Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses.

Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity. Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics. In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC50) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC50 6.6 µM and 1.9 µM respectively) and DENV (IC50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases' active sites.

Icing Condition Predictions Using FBGS.

Icing is a hazard which is important for the aerospace industry and which has grown over the last few years. Developing sensors that can detect the existence not only of standard icing conditions with typically small droplet size, but also of Supercooled Large Droplet (SLD) conditions is one of the most important aims in order to minimize icing hazards in the near future. In the present paper a study of the Fiber Bragg Grating Sensors' (FBGSs) performance as a flight icing detection system that predicts the conditions of an icing cloud is carried out. The test matrix was performed in the INTA Icing Wind Tunnel (IWT) with several icing conditions including SLD. Two optic fibers with 16 FBGS in total were integrated in the lower and upper surface of an airfoil to measure the temperature all over the chord. The results are compared with a Messinger heat and mass balance model and the measurements of the FBGS are used to predict the Liquid Water Content (LWC) and Ice Accretion Rate (IAR). Finally, the results are evaluated and a sensor assessment is made. A good correlation was observed between theoretical calculations and test results obtained with the FBGS in the IWT tests. FBGS proved to detect the beginning and end of ice accretion, LWC and IAR quickly and with good precision.

White Button Mushroom Extracts Modulate Hepatic Fibrosis Progression, Inflammation, and Oxidative Stress In Vitro and in LDLR-/- Mice.

Liver fibrosis can be caused by non-alcoholic steatohepatitis (NASH), among other conditions. We performed a study to analyze the effects of a nontoxic, water-soluble extract of the edible mushroom Agaricus bisporus (AB) as a potential inhibitor of fibrosis progression in vitro using human hepatic stellate cell (LX2) cultures and in vivo in LDLR-/- mice. Treatment of LX2 cells with the AB extract reduced the levels of fibrotic and oxidative-related markers and increased the levels of GATA4 expression. In LDLR-/- mice with high-fat diet (HFD)-induced liver fibrosis and inflammation, the progression of fibrosis, oxidative stress, inflammation, and apoptosis were prevented by AB extract treatment. Moreover, in the mouse model, AB extract could exert an antiatherogenic effect. These data suggest that AB mushroom extract seems to exert protective effects by alleviating inflammation and oxidative stress during the progression of liver fibrosis, possibly due to a decrease in Toll-like receptor 4 (TLR4) expression and a reduction in Nod-like receptor protein 3 (NLRP3) inflammasome activation. In addition, we observed a potential atheroprotective effect in our mouse model.

Quasicrystalline Coatings Exhibit Durable Low Interfacial Toughness with Ice.

Ice accretion can adversely impact many engineering structures in commercial and residential sectors. Although there are many reports of low-ice-adhesion-strength materials, a scalable and durable deicing solution remains elusive, as ice detachment is dominated by interfacial toughness for large interfaces. In this work, durable metallic coatings based on Al-rich quasicrystalline alloys were prepared and applied on aluminum substrates using high-velocity oxyfuel thermal spray. X-ray diffraction patterns confirmed the quasicrystalline phases of the coating, and its large-scale deicing capability was studied by evaluating the coating's ice detachment mechanics using long lengths of ice. A toughness-controlled regime of interfacial fracture was observed for ice lengths longer than ∼2 cm, and a low shear strength of ∼30 kPa was achieved for a 20 cm ice length. The metallic coatings exhibited excellent ice repellency even after being abraded, scratched, heated, UV-irradiated, and exposed to chemical contaminations, demonstrating promising durability for real-world, large-scale ice removal.

The Absence of NLRP3-inflammasome Modulates Hepatic Fibrosis Progression, Lipid Metabolism, and Inflammation in KO NLRP3 Mice during Aging.

