Molecular Pathways Leading to Induction of Cell Death and Anti-Proliferative Properties by Tacrolimus and mTOR Inhibitors in Liver Cancer Cells

Background/Aims: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells. Methods: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern. Results: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15 P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172 P-Cdk4/ Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors increased the protein expression of FKBP12 and FKBP51 that appeared to play pro-survival role. Interestingly, the administration of immunosuppressants yields a specific pattern of miRNAs. Tacrolimus and mTOR inhibitors decreased miR-92a-1-5p, miR-197-3p, miR-483-3p and miR- 720, and increased miR-22-3p, miR-376a-3p, miR-663b, miR-886-5p, miR-1300 and miR-1303 expressions in HepG2 cells. Conclusion: The more potent pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with an increased activation of PERK and p53 signaling, and p21 protein expression. FKBP12 and FKBP51 appeared to be the most relevant partners of Tacrolimus and mTOR inhibitors exerting a pro-survival effect in HepG2 cells. The observed effects of immunosuppressants were related to a specific miRNA signature in liver cancer cellsEspaña Ministry of Economy and Competitiveness (MINECO) cofinanced by the ERDF (BFU2016-75352-P AEI/FEDER, EU

Regulation of cell death receptor S-nitrosylation and apoptotic signaling by Sorafenib in hepatoblastoma cells

Nitric oxide (NO) plays a relevant role during cell death regulation in tumor cells. The overexpression of nitric oxide synthase type III (NOS-3) induces oxidative and nitrosative stress, p53 and cell death receptor expression and apoptosis in hepatoblastoma cells. S-nitrosylation of cell death receptor modulates apoptosis. Sorafenib is the unique recommended molecular-targeted drug for the treatment of patients with advanced hepatocellular carcinoma. The present study was addressed to elucidate the potential role of NO during Sorafenib-induced cell death in HepG2 cells. We determined the intra- and extracellular NO concentration, cell death receptor expression and their S-nitrosylation modifications, and apoptotic signaling in Sorafenib-treated HepG2 cells. The effect of NO donors on above parameters has also been determined. Sorafenib induced apoptosis in HepG2 cells. However, low concentration of the drug (10nM) increased cell death receptor expression, as well as caspase-8 and -9 activation, but without activation of downstream apoptotic markers. In contrast, Sorafenib (10 µM) reduced upstream apoptotic parameters but increased caspase-3 activation and DNA fragmentation in HepG2 cells. The shift of cell death signaling pathway was associated with a reduction of S-nitrosylation of cell death receptors in Sorafenib-treated cells. The administration of NO donors increased S-nitrosylation of cell death receptors and overall induction of cell death markers in control and Sorafenib-treated cells. In conclusion, Sorafenib induced alteration of cell death receptor S-nitrosylation status which may have a relevant repercussion on cell death signaling in hepatoblastoma cells.Instituto de Salud Carlos III PI13/00021Ministerio de Economía y Competitividad BFU2012-32056Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía BIO-0216Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía CTS-6264Consejería de Salud, Junta de Andalucía PI13/ 0002

Development of a liver graft assessment expert machine-learning system: when the artificial intelligence helps liver transplant surgeons

