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

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, Ser15P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172P-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 cells.We thank the Institute of Health Carlos III (ISCiii) cofinanced by the European Regional Development Fund “A way to achieve Europe” (ERDF) (PI13/00021, P16/00090 and PI19/01266), as well as the Andalusian Ministry of the Economy, Innovation, Science and Employment (CTS-6264) and Andalusian Ministry of Health (PI13/00025, PI16/0198, PIP-0215-2020 and PI-0216-2020) for their financial support to J.M. We also thank the Spanish Ministry of Economy and Competitiveness (MINECO) cofinanced by the ERDF (BFU2016-75352-P AEI/FEDER, EU) for their financial support to J.d.l.C. We thank Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCiii and cofinanced by the ERDF for their financial support. E.N-V. acknowledges IFI18/00014 fellowship from the ISCiii. P.d.l.C-O. acknowledges FPU17/00026 fellowship from the Spanish Ministry of Education (MEC). L.C. acknowledges FPU16/05127 fellowship from the MEC

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

Incidence and management of inguinodynia after inguinal plasty

Background: Hernia is defined as a defect of fascial and muscle-aponeurotic structures, allowing the protrusion of elements. The most frequent is inguinal region, prevailing in men 3:1 vs female. The most frequent complications are persistent chronic pain.Methods: A descriptive, prospective and cross-sectional study was performed in postoperative inguinal plasty patients, using a laparoscopic approach and open approach, the presence or absence of inguinodynia was studied using the visual analogue pain scale (VAS) and the Semmes-Weinstein monofilament, in addition to a systematic investigation in the following PubMed, Medline, Clinical Key and Index Medicus databases, with articles from July 2019 to April 2020.Results: Inguinodynia was present in laparoscopic surgery and open approach, 58 patients had inguinodynia at two weeks associated with the inflammatory response of the tissues and the presence of a foreign body (mesh), 77% of the patients with persistence of pain at 3 months reported mild pain (VAS 1-4), 21% moderate pain that did not limit their daily activities (VAS 5-8) and 2% of the patients reported severe pain which limited physical activity and effort   (VAS 9-10).Conclusions: Inguinodynia has an impact on hospital costs and quality life, we consider it is essential to domain the anatomical variants of the region. We propose an extensive follow-up of this group of patients, to make a comparison of diagnostic methods, as well as conservative management vs. modern techniques for pain control

Nutritional characteristics of different types of eggs

Objective: To analyze 5 types of poultry eggs (chicken, turkey, ostrich, duck and quail) to compare their nutritional characteristics and sensory properties. Design/ Methodology/ Approach: A physical analysis was performed: weight of the entire egg (weight and proportion of the albumin, yolk and shell) length and width of the entire egg, shape index, shell color, and yolk color, nutritional (determination of raw fat, protein, dry matter and ashes) and in sensory adaptation (measured through hedonic testing of adaptability through the arrangement of nine points to an individualized quantity of 97). Different types of egg used: chicken, turkey, ostrich, duck and quail. Results: The egg containing the most amount of protein was that of the duck (13.02 ± 0.46 %), while the sample containing the lowest result was that of the ostrich (9.47 ± 0.27 %). The type of egg that contained the fattest level was the duck (10.31 ± 0.75 %); on the other hand, the type of egg that demonstrated the least amount of fat was that of the chicken egg (8.28 ± 0.39 %). Results/ Findings/ Conclusion: Even though some physical differences exist in all types of eggs, they are similar and there is minimal variation in terms of their nutritional value. Therefore, these different types of eggs can be applied for consumption as substitutes for chicken eggs and as an alternative source of protein. Limitations of the study/ Implications: Lack of previous research in regard to comparisons of the types of analyzed eggs

Trained immunity is not universal: oral heat-inactivated Mycobacterium bovis confers no protection against the non-enveloped Porcine Circovirus 2