Aging is associated with metabolic changes and low-grade inflammation in several organs, which may be due to NLRP3 inflammasome activation. Methods: Here, we asked whether age-related liver changes such as lipid metabolism and fibrosis are reduced in aged mice lacking the NLRP3 inflammasome. We report reduced protein levels of lipid markers (MTP, FASN, DGAT1), SOD activity, oxidative stress marker PTPRG, and the fibrotic markers TPM2ß, COL1-α1 associated with increased GATA4, in NLRP3 deficient mice. Fibrotic, lipid, and oxidative reduction in liver tissues of mice was more pronounced in those old KO NLRP3 mice than in the younger ones, despite their greater liver damage. These results suggest that absence of the NLRP3 inflammasome attenuates age-related liver fibrotic pathology in mice, suggesting that pharmacological targeting may be beneficial.

Integrated molecular signaling involving mitochondrial dysfunction and alteration of cell metabolism induced by tyrosine kinase inhibitors in cancer.

Cancer cells have unlimited replicative potential, insensitivity to growth-inhibitory signals, evasion of apoptosis, cellular stress, and sustained angiogenesis, invasiveness and metastatic potential. Cancer cells adequately adapt cell metabolism and integrate several intracellular and redox signaling to promote cell survival in an inflammatory and hypoxic microenvironment in order to maintain/expand tumor phenotype. The administration of tyrosine kinase inhibitor (TKI) constitutes the recommended therapeutic strategy in different malignancies at advanced stages. There are important interrelationships between cell stress, redox status, mitochondrial function, metabolism and cellular signaling pathways leading to cell survival/death. The induction of apoptosis and cell cycle arrest widely related to the antitumoral properties of TKIs result from tightly controlled events involving different cellular compartments and signaling pathways. The aim of the present review is to update the most relevant studies dealing with the impact of TKI treatment on cell function. The induction of endoplasmic reticulum (ER) stress and Ca2+ disturbances, leading to alteration of mitochondrial function, redox status and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways that involve cell metabolism reprogramming in cancer cells will be covered. Emphasis will be given to studies that identify key components of the integrated molecular pattern including receptor tyrosine kinase (RTK) downstream signaling, cell death and mitochondria-related events that appear to be involved in the resistance of cancer cells to TKI treatments.

Dose-dependent regulation of mitochondrial function and cell death pathway by sorafenib in liver cancer cells.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth most frequent cause of cancer-related death worldwide. Sorafenib is the first line recommended therapy for patients with locally advanced/metastatic HCC. The low response rate is attributed to intrinsic resistance of HCC cells to Sorafenib. The potential resistance to Sorafenib-induced cell death is multifactorial and involves all hallmarks of cancer. However, the presence of sub-therapeutic dose can negatively influence the antitumoral properties of the drug. In this sense, the present study showed that the sub-optimal Sorafenib concentration (10 nM) was associated with activation of caspase-9, AMP-activated protein kinase (AMPK), sustained autophagy, peroxisome proliferator-activated receptor-coactivator 1α (PGC-1α) and mitochondrial function in HepG2 cells. The increased mitochondrial respiration by Sorafenib (10 nM) was also observed in permeabilized HepG2 cells, but not in isolated rat mitochondria, which suggests the involvement of an upstream component in this regulatory mechanism. The basal glycolysis was dose dependently increased at early time point studied (6 h). Interestingly, Sorafenib increased nitric oxide (NO) generation that played an inhibitory role in mitochondrial respiration in sub-therapeutic dose of Sorafenib. The administration of sustained therapeutic dose of Sorafenib (10 µM, 24 h) induced mitochondrial dysfunction and dropped basal glycolysis derived acidification, as well as increased oxidative stress and apoptosis in HepG2. In conclusion, the accurate control of the administered dose of Sorafenib is relevant for the potential prosurvival or proapoptotic properties induced by the drug in liver cancer cells.

Water-soluble extracts from edible mushrooms (Agaricus bisporus) as inhibitors of hepatitis C viral replication.

