Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients

1,3-diphenylpropan-1-ones as allosteric modulators of α7 nACh receptors with analgesic and antioxidant properties

Nicotine acethylcholine receptors (nAChRs) play critical roles in cognitive processes, neuroprotection and inflammation. Results: According to their substituents, 1,3-diphenylpropan-1-one derivatives act as α7 nAChRs negative allosteric modulators (NAM, OMe) or Type I positive allosteric modulators (PAMs, OH). Compounds 7 and 31 were the most effective (989 and 666% enhancement of ACh-induced currents) and potent (EC: 12.9 and 6.85 μM) PAMs. They exhibited strong radical scavenging values. Compound 31, selective over other neuronal nAChR subtypes and with acceptable pharmacokinetic profile, showed antinociceptive effects in a model of inflammatory pain. Conclusion: Compound 31 is a novel, potent and selective α7 nAChR PAM, displaying antioxidant and analgesic activities. The 1,3-diphenylpropan-1-one scaffold could be the base toward more advanced type I PAMs for the treatment of nAChR-mediated diseases.This work was supported by the Spanish MINECO: CSD2008-00005, The Spanish Ion Channel Initiative-CONSOLIDER INGENIO 2010, SAF2011-22802 and BFU2012-39092-C02. The Instituto de Neurociencias is a “Centre of Excellence Severo Ochoa”. We thank Susana Cámara Garrido for her assistance in the synthesis of some starting compounds and Susana Gerber for technical assistance. BBP thanks the CSIC for a predoctoral fellowship (JAE-Predoc from Junta para la Ampliación de Estudios, co-financed by FSE). Alpha7 nAChRPeer Reviewe

A comprehensive biomarker analysis of microsatellite unstable/mismatch repair deficient colorectal cancer cohort treated with immunotherapy

The search for immunotherapy biomarkers in Microsatellite Instability High/Deficient Mismatch Repair system (MSI-H/dMMR) metastatic colorectal cancer (mCRC) is an unmet need. Sixteen patients with mCRC and MSI-H/dMMR (determined by either immunohistochemistry or polymerase chain reaction) treated with PD-1/PD-L1 inhibitors at our institution were included. According to whether the progression-free survival with PD-1/PD-L1 inhibitors was longer than 6 months or shorter, patients were clustered into the IT-responder group (n: 9 patients) or IT-resistant group (n: 7 patients), respectively. In order to evaluate determinants of benefit with PD-1/PD-L1 inhibitors, we performed multimodal analysis including genomics (through NGS panel tumour-only with 431 genes) and the immune microenvironment (using CD3, CD8, FOXP3 and PD-L1 antibodies). The following mutations were more frequent in IT-resistant compared with IT-responder groups: B2M (4/7 versus 2/9), CTNNB1 (2/7 versus 0/9), and biallelic PTEN (3/7 versus 1/9). Biallelic ARID1A mutations were found exclusively in the IT-responder group (4/9 patients). Tumour mutational burden did not correlate with immunotherapy benefit, neither the rate of indels in homopolymeric regions. Of note, biallelic ARID1A mutated tumours had the highest immune infiltration and PD-L1 scores, contrary to tumours with CTNNB1 mutation. Immune microenvironment analysis showed higher densities of different T cell subpopulations and PD-L1 expression in IT-responders. Misdiagnosis of MSI-H/dMMR inferred by discordances between immunohistochemistry and polymerase chain reaction was only found in the IT-resistant population (3/7 patients). Biallelic ARID1A mutations and Wnt signalling activation through CTNNB1 mutation were associated with high and low T cell immune infiltrates, respectively, and deserve special attention as determinants of response to PD-1/PD-L1 inhibitors. The non-MSI-H phenotype in dMMR is associated with poor benefit to immunotherapy. Our results suggest that mechanisms of resistance to immunotherapy are multi-factorial

Plan Local de prevención de incendios forestales del término municipal de Gata de Gorgos

Los incendios forestales constituyen un grave problema, que en la actualidad se acentúa en las zonas mediterráneas, y muy especialmente en el territorio valenciano. De forma recurrente, los incendios provocan graves daños en las masas forestales con la consiguiente repercusión negativa sobre el medio natural, económico y social. Dichos incendios pueden dar lugar a situaciones de grave riesgo, catástrofe o calamidad pública, por lo cual se hace necesario el empleo coordinado de los recursos y medios disponibles, así como la implementación de un plan de prevención de incendios forestales. El Plan Municipal de Prevención de Incendios Forestales establecerá las medidas generales para la prevención de incendios forestales, la defensa de los montes y terrenos forestales incluidos en el término municipal, la protección de la personas, bienes y núcleos rurales así como la promoción y adopción de una política de prevención adecuada según las necesidades y posibilidades de la población de Gata de Gorgos.Mulet Soler, JJ. (2012). Plan Local de prevención de incendios forestales del término municipal de Gata de Gorgos. Universitat Politècnica de València. http://hdl.handle.net/10251/18455Archivo delegad

