el mejor inhibidor fue quercetina, seguido de rutina > luteolina > catequina > hesperetina, con valores de IC50 de 10.30, 13.50, 14.70, 28.50 y 30.50 μM, respectivamente. Todos los flavonoides mostraron una inhibición mixta, excepto catequina que mostró una inhibición acompetitiva. La capacidad inhibitoria de los flavonoides se relacionó con propiedades estructurales compartidas entre los distintos flavonoides, como la hidroxilación en las posiciones C5, C7 (anillo A), C2’ y C3’ (anillo B), y el doble enlace entre C2 y C3 (anillo C). Los resultados de inhibición coincidieron con el análisis de la fluorescencia extrínseca. Los estudios de docking molecular indicaron que la interacción entre lipasa pancreática y los flavonoides fue principalmente mediante interacciones hidrofóbicas (pi-stacking). Las interacciones de todos los flavonoides, excepto rutina, se dieron en el mismo sitio (subsitio 1) de la enzima. La insaturación entre C2 y C3 fue determinante para el acomodo de los flavonoides con la enzima, principalmente por interacciones de pi-stacking.ABSTRACTPancreatic lipase is a key enzyme in lipid metabolism. Flavonoids are bioactive compounds obtained from vegetables with big relevance, due to their intrinsic interaction with digestive enzymes. Pancreatic lipase activity was evaluated in the presence of flavonoids, through UV-Vis spectroscopy. All tested flavonoids showed a mixed-type inhibition, except catechin, which showed a uncompetitive inhibition. The best inhibitor was quercetin followed by rutin > luteolin > catechin > hesperetin, with IC50 values of 10.30, 13.50, 14.70, 28.50 and 30.50 μM, respectively. The flavonoids inhibitory capacity was related to structural properties shared between the different flavonoids, such as the hydroxylation at C5, C7 (ring A), C2’ and C3’ (ring B), and the double bond between C2 and C3 (ring C). The inhibition results are in agreement with the extrinsic fluorescence analysis. Molecular docking studies indicated that the interaction between pancreatic lipase and flavonoids was mainly through hydrophobic interactions (pi-stacking). The interactions of all flavonoids, except rutin, occurred at the same enzyme site (subsite 1). Instauration between C2 and C3 was decisive for the arrangement of flavonoids with the enzyme, mainly due to pi-stacking interactions

In vitro Inhibition of Pancreatic Lipase by Polyphenols: A Kinetic, Fluorescence Spectroscopy and Molecular Docking Study

Svrha je ovog istraživanja bila ispitati molekulsko uklapanje i inhibicijski učinak četiri fenolna spoja pronađena u ljutim papričicama, i to: kavene kiseline, p-kumarne kiseline, kvercetina i kapsaicina, na aktivnost lipaze izolirane iz svinjske gušterače. Najjači inhibicijski učinak imao je kvercetin (IC50=(6.1±2.4) μM), zatim p-kumarna (170.2±20.6) μM) i kavena kiselina (401.5±32.1) μM), dok su kapsaicin i ekstrakt ljute papričice imali iznimno slab učinak. Svi polifenolni spojevi imali su inhibicijski učinak miješanog tipa. Mjerenjem fluorescencije utvrđeno je da su polifenolni spojevi ugasili prirođenu fluorescenciju lipaze izolirane iz gušterače, i to pomoću statičkog mehanizma. Sekvencija Stern-Volmerove konstante bila je: kvercetin, kavena kiselina, te p-kumarna kiselina. Rezultati ispitivanja molekulskih uklapanja pokazali su da se kavena kiselina, kvercetin i p-kumarna kiselina vežu blizu, za razliku od kapsaicina koji se veže daleko od aktivnog mjesta. Vodikove veze i hidrofobne pi-interakcije glavni su načini međusobnog povezivanja polifenolnih spojeva u lipazi izoliranoj iz gušterače.The inhibitory activity and binding characteristics of caffeic acid, p-coumaric acid, quercetin and capsaicin, four phenolic compounds found in hot pepper, against porcine pancreatic lipase activity were studied and compared to hot pepper extract. Quercetin was the strongest inhibitor (IC50=(6.1±2.4) μM), followed by p-coumaric acid ((170.2±20.6) μM) and caffeic acid ((401.5±32.1) μM), while capsaicin and a hot pepper extract had very low inhibitory activity. All polyphenolic compounds showed a mixed-type inhibition. Fluorescence spectroscopy studies showed that polyphenolic compounds had the ability to quench the intrinsic fluorescence of pancreatic lipase by a static mechanism. The sequence of Stern-Volmer constant was quercetin, followed by caffeic and p-coumaric acids. Molecular docking studies showed that caffeic acid, quercetin and p-coumaric acid bound near the active site, while capsaicin bound far away from the active site. Hydrogen bonds and π-stacking hydrophobic interactions are the main pancreatic lipase-polyphenolic compound interactions observed

Krebs von den Lungen-6 glycoprotein circulating levels are not useful as prognostic marker in COVID-19 pneumonia : A large prospective cohort study