BackgroundThe complex process of liver graft assessment is one point for improvement in liver transplantation. The main objective of this study is to develop a tool that supports the surgeon who is responsible for liver donation in the decision-making process whether to accept a graft or not using the initial variables available to it.Material and methodLiver graft samples candidate for liver transplantation after donor brain death were studied. All of them were evaluated “in situ” for transplantation, and those discarded after the “in situ” evaluation were considered as no transplantable liver grafts, while those grafts transplanted after “in situ” evaluation were considered as transplantable liver grafts. First, a single-center, retrospective and cohort study identifying the risk factors associated with the no transplantable group was performed. Then, a prediction model decision support system based on machine learning, and using a tree ensemble boosting classifier that is capable of helping to decide whether to accept or decline a donor liver graft, was developed.ResultsA total of 350 liver grafts that were evaluated for liver transplantation were studied. Steatosis was the most frequent reason for classifying grafts as no transplantable, and the main risk factors identified in the univariant study were age, dyslipidemia, personal medical history, personal surgical history, bilirubinemia, and the result of previous liver ultrasound (p < 0.05). When studying the developed model, we observe that the best performance reordering in terms of accuracy corresponds to 76.29% with an area under the curve of 0.79. Furthermore, the model provides a classification together with a confidence index of reliability, for most cases in our data, with the probability of success in the prediction being above 0.85.ConclusionThe tool presented in this study obtains a high accuracy in predicting whether a liver graft will be transplanted or deemed non-transplantable based on the initial variables assigned to it. The inherent capacity for improvement in the system causes the rate of correct predictions to increase as new data are entered. Therefore, we believe it is a tool that can help optimize the graft pool for liver transplantation

Polarimetric imaging for the detection of synthetic models of SARS-CoV-2: A proof of concept

Objective: To conduct a proof-of-concept study of the detection of two synthetic models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using polarimetric imaging. Approach: Two SARS-CoV-2 models were prepared as engineered lentiviruses pseudotyped with the G protein of the vesicular stomatitis virus, and with the characteristic Spike protein of SARS-CoV-2. Samples were prepared in two biofluids (saline solution and artificial saliva), in four concentrations, and deposited as 5-µL droplets on a supporting plate. The angles of maximal degree of linear polarization (DLP) of light diffusely scattered from dry residues were determined using Mueller polarimetry from87 samples at 405 nm and 514 nm. A polarimetric camera was used for imaging several samples under 380–420 nm illumination at angles similar to those of maximal DLP. Per-pixel image analysis included quantification and combination of polarization feature descriptors in 475 samples. Main results: The angles (from sample surface) of maximal DLP were 3° for 405 nm and 6° for 514 nm. Similar viral particles that differed only in the characteristic spike protein of the SARS-CoV-2, their corresponding negative controls, fluids, and the sample holder were discerned at 10-degree and 15-degree configurations. Significance: Polarimetric imaging in the visible spectrum may help improve fast, non-contact detection and identification of viral particles, and/or other microbes such as tuberculosis, in multiple dry fluid samples simultaneously, particularly when combined with other imaging modalities. Further analysis including realistic concentrations of real SARS-CoV-2 viral particles in relevant human fluids is required. Polarimetric imaging under visible light may contribute to a fast, cost-effective screening of SARS-CoV-2 and other pathogens when combined with other imaging modalities.12 página

Hyperspectral image processing for the identification and quantification of lentiviral particles in fluid samples

Optical spectroscopic techniques have been commonly used to detect the presence of biofilm-forming pathogens (bacteria and fungi) in the agro-food industry. Recently, near-infrared (NIR) spectroscopy revealed that it is also possible to detect the presence of viruses in animal and vegetal tissues. Here we report a platform based on visible and NIR (VNIR) hyperspectral imaging for non-contact, reagent free detection and quantification of laboratory-engineered viral particles in fluid samples (liquid droplets and dry residue) using both partial least square-discriminant analysis and artificial feed-forward neural networks. The detection was successfully achieved in preparations of phosphate buffered solution and artificial saliva, with an equivalent pixel volume of 4 nL and lowest concentration of 800 TU.mu L-1. This method constitutes an innovative approach that could be potentially used at point of care for rapid mass screening of viral infectious diseases and monitoring of the SARS-CoV- 2 pandemic.This research was funded by grants number COV20-00080 and COV20-00173 of the 2020 Emergency Call for Research Projects about the SARS-CoV-2 virus and the COVID-19 disease of the Institute of Health 'Carlos III', Spanish Ministry of Science and Innovation, and by grant number EQC2019-006240-P of the 2019 Call for Acquisition of Scientific Equipment, FEDER Program, Spanish Ministry of Science and Innovation. This work has been supported by the European Commission through the JRC HUMAINT project. ABR was supported by grant number RTI2018-094465-J funded by the Spanish National Agency of Research. The authors would like to gratefully acknowledge the assistance of the members of the EOD-CBRN Group of the Spanish National Police, whose identities cannot be disclosed, and who are represented here by JMNG. Authors thank continuous support from their institutions

