Browsing by Author "Saez, Juan"
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Item ATP is required and advances cytokine-induced gap junction formation in microglia in vitro(Hindawi Publishing Corp., 2013) Saez, Pablo; Shoji, Kenji; Retamal, Mauricio; Harcha, Paloma; Ramirez, Gigliola; Jiang, Jean; von Bernhardi, Rommy; Saez, JuanMicroglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs) and gap junction channels (GJCs), affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF-/IFN--induced dye coupling, probably through the induction of IL-1 release. Moreover, TNF-/IFN-, but not TNF- plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-/IFN-, but not TNF- plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-/IFN- in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.Item Connexin hemichannels explain the ionic imbalance and lead to atrophy in denervated skeletal muscles(Elsevier, 2016) Cisterna, Bruno; Vargas, Anibal; Puebla, Carlos; Saez, JuanDenervated fast skeletal muscles undergo atrophy, which is associated with an increase in sarcolemma permeability and protein imbalance. However, the mechanisms responsible for these alterations remain largely unknown. Recently, a close association between de novo expression of hemichannels formed by connexins 43 and 45 and increase in sarcolemma permeability of denervated fast skeletal myofibers was demonstrated. However, it remains unknown whether these connexins cause the ionic imbalance of denervates fast myofibers. To elucidate the latter and the role of hemichannels formed by connexins (Cx HCs) in denervation-induced atrophy, skeletal myofibers deficient in Cx43 and Cx45 expression (Cx43fl/flCx45fl/fl:Myo-Cre mice) and control (Cx43fl/flCx45fl/fl mice) were denervated and several muscle features were systematically analyzed at different post-denervation (PD) times (1, 3, 5, 7 and 14days). The following sequence of events was found in denervated myofibers of Cx43fl/flCx45fl/fl mice: 1) from day 3 PD, increase in sarcolemmal permeability, 2) from day 5 PD, increases of intracellular Ca2+ and Na+ signals as well as a significant increase in protein synthesis and degradation, yielding a negative protein balance and 3) from day 7 PD, a fall in myofibers cross-section area. All the above alterations were either absent or drastically reduced in denervated myofibers of Cx43fl/flCx45fl/fl:Myo-Cre mice. Thus, the denervation-induced Cx HCs expression is an early event that precedes the electrochemical gradient dysregulation across the sarcolemma and critically contributes to the progression of skeletal muscle atrophy. Consequently, Cx HCs could be a therapeutic target to drastically prevent the denervation-induced atrophy of fast skeletal muscles.Item Hemichannels; from the molecule to the function(Frontiers Research Foundation, 2014) Retamal, Mauricio; Saez, JuanCoordinated cell interactions are required to accomplish diverse complex and dynamic tasks of several tissues in vertebrates and invertebrates. Cell functions, such as intercellular propagation of calcium waves and spread of electrotonic potentials, are coordinated by cell-to-cell communication through gap junction channels (GJCs). These channels are formed by the serial docking of two hemichannels (HCs), which in vertebrates are formed by six protein subunits called connexins (Cxs). In humans, a gene family encodes 21 different proteins with a highly variable C-terminal where most posttranslational modifications occur. Among them protein phosphorylation and/or oxidation (e.g., nitrosylation) induces functional changes. Currently, it is believed that undocked HCs may have functional relevance in cell physiology allowing diffusional exchange of ions and small molecules between intra- and extra-cellular compartments. In support to this new concept, it has been shown that controlled HC opening allows the release of small signaling molecules (e.g., ATP, glutamate, NAD+, adenosine, and cyclic nucleotides) and uptake of metabolically relevant molecules (e.g., glucose). Additionally, a growing body of evidences shows that HCs are involved in important and diverse processes, such PGE2 release from osteocytes, glucose detection in tanicytes, T cell infection with AIDS virus, memory consolidation in the basolateral amygdala and release of nitric oxide from endothelial cells, among others. However, HCs can also play an important role in the homeostatic imbalance observed in diverse diseases. In fact, enhanced HCs opening induces or accelerates cell death in several pathological conditions. Hemichannel-mediated cell death is due mainly to Ca+2 influx and cellular overload. The latter activates proteases, nucleases and lipases, causing irreversible cell damage. Accordingly, blockade of HCs reduces the cellular damage observed in several animal models of human diseases. Additionally, another family of proteins called pannexins (Panxs) also forms channels at the plasma membrane and some of their functional and pharmacological sensitivities overlap with those of Cx HCs. Recently, Panx channels have been involved in both pathological and physiological processes. Therefore, Cx HCs and Panx channels appear as promising drug targets for clinical treatment of several inherited and acquired human diseases. This research topic gathers 11 articles that give a broad view about the role of Cx- and Panx–based channels from purified molecules reconstituted in a lipid environment and posttranslational regulation, to physiological and pathological implications. In addition, it proposes a putative molecular explanation of HC malfunctioning in specific diseases.Item Total mutational load and clinical features as predictors of the metastatic status in lung adenocarcinoma and squamous cell carcinoma patients(2022) Oróstica, Karen; Saez, Juan; De Santiago, Pamela; Rivas, Solange; Contreras, Sebastián; Navarro, Gonzalo; Asenjo, Juan; Olivera, Álvaro; Armisén, RicardoAbstract Background: Recently, extensive cancer genomic studies have revealed mutational and clinical data of large cohortsof cancer patients. For example, the Pan-Lung Cancer 2016 dataset (part of The Cancer Genome Atlas project), sum‑marises the mutational and clinical profles of diferent subtypes of Lung Cancer (LC). Mutational and clinical signa‑ tures have been used independently for tumour typifcation and prediction of metastasis in LC patients. Is it then possible to achieve better typifcations and predictions when combining both data streams? Methods: In a cohort of 1144 Lung Adenocarcinoma (LUAD) and Lung Squamous Cell Carcinoma (LSCC) patients, we studied the number of missense mutations (hereafter, the Total Mutational Load TML) and distribution of clinical variables, for diferent classes of patients. Using the TML and diferent sets of clinical variables (tumour stage, age, sex, smoking status, and packs of cigarettes smoked per year), we built Random Forest classifcation models that calculate the likelihood of developing metastasis. Results: We found that LC patients diferent in age, smoking status, and tumour type had signifcantly diferent mean TMLs. Although TML was an informative feature, its efect was secondary to the "tumour stage" feature. However, its contribution to the classifcation is not redundant with the latter; models trained using both TML and tumour stage performed better than models trained using only one of these variables. We found that models trained in the entire dataset (i.e., without using dimensionality reduction techniques) and without resampling achieved the highest perfor‑mance, with an F1 score of 0.64 (95%CrI [0.62, 0.66]). Conclusions: Clinical variables and TML should be considered together when assessing the likelihood of LC patients progressing to metastatic states, as the information these encode is not redundant. Altogether, we provide new evi‑ dence of the need for comprehensive diagnostic tools for metastasis.