Altres ajuts: Departament de Salut, Generalitat de Catalunya (COVID-PoC BioCAT).Coronavirus disease 2019 (COVID-19) has rapidly expanded worldwide. Currently, there are no biomarkers to predict respiratory worsening in patients with mild to moderate COVID-19 pneumonia. Small studies explored the use of Krebs von de Lungen-6 circulating serum levels (sKL-6) as a prognostic biomarker of the worsening of COVID-19 pneumonia. We aimed at a large study to determine the prognostic value of sKL-6 in predicting evolving trends in COVID-19. We prospectively analyzed the characteristics of 836 patients with COVID-19 with mild lung disease on admission. sKL-6 was obtained in all patients at least at baseline and compared among patients with or without respiratory worsening. The receiver operating characteristic curve was used to find the optimal cutoff level. A total of 159 (19%) patients developed respiratory worsening during hospitalization. Baseline sKL-6 levels were not higher in patients who had respiratory worsening (median {IQR} 315.5 {209-469} vs. 306 {214-423} U/ml p = 0.38). The last sKL-6 and the change between baseline and last sKL-6 were higher in the respiratory worsening group (p = 0.02 and p < 0.0001, respectively). The best sKL-6 cutoff point for respiratory worsening was 497 U/ml (area under the curve 0.52; 23% sensitivity and 85% specificity). sKL-6 was not found to be an independent predictor of respiratory worsening. A conditional inference tree (CTREE) was not useful to discriminate patients at risk of worsening. We found that sKL-6 had a low sensibility to predict respiratory worsening in patients with mild-moderate COVID-19 pneumonia and may not be of use to assess the risk of present respiratory worsening in inpatients with COVID-19 pneumonia

Phosphorylation disrupts long-distance electron transport in cytochrome c

It has been recently shown that electron transfer between mitochondrial cytochrome c and the cytochrome c1 subunit of the cytochrome bc1 can proceed at long-distance through the aqueous solution. Cytochrome c is thought to adjust its activity by changing the affinity for its partners via Tyr48 phosphorylation, but it is unknown how it impacts the nanoscopic environment, interaction forces, and long-range electron transfer. Here, we constrain the orientation and separation between cytochrome c1 and cytochrome c or the phosphomimetic Y48pCMF cytochrome c, and deploy an array of single-molecule, bulk, and computational methods to investigate the molecular mechanism of electron transfer regulation by cytochrome c phosphorylation. We demonstrate that phosphorylation impairs long-range electron transfer, shortens the long-distance charge conduit between the partners, strengthens their interaction, and departs it from equilibrium. These results unveil a nanoscopic view of the interaction between redox protein partners in electron transport chains and its mechanisms of regulation

Guanidinium and spermidinium decavanadates: as small biomimetic models to understand non-covalent interactions between decavanadate and arginine and lysine side chains in proteins

During the last three decades, numerous investigations have been conducted on polyoxidovanadates to treat several illnesses and inhibit enzymes. Numerous decavanadate compounds have been proposed as potential therapies for Diabetes mellitus, Cancer, and Alzheimer’s disease. Only six relevant functional proteins interacting with decavanadate, V10, have been deposited in the PDB. These are acid phosphatase, tyrosine kinase, two ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), the human transient receptor potential cation channel (TRPM4), and the human cell cycle protein CksHs1. The interaction sites in these proteins mainly consist of Arginine and Lysine, side chains binding to the decavanadate anion. To get further knowledge regarding non-covalent interactions of decavanadate in protein environments, guanidinium and spermidinium decavanadates were synthesized, crystallized, and subjected to analysis utilizing various techniques, including FTIR, Raman, 51V-NMR, TGA, and X-ray diffraction. The DFT calculations were employed to calculate the interaction energy between the decavanadate anion and the organic counterions. Furthermore, the Quantum Theory of Atoms in Molecules (QTAIM) and Non-covalent Interaction-Reduced Density Gradient (NCI-RDG) analyses were conducted to understand the non-covalent interactions present in these adducts. Decavanadate can engage in electrostatic forces, van der Waals, and hydrogen bond interactions with guanidinium and spermidinium, as shown by their respective interaction energies. Both compounds were highly stabilized by strong hydrogen bond interactions N−H···O and weak non-covalent interactions C−H···O. In addition, the interactions between guanidinium and spermidinium cations and decavanadate anion form several stable rings. This study provides new information on non-covalent intermolecular interactions between decavanadate and small biomimetic models of arginine and lysine lateral chains in protein environments

BAYESIAN PREDICTION METHOD FOR SHADOW DETECTION AND RECONSTRUCTION IN HSR IMAGES USING MORPHOLOGICAL FILTER

Several approaches are exists today according to color, intensity and saturation value etc that are very less accurate. Within this paper, we advise alternative shadow recognition formula according to thresholding and morphological filtering, along with an alternate shadow renovation formula in line with the example learning method and Markov random field (MRF). The primary purpose of this project is recognition and renovation of shadows from VHSR images. Removing or alleviating the instants while using shadows in HSR images for more processing is an extremely important task because the shadows are induce to loss or miss conjecture of radiometric information and induce to image interpretation. Throughout the shadow recognition procedure, the bimodal distributions of pixel values within the near-infrared (NIR) band and also the panchromatic band are adopted for thresholding. Throughout the shadow renovation procedure, we model the connection between non shadow and also the corresponding shadow pixels and between neighboring no shadow pixels by using MRF. With extension for this paper we advise Bayesian conjecture way of accurate conjecture of shadow. Within this paper for accurate shadow recognition we combine thresholding and morphological filtering concepts. This shadow recognition includes Thresholding, Morphological filtering and edge compensation stages