Hepatitis C virus (HCV) is the main agent responsible for chronic liver disease. Recent advances in anti-HCV treatment strategies have significantly increased the viral clearance rate (>90%). However, sustained antiviral responses vary in different cohorts, and high costs limit the broad use of direct-acting antivirals (DAAs). The goal of this study is to evaluate the inhibitory ability of well characterized (LC-QTOF-MS/MS) aqueous extracts obtained from edible mushrooms (Agaricus bisporus) to diminish HCV viral replication. Our data have demonstrated an in vitro inhibitory effect of A. bisporus extracts on NS3/4A protease and HCV replication. Fractionation by ultra-filtration and sequential liquid-liquid extraction showed that the compounds responsible for the inhibition are water-soluble with low molecular weights (<3 kDa) and that action could be through the following five compounds: ergothioneine, adenine, guanine, hypoxanthine, and xanthine, which are present in all fractions (UF-3, AqF-3 kDa and organic fractions) showing NS3/4A inhibition. Low molecular weight aqueous extracts (<3 kDa) from A. bisporus have potential applications in the prophylaxis and treatment of HCV, especially for patients who do not have access to the last generation of DAAs. They may be useful as well for other flaviviruses, which also possess a NS3 serine protease.

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 Ser51 P-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,Tyr185 P-JNK1/2/JNK1/2, Thr172 P-AMPKα, Ser413 P-Foxo3a, Thr308 P-AKt/AKt and Thr32 P-Foxo3a/Foxo3a ratios, and reduction of Ser2481 P-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 Thr308 P-AKt/AKt and Ser473 P-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.

Genetic and Epigenetic Regulation in Nonalcoholic Fatty Liver Disease (NAFLD).

Genetics and epigenetics play a key role in the development of several diseases, including nonalcoholic fatty liver disease (NAFLD). Family studies demonstrate that first degree relatives of patients with NAFLD are at a much higher risk of the disease than the general population. The development of the Genome Wide Association Study (GWAS) technology has allowed the identification of numerous genetic polymorphisms involved in the evolution of diseases (e.g., PNPLA3, MBOAT7). On the other hand, epigenetic changes interact with inherited risk factors to determine an individual's susceptibility to NAFLD. Modifications of the histones amino-terminal ends are key factors in the maintenance of chromatin structure and gene expression (cAMP-responsive element binding protein H (CREBH) or SIRT1). Activation of SIRT1 showed potential against the physiological mechanisms related to NAFLD. Abnormal DNA methylation represents a starting point for cancer development in NAFLD patients. Besides, the evaluation of circulating miRNA profiles represents a promising approach to assess and non-invasively monitor liver disease severity. To date, there is no approved pharmacologic therapy for NAFLD and the current treatment remains weight loss with lifestyle modification and exercise. In this review, the status of research into relevant genetic and epigenetic modifiers of NAFLD progression will be discussed.

Role of inflammatory response in liver diseases: Therapeutic strategies.

Inflammation and tumorigenesis are tightly linked pathways impacting cancer development. Inflammasomes are key signalling platforms that detect pathogenic microorganisms, including hepatitis C virus (HCV) infection, and sterile stressors (oxidative stress, insulin resistance, lipotoxicity) able to activate pro-inflammatory cytokines interleukin-1ß and IL-18. Most of the inflammasome complexes that have been described to date contain a NOD-like receptor sensor molecule. Redox state and autophagy can regulate inflammasome complex and, depending on the conditions, can be either pro- or anti-apoptotic. Acute and chronic liver diseases are cytokine-driven diseases as several proinflammatory cytokines (IL-1α, IL-1ß, tumor necrosis factor-alpha, and IL-6) are critically involved in inflammation, steatosis, fibrosis, and cancer development. NLRP3 inflammasome gain of function aggravates liver disease, resulting in severe liver fibrosis and highlighting this pathway in the pathogenesis of non-alcoholic fatty liver disease. On the other hand, HCV infection is the primary catalyst for progressive liver disease and development of liver cancer. It is well established that HCV-induced IL-1ß production by hepatic macrophages plays a critical and central process that promotes liver inflammation and disease. In this review, we aim to clarify the role of the inflammasome in the aggravation of liver disease, and how selective blockade of this main pathway may be a useful strategy to delay fibrosis progression in liver diseases.

Simvastatin and metformin inhibit cell growth in hepatitis C virus infected cells via mTOR increasing PTEN and autophagy.