Unidad de Ómicas

Póster presentado al Encuentro de Investigación: Buscando Sinergias, celebrado el 7 de junio de 2022 en el Salón de Actos del Hospital General Dr. Balmis (Alicante).Peer reviewe

Role of loop 9 on the function of neuronal nicotinic receptors

We have studied the role of loop 9 in the function of neuronal nicotinic receptors. By systematically mutating the residues in the loop we have determined that the most important amino acids determining the coupling of binding to gating are the ones closer to the transmembrane region. Single mutations at location E173 in homomeric alpha 7 receptors destroyed their function by completely abolishing the current while preserving the expression at the membrane. In contrast, heteromeric receptor alpha 3 beta 4 with the same mutations retained some function. We conclude that loop 9 has a different role in the function of homomeric and heteromeric receptors. (C) 2009 Elsevier B.V. All rights reserved.This work was supported by grants from the Ministerio de Educación y Ciencia of Spain and FEDER (SAF2005-00534, SAF2005- 02045 and SAF2006-03933) and grants BFU2008-02160 and CSD2008-00005 (The Spanish Ion Channel Initiative-CONSOLIDER INGENIO 2010) from the Ministerio de Ciencia e Innovación of Spain.Peer Reviewe

A small cytoplasmic region adjacent to the fourth transmembrane segment of the α7 nicotinic receptor is essential for its biogenesis

Deletion of a small cytoplasmic fragment close to the fourth transmembrane segment of the nicotinic α7 receptor (Glu437 to Arg447) abolished membrane expression. Different single mutants showed moderate to strong decreases in expression whereas the latter was totally abolished upon proline substitutions. We hypothesize that preservation of an α-helix formed by the fourth transmembrane segment and the adjacent cytoplasmic region is essential for membrane receptor expression. Moreover, in selected mutants with low or null membrane expression, a significant proportion of mature receptors was present inside the cell. Hence, elements in this cytoplasmic fragment might influence receptor transport to the membrane. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.This work was supported by grants BFU2008-02160 and CSD2008-00005 (The Spanish Ion Channel Initiative-CONSOLIDER INGENIO 2010) from the Ministry of Science and Innovation of Spain.Peer Reviewe

Mutants of β-strand β3 and the loop B in the interface between α7 subunits of a homomeric acetylcholine receptor show functional and pharmacological alterations

11 p., 7 figures, 2 tables and references.Activation of nicotinic acetylcholine receptors (nAChR) requires a global conformational change involving a number of domains of the protein. Structural data from Torpedo nAChR suggest that adjacent subunits might be functionally coupled at the interface between the β-strand β3 and the loop B through a salt bridge between α1Asp152 and γArg78. We have checked this hypothesis in homomeric α7 nAChRs by mutating residues at these (Gly152 and Arg79) and neighboring locations and analyzing the results obtained after expression of single and double mutants in Xenopus oocytes. We found that Arg79 mutants showed a decreased gating function when challenged with different agonists, being the reduction more important for dimethylphenylpiperazinium. EC 50 values in these mutants were also increased up to 30-fold. In contrast, mutating Gly152 only showed significant higher EC 50 values for ACh. However, all Gly153 mutants presented increased gating function and lower EC 50 values with no significant differences among them. When analyzing several mutant cycles it is concluded that Arg79 is functionally coupled to Gly152, but neither to Gly153 nor to Asp157. These data suggest an involvement of the minus side of homomeric α7 nAChRs in their gating function, reinforcing the significance of complementary subunits in the gating of neuronal nAChRs.This work was supported by grants from the Ministry of Education and Science of Spain and FEDER (SAF2006-03933) and grants BFU2008-02160 and CSD2008-00005 (The Spanish Ion Channel Initiative-CONSOLIDER INGENIO 2010) from the Ministry of Science and Innovation of Spain.Peer reviewe

Charged amino acids of the N-terminal domain are involved in coupling binding and gating in α7 nicotinic receptors

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the α7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.This work was supported by Grants BMC2002-00972 and SAF200200209 from the Ministry of Science and Technology of Spain and Grants CTIDIB/2002/138 and GRUPOS03/038 from the Generalitat Valenciana.Peer reviewe