USP49 deubiquitinase regulates the mitotic spindle checkpoint and prevents aneuploidy.

The spindle assembly checkpoint (SAC) is an essential mechanism that ensures the accurate chromosome segregation during mitosis, thus preventing genomic instability. Deubiquitinases have emerged as key regulators of the SAC, mainly by determining the fate of proteins during cell cycle progression. Here, we identify USP49 deubiquitinase as a novel regulator of the spindle checkpoint. We show that loss of USP49 in different cancer cell lines impairs proliferation and increases aneuploidy. In addition, USP49-depleted cells overcome the arrest induced by the SAC in the presence of nocodazole. Finally, we report new binding partners of USP49, including ribophorin 1, USP44, and different centrins.We thank A.P. Ugalde, D. Rodríguez, J.G. Pérez-Silva, Y. Español and F. Rodríguez for helpful comments and advice. We also thank S.A. Miranda, D. Álvarez-Puente and C. Garabaya for excellent technical assistance, as well as the staff of the scientific core facilities from the University of Oviedo (Unidad de Ensayos Biotecnológicos y Biomédicos, Servicios Científico-Técnicos). This work was supported by the Ministerio de Ciencia e Innovación (SAF2017-87655-R, PDI2020-118394RB-100 and PGC2018- 097019-B-I00), “la Caixa” Banking Foundation (project code HR17-00247), and the European Research Council (742067, DeAge). The IUOPA is funded by the Asturian Government and Fundación Cajastur-Liberbank.S

Dinámica de septinas y regulación de la separación celular durante el crecimiento hifal de Candida albicans ¿una diana de antifúngicos?

Resumen del trabajo presentado al "IV Congreso GEBMP: Simposio Hongos Patógenos" celebrado en Badajoz el 6 de julio del 2012.Peer Reviewe

A membrane-associated form of the transcription factor Ace2 controls septin ring dynamics during Candida albicans hyphal growth

Resumen del trabajo presentado a la Vth International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis celebrada en Primosten (Croacia) del 6 al 9 de Junio de 2012.Peer Reviewe

Akt inhibitors induce apoptosis in chronic lymphocytic leukemia cells

Background: The phosphatidylinositol-3-kinase/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia cells. In this study we analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) on the survival of chronic lymphocytic leukemia cells. Design and Methods: Using cytometry we studied the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with chronic lymphocytic leukemia and from healthy donors. We studied the changes induced by Akti-1/2 and A-443654 at the mRNA level by performing reverse transcriptase multiplex ligation-dependent probe amplification. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on chronic lymphocytic leukemia cells by western blotting. Moreover, we analyzed the cytotoxic effect of Akt inhibitors in patients' cells with deleted/mutated TP53. Results: Both inhibitors induced apoptosis in chronic lymphocytic leukemia cells in a dose-dependent manner. Moreover, B cells from patients with chronic lymphocytic leukemia were more sensitive to Akt inhibitors than T cells from leukemic patients, and B or T cells from healthy donors. Survival factors for chronic lymphocytic leukemia cells, such as interleukin-4 and stromal cell-derived factor-1 alpha, were not able to block the apoptosis induced by either Akt inhibitor. Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. Conclusions: These results demonstrate that Akt inhibitors induce apoptosis of chronic lymphocytic leukemia cells and might be a new therapeutic option for the treatment of chronic lymphocytic leukemia

The potential anticancer agent PK11195 induces apoptosis irrespective of p53 and ATM status in chronic lymphocytic leukemia cells