Hepatitis C virus (HCV) infection has been related to increased risk of development of hepatocellular carcinoma (HCC) while metformin (M) and statins treatment seemed to protect against HCC development. In this work, we aim to identify the mechanisms by which metformin and simvastatin (S) could protect from liver cancer. Huh7.5 cells were infected with HCV particles and treated with M+S. Human primary hepatocytes were treated with M+S. Treatment with both drugs inhibited Huh7.5 cell growth and HCV infection. In non-infected cells S increased translational controlled tumor protein (TCTP) and phosphatase and tensin homolog (PTEN) proteins while M inhibited mammalian target of rapamycin (mTOR) and TCTP. Simvastatin and metformin co-administered down-regulated mTOR and TCTP, while PTEN was increased. In cells infected by HCV, mTOR, TCTP, p62 and light chain 3B II (LC3BII) were increased and PTEN was decreased. S+M treatment increased PTEN, p62 and LC3BII in Huh7.5 cells. In human primary hepatocytes, metformin treatment inhibited mTOR and PTEN, but up-regulated p62, LC3BII and Caspase 3. In conclusion, simvastatin and metformin inhibited cell growth and HCV infection in vitro. In human hepatocytes, metformin increased cell-death markers. These findings suggest that M+S treatment could be useful in therapeutic prevention of HCV-related hepatocellular carcinoma.

Natural Extracts Abolished Lipid Accumulation in Cells Harbouring non-favourable PNPLA3 genotype.

Background & aims. G-allele of PNPLA3 (rs738409) favours triglycerides accumulation and steatosis. In this study, we examined the effect of quercetin and natural extracts from mushroom and artichoke on reducing lipid accumulation in hepatic cells. MATERIAL AND METHODS: Huh7.5 cells were exposed to oleic acid (OA) and treated with quercetin and extracts to observe the lipid accumulation, the intracellular-TG concentration and the LD size. Sterol regulatory element binding proteins-1 (SREBP-1), peroxisome proliferator-activated receptor (PPARα-γ) and cholesterol acyltransferase (ACAT) gene expression levels were analysed. RESULTS: Quercetin decreased the intracellular lipids, LD size and the levels of intracellular-TG through the down-regulation of SREBP-1c, PPARγ and ACAT1 increasing PPARα. The natural-extracts suppressed OA-induced lipid accumulation and the intracellular-TG. They down-regulate the hepatic lipogenesis through SREBP-1c, besides the activation of lipolysis through the increasing of PPARα expression. CONCLUSIONS: Quercetin and the aqueous extracts decrease intracellular lipid accumulation by down-regulation of lipogenesis and up-regulation of lipolysis.

[Prognostic value of electrocardiographic findings in hemodynamically stable patients with acute symptomatic pulmonary embolism]. / Valor pronóstico de los hallazgos electrocardiográficos en pacientes estables hemodinámicamente con tromboembolia de pulmón aguda sintomática.

INTRODUCTION AND OBJECTIVES: The aim of this study was to determine the prognostic value of electrocardiography in hemodynamically stable patients with a diagnosis of acute symptomatic pulmonary embolism (PE). METHODS: This prospective study included all hemodynamically stable outpatients who were diagnosed with PE at a university hospital. The electrocardiographic abnormalities investigated were: a) sinus tachycardia (>100 beats/min); b) ST-segment or T-wave abnormalities; c) right bundle branch block; d) an S1Q3T3 pattern, and e) recent-onset atrial arrhythmia. RESULTS: The study included 644 patients. Overall, 5% of those with an ECG abnormality died due to PE in the 15 days after diagnosis compared with 2% of those with normal ECG findings (relative risk [RR]=2.4; 95% confidence interval [CI], 1-5,8; P=.05). Multivariate analysis showed that sinus tachycardia was associated with a 2.2-fold increased risk of death due to all causes in the month after PE diagnosis. After adjusting for age, a history of cancer, immobility, ECG abnormalities, and sinus tachycardia, the presence of recent-onset atrial arrhythmia was significantly associated with death due to PE in the first 15 days (RR=2.8; 95% CI, 1-8.3; P=.05). The negative predictive value of atrial arrhythmia for 15-day PE-related mortality was 97%, while the negative likelihood ratio was 0.79. CONCLUSIONS: In hemodynamically stable patients with acute symptomatic PE, the presence of sinus tachycardia and atrial arrhythmia were independent predictors of a poor prognosis. However, the usefulness of these factors for stratifying risk in PE patients is limited.