[Background] Trained immunity, the enhanced response of innate cells leading to an improved innate immune response, and antibodies against the glycan galactose-α-1,3-galactose (α-Gal), produced by animals unable to synthesize α-Gal epitopes, have been suggested to provide the host certain advantage in infections with enveloped viruses. Conversely, the evidence of protection against non-enveloped viruses attributed to the referred mechanisms remains scarce. Aiming to evaluate whether a heat-inactivated Mycobacterium bovis (HIMB) immunostimulant, which had proven to protect against related and non-related pathogens, confers an advantage against non-enveloped viruses, we performed an immunization and challenge experiment with porcine circovirus 2 (PCV-2) in swine. Sixteen piglets were randomly assigned to the immunized group (n = 8), which received two oral doses of HIMB with an interval of three weeks, or to the control group (n = 8). All animals were infected by intranasal inoculation with PCV-2 21 days later and euthanized at day 21 post-challenge.[Results] No differences in body weight and body temperature, viremia and viral burden in target tissues, antibody production and histopathological changes in target tissues were observed between the immunized and the control group. Overall, oral immunization with HIMB did not protect pigs against PCV-2 infection.[Conclusions] Our study suggests that HIMB confers no advantage against pathogens lacking α-Gal, mainly non-enveloped viruses such as PCV-2, in α-Gal-producing hosts, such as the swine.The present study has been funded by project MYCOTRAINING SBPLY/19/180501/000174 (Junta de Castilla-La Mancha, Spain, and EU-FEDER). E. Ferreras-Colino (2020/3836) and R. Vaz-Rodrigues (2022/20675) were supported by the predoctoral contract from Universidad de Castilla-La Mancha (UCLM), co-financed by the European Social Fund (ESF). Marinela Contreras was supported by the Ministerio de Ciencia, Innovación y Universidades, Spain, grant IJC2020-042710-I.Peer reviewe

Shallow magma convection evidenced by excess degassing and thermal radiation during the dome-forming Sabancaya eruption (2012–2020)

We used a large set of satellite- (visible, infrared, and radar images from Planetscope, MODIS, VIIRS, Sentinel2, Landsat 8, and Sentinel 1) and ground-based data (optical images, SO2 flux, shallow seismicity) to describe and characterize the activity of the Sabancaya volcano during the unrest and eruption phases that occurred between 2012 and 2020. The unrest phase (2012–2016) was characterized by increasing gas and thermal flux, sourced by a convective magma column rising along with the remnants of a buried plug still permeable to fluid flow. Conversely, a new conduit, adjacent to the previous one, fed the eruptive phase (2016–2020) which was instead characterized by a discontinuous extrusive activity, with phases of dome growth (at rates from 0.04 to 0.75 m3 s−1) and collapse. The extrusive activity was accompanied by fluctuating thermal anomalies (0.5–25 MW), by irregular SO2 degassing (700–7000 tons day−1), and by variable explosive activity (4–100 events d−1) producing repeated vulcanian ash plumes (500–5000 m above the crater). Magma budget calculation during the eruptive phase indicates a large excess of degassing, with the volume of degassed magma (0.25–1.28 km3) much higher than the volume of erupted magma (< 0.01 km3). Similarly, the thermal energy radiated by the eruption was much higher than that sourced by the dome itself, an unbalance that, by analogy with the degassing, we define as “excess thermal radiation”. Both of these unbalances are consistent with the presence of shallow magma convection that fed the extrusive and explosive activity of the Sabancaya dome