Priority index on road sections for noise action plan (Índice para determinación de la prioridad de actuación en los tramos de carretera de un plan de acción contra el ruido)

It is well known that the road traffic noise is one of the most important contributors to the environmental impact generated by roads. There no exists hitherto any regulated guidance or procedure that establishes how to undertake a well-founded prioritization among the diverse road sections which are included in the corresponding Action Plans against noise according to the Environmental Noise Directive (2002/49/EC). In this work, the so-called “priority road stretch index” (PRSI) is developed. This index is based on a number of variables (called “priority road stretch variables”, PRSV), which are weighted according to their influence on the road traffic noise problem. This developed index allows sorting out by priority action the road sections included in the Noise Action Plans. Also, in order to test the validity of the proposed methodology, the “priority road stretch index” is applied to the Action Plan against Noise 2008-2012 of the road network of the regional government of Andalusia in the province of Almería

Una metodología para determinar la importancia de los criterios para priorizar las actuaciones contra el ruido del tráfico de carreteras A methodology to determine the significance of criteria for prioritizing road stretches for action against traffic noise

El ruido del tráfico destaca de entre los impactos ambientales de las carreteras, situación que exige medidas para su reducción. El estudio crítico de los planes de acción contra el ruido publicados en España deja patente la generalizada falta de metodologías y de criterios tenidos en cuenta en la toma de decisiones sobre el problema de ordenar por prioridad las actuaciones incluidas en ellos. El presente artículo define estos principales criterios y desarrolla una metodología objetiva y razonada, denominada PATRON, en la cual se determina su importancia relativa dentro de un índice numérico normalizado ponderado, el Índice de Prioridad de Tramo. Para ello, se emplea con éxito el Proceso Analítico Jerárquico Difuso a los resultados de un panel de expertos. La aplicación de esta metodología a un caso de estudio permitió la validación de la asignación de los pesos a estos criterios de decisión en el problema estudiado

A methodology for classification by priority for action: Selecting road stretches for network noise action plans

Road traffic noise is an element of outstanding importance within the overall context of environmental impact. This problem must be technically addressed from an efficient viewpoint, and solutions or alternatives should be considered by means of appropriate and consolidated procedures. Up to now, there is no regulated guideline for establishing well-founded priorities when dealing with the diverse road stretches included in the corresponding Action Plans against noise under the Environmental Noise Directive (2002/49/EC). To this end, the present study proposes a methodology to sort, by priority, road stretches identified by their noise problems and therefore requiring appropriate action. The methodology is based on the so-called ‘‘road stretch priority index’’ (henceforth referred to as RSPI). This index involves a number of variables (called ‘‘road stretch priority variables’’) that are weighted according to their influence on the road traffic noise problem. Thus, the RSPI makes it possible to prioritize different stretches of the Action Plan. To illustrate the application of the proposed methodology, this paper also describes a real case entailing a difficult choice, applying the proposed methodology to a review of the Action Plan against Noise 2008–2012 in the province of Almería, for the road network of the regional government of Andalusia, Spain

Laparoscopic aproach in post-traumatic chylous ascites.

Chylous ascites is a rare condition little reported in literature, with even more exceptional traumatic origin. Its production mechanism is not known exactly but has been attributed to hyperflexion or hyperextension of the spine leading to a rupture of lymph ducts. We present a case of post-traumatic chyloperitoneum in a young patient after abdominal blunt trauma. Conservative treatment should be the first therapeutic option, especially if the origin is traumatic, reserving more aggressive possibilities for cases that do not respond after 15 days, if debt is greater than 1.5 litres/day in adult patients or in those with metabolic complications. A laparoscopic approach is indicated to confirm the diagnosis and to rule out other associated injuries that may require other surgical procedures