Valor pronóstico de los hallazgos electrocardiográficos en pacientes estables hemodinámicamente con tromboembolia de pulmón aguda sintomática / Prognostic value of electrocardiographic findings in hemodynamically stable patients with acute symptomatic pulmonary embolism

Introducción y objetivos. El objetivo de este estudio es evaluar el valor pronóstico del electrocardiograma (ECG) en pacientes estables hemodinámicamente con diagnóstico de tromboembolia pulmonar (TEP) aguda sintomática. Métodos. Se incluyó de forma prospectiva a todos los pacientes ambulatorios estables hemodinámicamente diagnosticados de TEP aguda sintomática en un hospital universitario terciario. Las anomalías electrocardiográficas consideradas fueron: a) taquicardia sinusal (> 100 lat/min); b) alteraciones del segmento ST o de la onda T; c) bloqueo de la rama derecha del haz de His (BRDHH); d) patrón S1Q3T3, y e) arritmias auriculares de reciente comienzo. Resultados. Se incluyó a 644 pacientes en el estudio. Un 5% de los pacientes con ECG anormal fallecieron por TEP en los 15 días posteriores al diagnóstico, comparado con un 2% de los pacientes con ECG normal (razón de riesgo [RR] = 2,4; intervalo de confianza [IC] del 95%, 1-5,8; p = 0,05). En el análisis multivariable, la taquicardia sinusal multiplicó por 2,2 el riesgo de muerte por todas las causas en el mes posterior al diagnóstico de TEP. Tras ajustar por edad, antecedentes de cáncer, inmovilización, un ECG alterado y la presencia de taquicardia sinusal, las arritmias auriculares de reciente diagnóstico se asociaron de forma significativa a la muerte por TEP durante los primeros 15 días (RR = 2,8; IC del 95%, 1-8,3; p = 0,05). Las arritmias auriculares mostraron un alto valor predictivo negativo de muerte por TEP a los 15 días (97%), pero la razón de probabilidad negativa fue 0,79. Conclusiones. En pacientes estables hemodinámicamente con TEP aguda sintomática, la taquicardia sinusal y las arritmias auriculares son predictoras independientes de mal pronóstico. Sin embargo, su utilidad en la estratificación pronóstica de estos pacientes es limitada

Introduction and objectives. The aim of this study was to determine the prognostic value of electrocardiography in hemodynamically stable patients with a diagnosis of acute symptomatic pulmonary embolism (PE). Methods. This prospective study included all hemodynamically stable outpatients who were diagnosed with PE at a university hospital. The electrocardiographic abnormalities investigated were: a) sinus tachycardia (>100 beats/min); b) ST-segment or T-wave abnormalities; c) right bundle branch block; d) an S1Q3T3 pattern, and e) recent-onset atrial arrhythmia. Results. The study included 644 patients. Overall, 5% of those with an ECG abnormality died due to PE in the 15 days after diagnosis compared with 2% of those with normal ECG findings (relative risk [RR]=2.4; 95% confidence interval [CI], 1­5,8; P=.05). Multivariate analysis showed that sinus tachycardia was associated with a 2.2-fold increased risk of death due to all causes in the month after PE diagnosis. After adjusting for age, a history of cancer, immobility, ECG abnormalities, and sinus tachycardia, the presence of recent-onset atrial arrhythmia was significantly associated with death due to PE in the first 15 days (RR=2.8; 95% CI, 1­8.3; P=.05). The negative predictive value of atrial arrhythmia for 15-day PE-related mortality was 97%, while the negative likelihood ratio was 0.79. Conclusions. In hemodynamically stable patients with acute symptomatic PE, the presence of sinus tachycardia and atrial arrhythmia were independent predictors of a poor prognosis. However, the usefulness of these factors for stratifying risk in PE patients is limited