Background and Objectives The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein (18KDa) (TSPO) ligand, facilitates the induction of cell death by a variety of cytotoxic and chemotherapeutic agents. Primary chronic lymphocytic leukemia (CLL) cells overexpress TSPO. The aim of this study was to examine the effects of PK11195 on CLL cells. Design and Methods Using cytometric analysis, we studied the cytotoxic effects of PK11195 on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Western blot and cytometric analyses were used to study the mitochondrial effects of PK11195 on CLL cells. Moreover, we analyzed the cytotoxic effect of PK11195 in patients' cells with mutated p53 or ATM. Results PK11195 induces apoptosis and had additive effects with chemotherapeutic drugs in primary CLL cells. Other TSPO ligands such as RO 5-4864 and FGIN-1-27 also induce apoptosis in CLL cells. PK11195 induces mitochondrial depolarization and cytochrome c release upstream of caspase activation, and dithiocyana-tostilbene-2,2-disulfonic acid (DIDS), a voltage-dependent anion channel (VDAC) inhibitor, inhibits PK11195-induced apoptosis, demonstrating a direct involvement of mitochondria. CLL cells and normal B cells are more sensitive than T cells to PK11195-induced apoptosis. Interestingly, PK11195 induced apoptosis in CLL cells irrespective of their p53 or ATM status. Interpretation and Conclusions These results suggest that PK11195 alone or in combination with chemotherapeutic drugs might be a new therapeutic option for the treatment of CLL

The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells through the upregulation of NOXA and synergizes with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In the study herein, the pro-apoptotic effect of fluorizoline was assessed in 34 primary samples from patients with chronic lymphocytic leukemia. Fluorizoline induced apoptosis in chronic lymphocytic leukemia cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespective of patients' clinical or genetic features, whereas normal T lymphocytes were less sensitive. Fluorizoline increased the protein levels of the pro-apoptotic B-cell lymphoma 2 family member NOXA in chronic lymphocytic leukemia cells. Furthermore, fluorizoline synergized with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax to induce apoptosis. These results suggest that targeting prohibitins could be a new therapeutic strategy for chronic lymphocytic leukemia

A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation

We previously described diaryl trifluorothiazoline compound 1a (hereafter referred to as fluorizoline) as a first-in-class small molecule that induces p53-independent apoptosis in a wide range of tumor cell lines. Fluorizoline directly binds to prohibitin 1 and 2 (PHBs), two proteins involved in the regulation of several cellular processes, including apoptosis. Here we demonstrate that fluorizoline-induced apoptosis is mediated by PHBs, as cells depleted of these proteins are highly resistant to fluorizoline treatment. In addition, BAX and BAK are necessary for fluorizoline-induced cytotoxic effects, thereby proving that apoptosis occurs through the intrinsic pathway. Expression analysis revealed that fluorizoline induced the upregulation of Noxa and Bim mRNA levels, which was not observed in PHB-depleted MEFs. Finally, Noxa-/-/Bim-/- MEFs and NOXA-downregulated HeLa cells were resistant to fluorizoline-induced apoptosis. All together, these findings show that fluorizoline requires PHBs to execute the mitochondrial apoptotic pathway

Methods for economic evaluation of a factorial-design cluster randomised controlled trial of a nutrition supplement and an exercise programme among healthy older people living in Santiago, Chile: the CENEX study

BACKGROUND: In an effort to promote healthy ageing and preserve health and function, the government of Chile has formulated a package of actions into the Programme for Complementary Food in Older People (Programa de Alimentación Complementaria para el Adulto Mayor - PACAM). The CENEX study was designed to evaluate the impact, cost and cost-effectiveness of the PACAM and a specially designed exercise programme on pneumonia incidence, walking capacity and body mass index in healthy older people living in low- to medium-socio-economic status areas of Santiago. The purpose of this paper is to describe in detail the methods that will be used to estimate the incremental costs and cost-effectiveness of the interventions. METHODS AND DESIGN: The base-case analysis will adopt a societal perspective, including the direct medical and non-medical costs borne by the government and patients. The cost of the interventions will be calculated by the ingredients approach, in which the total quantities of goods and services actually employed in applying the interventions will be estimated, and multiplied by their respective unit prices. Relevant information on costs of interventions will be obtained mainly from administrative records. The costs borne by patients will be collected via exit and telephone interviews. An annual discount rate of 8% will be used, consistent with the rate recommended by the Government of Chile. All costs will be converted from Chilean Peso to US dollars with the 2007 average period exchange rate of US$1 = 522.37 Chilean Peso. To test the robustness of model results, we will vary the assumptions over a plausible range in sensitivity analyses. DISCUSSION: The protocol described here indicates our intent to conduct an economic evaluation alongside the CENEX study. It provides a detailed and transparent statement of planned data collection methods and analyses. TRIAL REGISTRATION: ISRCTN48153354