The Thyroid Hormone Receptors Modulate the Skin Response to Retinoids

[Background]: Retinoids play an important role in skin homeostasis and when administered topically cause skin hyperplasia, abnormal epidermal differentiation and inflammation. Thyroidal status in humans also influences skin morphology and function and we have recently shown that the thyroid hormone receptors (TRs) are required for a normal proliferative response to 12-O-tetradecanolyphorbol-13-acetate (TPA) in mice. [Methodology/Principal Findings]: We have compared the epidermal response of mice lacking the thyroid hormone receptor binding isoforms TRα1 and TRβ to retinoids and TPA. Reduced hyperplasia and a decreased number of proliferating cells in the basal layer in response to 9-cis-RA and TPA were found in the epidermis of TR-deficient mice. Nuclear levels of proteins important for cell proliferation were altered, and expression of keratins 5 and 6 was also reduced, concomitantly with the decreased number of epidermal cell layers. In control mice the retinoid (but not TPA) induced parakeratosis and diminished expression of keratin 10 and loricrin, markers of early and terminal epidermal differentiation, respectively. This reduction was more accentuated in the TR deficient animals, whereas they did not present parakeratosis. Therefore, TRs modulate both the proliferative response to retinoids and their inhibitory effects on skin differentiation. Reduced proliferation, which was reversed upon thyroxine treatment, was also found in hypothyroid mice, demonstrating that thyroid hormone binding to TRs is required for the normal response to retinoids. In addition, the mRNA levels of the pro-inflammatory cytokines TNFα and IL-6 and the chemotactic proteins S1008A and S1008B were significantly elevated in the skin of TR knock-out mice after TPA or 9-cis-RA treatment and immune cell infiltration was also enhanced. [Conclusions/significance]: Since retinoids are commonly used for the treatment of skin disorders, these results demonstrating that TRs regulate skin proliferation, differentiation and inflammation in response to these compounds could have not only physiological but also therapeutic implications.This work was supported by grants BFU2007-62402 and SAF2008-00121 from Ministerio de Ciencia e Innovación, RD06/0020/0036 and RD06/0020/0029 from the Fondo de Investigaciones Sanitarias and by the European Grant CRESCENDO (FP-018652).Peer reviewe

IL-6 serum levels predict severity and response to tocilizumab in COVID-19: An observational study

Background: Patients with coronavirus disaese 2019 (COVID-19) can develop a cytokine release syndrome that eventually leads to acute respiratory distress syndrome requiring invasive mechanical ventilation (IMV). Because IL-6 is a relevant cytokine in acute respiratory distress syndrome, the blockade of its receptor with tocilizumab (TCZ) could reduce mortality and/or morbidity in severe COVID-19. Objective: We sought to determine whether baseline IL-6 serum levels can predict the need for IMV and the response to TCZ. Methods: A retrospective observational study was performed in hospitalized patients diagnosed with COVID-19. Clinical information and laboratory findings, including IL-6 levels, were collected approximately 3 and 9 days after admission to be matched with preadministration and postadministration of TCZ. Multivariable logistic and linear regressions and survival analysis were performed depending on outcomes: need for IMV, evolution of arterial oxygen tension/fraction of inspired oxygen ratio, or mortality. Results: One hundred forty-six patients were studied, predominantly males (66%); median age was 63 years. Forty-four patients (30%) required IMV, and 58 patients (40%) received treatment with TCZ. IL-6 levels greater than 30 pg/mL was the best predictor for IMV (odds ratio, 7.1; P < .001). Early administration of TCZ was associated with improvement in oxygenation (arterial oxygen tension/fraction of inspired oxygen ratio) in patients with high IL-6 (P = .048). Patients with high IL-6 not treated with TCZ showed high mortality (hazard ratio, 4.6; P = .003), as well as those with low IL-6 treated with TCZ (hazard ratio, 3.6; P = .016). No relevant serious adverse events were observed in TCZ-treated patients. Conclusions: Baseline IL-6 greater than 30 pg/mL predicts IMV requirement in patients with COVID-19 and contributes to establish an adequate indication for TCZ administrationThis study was funded by Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and Instituto de Salud Carlos III (grant nos. RD16/0011/0012 and PI18/ 0371 to I.G.A., grant no. PI19/00549 to A.A., and grant no. SAF2017-82886-R to F.S.-M.) and co-funded by the European Regional Development Fund. The study was also funded by ‘‘La Caixa Banking Foundation’’ (grant no. HR17-00016 to F.S.-M.) and ‘‘Fondos Supera COVID19’’ by Banco de Santander and CRUE. None of these sponsors have had any role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publicatio