The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells through the upregulation of NOXA and synergizes with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax

Fluorizoline is a new synthetic molecule that induces apoptosis by selectively targeting prohibitins. In the study herein, the pro-apoptotic effect of fluorizoline was assessed in 34 primary samples from patients with chronic lymphocytic leukemia. Fluorizoline induced apoptosis in chronic lymphocytic leukemia cells at concentrations in the low micromolar range. All primary samples were sensitive to fluorizoline irrespective of patients’ clinical or genetic features, whereas normal T lymphocytes were less sensitive. Fluorizoline increased the protein levels of the pro-apoptotic B-cell lymphoma 2 family member NOXA in chronic lymphocytic leukemia cells. Furthermore, fluorizoline synergized with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax to induce apoptosis. These results suggest that targeting prohibitins could be a new therapeutic strategy for chronic lymphocytic leukemia

Evaluation of Nutritional Practices in the Critical Care Patient (The ENPIC Study): Does Nutrition Really Affect ICU Mortality?

Background & aims: The importance of artificial nutritional therapy is underrecognized, typically being considered an adjunctive rather than a primary therapy. We aimed to evaluate the influence of nutritional therapy on mortality in critically ill patients. Methods: This multicenter prospective observational study included adult patients needing artificial nutritional therapy for >48 h if they stayed in one of 38 participating intensive care units for >= 72 h between April and July 2018. Demographic data, comorbidities, diagnoses, nutritional status and therapy (type and details for <= 14 days), and outcomes were registered in a database. Confounders such as disease severity, patient type (e.g., medical, surgical or trauma), and type and duration of nutritional therapy were also included in a multivariate analysis, and hazard ratios (HRs) and 95% confidence intervals (95% CIs) were reported. Results: We included 639 patients among whom 448 (70.1%) and 191 (29.9%) received enteral and parenteral nutrition, respectively. Mortality was 25.6%, with non-survivors having the following char-acteristics: older age; more comorbidities; higher Sequential Organ Failure Assessment (SOFA) scores (6.6 +/- 3.3 vs 8.4 +/- 3.7; P < 0.001); greater nutritional risk (Nutrition Risk in the Critically Ill [NUTRIC] score: 3.8 +/- 2.1 vs 5.2 +/- 1.7; P < 0.001); more vasopressor requirements (70.4% vs 83.5%; P=0.001); and more renal replacement therapy (12.2% vs 23.2%; P=0.001). Multivariate analysis showed that older age (HR: 1.023; 95% CI: 1.008-1.038; P=0.003), higher SOFA score (HR: 1.096; 95% CI: 1.036-1.160; P=0.001), higher NUTRIC score (HR: 1.136; 95% CI: 1.025-1.259; P=0.015), requiring parenteral nutrition after starting enteral nutrition (HR: 2.368; 95% CI: 1.168-4.798; P=0.017), and a higher mean Kcal/Kg/day intake (HR: 1.057; 95% CI: 1.015-1.101; P=0.008) were associated with mortality. By contrast, a higher mean protein intake protected against mortality (HR: 0.507; 95% CI: 0.263-0.977; P=0.042). Conclusions: Old age, higher organ failure scores, and greater nutritional risk appear to be associated with higher mortality. Patients who need parenteral nutrition after starting enteral nutrition may represent a high-risk subgroup for mortality due to illness severity and problems receiving appropriate nutritional therapy. Mean calorie and protein delivery also appeared to influence outcomes. (C) 2021 The Author(s). Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism