Deletion of the Tetrahymena thermophila rDNA replication fork barrier region disrupts macronuclear rDNA excision and creates a fragile site in the micronuclear genome

During macronuclear development the Tetrahymena thermophila ribosomal RNA gene is excised from micronuclear chromosome 1 by site-specific cleavage at chromosome breakage sequence (Cbs) elements, rearranged into a ‘palindromic’ 21 kb minichromosome and extensively amplified. Gene amplification initiates from origins in the 5′ non-transcribed spacer, and forks moving toward the center of the palindrome arrest at a developmentally regulated replication fork barrier (RFB). The RFB is inactive during vegetative cell divisions, suggesting a role in the formation or amplification of macronuclear rDNA. Using micronuclear (germline) transformation, we show that the RFB region facilitates Cbs-mediated excision. Deletion of the RFB inhibits chromosome breakage in a sub-population of developing macronuclei and promotes alternative processing by a Cbs-independent mechanism. Remarkably, the RFB region prevents spontaneous breakage of chromosome 1 in the diploid micronucleus. Strains heterozygous for ΔRFB and wild-type rDNA lose the ΔRFB allele and distal left arm of chromosome 1 during vegetative propagation. The wild-type chromosome is subsequently fragmented near the rDNA locus, and both homologs are progressively eroded, suggesting that broken micronuclear chromosomes are not ‘healed’ by telomerase. Deletion of this 363 bp segment effectively creates a fragile site in the micronuclear genome, providing the first evidence for a non-telomere cis-acting determinant that functions to maintain the structural integrity of a mitotic eukaryotic chromosome

Direct and indirect air particle cytotoxicity in human alveolar epithelial cells

Air particulate matter has been associated with adverse impact on the respiratory system leading to cytotoxic and proinflammatory effects. The biological mechanisms behind these associations may be initiated by inhaled small size particles, particle components (soluble fraction) and/or mediators released by particle-exposed cells (conditioned media). The effect of Urban Air Particles from Buenos Aires (UAP-BA) and Residual Oil Fly Ash (ROFA) a surrogate of ambient air pollution, their soluble fractions (SF) and conditioned media (CM) on A549 lung epithelial cells was examined. After 24h exposure to TP (10 and 100 μg/ml), SF or CM, several biological parameters were assayed on cultured A549 cells. We tested cell viability by MTT, superoxide anion (O2-) generation by NBT and proinflammatory cytokine (TNF α, IL-6 and IL-8) production by ELISA. UAP-BA particles or its SF (direct effect) did not modify cell viability and generation of O2- for any of the doses tested. On the contrary, UAP-BA CM (indirect effect) reduced cell viability and increased both generation of O2- and IL-8 production. Exposure to ROFA particles, SF or ROFA CM reduced proliferation and O2- but, stimulated IL-8. It is worth to note that UAP-BA and ROFA depicted distinct effects on particle-exposed A549 cells implicating morphochemical dependence. These in vitro findings support the hypothesis that particle-induced lung inflammation and disease may involve lung-derived mediators.Fil: Orona, Nadia Soledad. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Astort, Francisco. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Maglione, Guillermo Alberto. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Saldiva, P. H. N.. Iira, National Research Council, Brazil; . Institute of Integrated Risk Analysis; BrasilFil: Yakisich, J. S.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Tasat, Deborah Ruth. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentina. Universidad de Buenos Aires. Facultad de Odontología; Argentin

Particulate matter cytotoxicity in cultured SH-SY5Y cells is modulated by simvastatin: Toxicological assessment for oxidative damage

Epidemiological studies have shown a positive correlation between environmental particulate matter and adverse health effects. In particular, residual oil fly ash (ROFA) induces inflammation and reactive oxygen species (ROS), exerting not only local, but also systemic adverse effects. Previously, in an experimental animal model, we found that simvastatin (Sv) pretreatment was effective in preventing ROFA induced lung inflammation. Herein, using the human neuroblastoma SH-SY5Y cell line as a neurotoxicity in vitro model, we studied the potential Sv protective effect on ROFA cytotoxicity. We evaluated cell viability by the MTT assay, superoxide anion generation by NBT test, Nrf2 activation by immunofluorescence, apoptosis by cleaved-PARP and active-caspase 3 expressions, and senescence by b-galactosidase activity. SH-SY5Y cells exposed to ROFA (10 and 50mg/ml) for 24 h showed decreased cell viability, increased superoxide anion generation, apoptosis and senescence. Pretreatment with Sv (1mM) for 6 days, restored cell viability to basal levels, reduced ROFA-induced O2 generation as well as the number of apoptotic and senescent cells. Sv pretreatment stimulated the basal and ROFA-induced levels of Nrf2 nuclear translocation suggesting that activation of the cellular antioxidant defense system prevented particle-induced oxidative stress. In parallel, rescue experiments with mevalonate did not modify the effects of SV pretreatment in any of the parameters evaluated in this study. We conclude that simvastatin may provide neuroprotection against air particulate matter-induced neurotoxicity independently of its ability to inhibit cholesterol synthesis.Fil: Ferraro, Sebastián Ariel. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Astort, Francisco. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Yakisich, Juan Sebastian. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; ArgentinaFil: Tasat, Deborah Ruth. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología. Centro de Estudios en Salud y Medio Ambiente; Argentina. Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Histología y Embriología; Argentin

Chemoresistance of Lung Cancer Cells: 2D and 3D In Vitro Models for Anticancer Drug Screening

Chemoresistance of lung cancer cells is a key factor that limits the treatment of lung cancer patients. Patients may initially respond to standard chemotherapy, but this is often followed by rapid development of drug resistance and disease progression. Tumor heterogeneity and the presence of putative cancer stem-like cells (CS-LCs) provide a viable explanation for the chemoresistance of several types of tumors. In this book chapter, we will first describe the current knowledge of the role of both tumor heterogeneity and CS-LCs in lung cancer chemoresistance, tumor progression and metastasis. Next, we will discuss ongoing strategies at the in vitro level to screen for more effective anticancer drugs. We will specifically focus in three-dimensional (3D) culture systems (Spheroids and tumorspheres) and their application in anticancer drug discovery for lung cancer

Use of Vagus Nerve Stimulation and Vagal Maneuvers as Adjuvant Therapy for COVID-19 Patients

At present there is no effective specific antiviral drug to treat the ongoing COVID-19 pandemic that has already infected millions of individual and caused hundreds of thousand deaths worldwide. There is strong indication that a cytokine storm is responsible for the severity of COVID-19 patients. Pilot studies using IL-6 receptor inhibitors such as Tolicizumab have shown promising results. However, since the cytokine storm is a complex systemic inflammatory response involving multiple cytokines it can be hypothesized that a “paninhibition” of cytokines and/or cytokine receptors will be more effective. However, at the same time this strategy may cause more adverse effects. In this article, we propose the application of Vagus Nerve Stimulation (NVS) and/or some forms of vagal maneuvers as adjuvant therapies to prevent and/or mitigate the cytokine response in COVID-19 patients. This proposal is based on the ability of NVS and to decrease the production of IL-6 and other cytokines. The potential application of the diving response (one form of vagal maneuver), that has been shown to confer intrinsic resistance to inflammation in the blood of diving mammals, is also discussed as adjuvant therapy for COVID-19 patients.   Doi: 10.28991/SciMedJ-2021-03-SI-2 Full Text: PD

EMPLEABILIDAD Y GÉNERO EN UNA UNIVERSIDAD PRIVADA DE ASUNCIÓN

La empleabilidad en función al género, ha sido un tema que ocasionó y sigue generando mucha controversia. Actualmente constituye no sólo una discriminación positiva hacia un ser humano, sino que se traduce en un perjuicio y en un retroceso de la humanidad misma. En esta investigación se analiza la situación en la que se encuentra el género femenino en la Universidad Americana de Asunción, en cuanto a la igualdad de oportunidades al acceso de empleo. Para esto se ha analizado cuantitativamente la población de funcionarios que la conforman, en los distintos cargos ocupacionales. Se pudo constatar en base a los datos contrastados que en dicha institución educativa existe un techo de cristal para el género femenino ya que en las dos áreas ocupacionales de mayor relevancia, encargadas del trabajo estratégico y directivo, predominan los hombres y en la última área, encargada netamente del trabajo operativo, predomina una mayoría de mujeres

Chemical Biology Drug Sensitivity Screen Identifies Sunitinib as Synergistic Agent with Disulfiram in Prostate Cancer Cells

Peer reviewe

Regulation of ongoing DNA synthesis in normal and neoplastic brain tissue

The treatment of human brain tumour is challenging in part due to the blood brain barrier and in part due to the specific biology of brain tumours that confer resistance to chemotherapy. For instance, the 5 years survival rate for patients carrying intracranial glioblastoma multiforme has remained at 4-5 % for the last 30 years. The knowledge of the brain tumour biology as well as the biology of the normal brain tissue would help to design new therapeutic strategies and to develop new and less toxic antineoplastic drugs for brain tumour treatment. Normal tissue must be studied in order to identify tumour-specific vulnerabilities and ways to inhibit toxicity in the host. The present thesis describes a series of investigations of potential antineoplastic drugs performed in normal rat cerebral cortex, human brain tumour specimens and RG2 gliomas, performed "in vitro" in order to 1) better understand factors controlling the cell cycle and DNA replication in normal and neoplastic brain tissue, and thus, exploiting potential targets for new drugs 2) better apply the available antineoplastic drugs for the treatment of human brain tumours while producing no or low side effect on normal tissue. A novel assay, which preserves the metabolic and proliferative properties of the tissue was developed and used to study ongoing DNA synthesis and its regulation by protein phosphorylation and proteolysis. The effect of low MW drugs (protein kinase and protease inhibitors) on these processes was evaluated, By analyzing the effects of different chemically unrelated inhibitors of protein kinases we found that many of these inhibitors might act through long term mechanism of action (e.g. inhibiting cell cycle transitions) rather than a direct effect on the DNA replication machinery, although some of these drugs are currently used as "DNA synthesis inhibitors". We suggest that, from the clinical point of view, it would be important to distinguish between these long and short-term mechanism of action. Our results also suggest that different sets of protein kinases and proteases yet not clearly identified regulate "ongoing DNA replication". A more detailed study was carried out using roscovitine, a highly specific cyclin-dependent kinase inhibitor. The effect of roscovitine on DNA synthesis was evaluated in normal rat cerebral cortex, specimens obtained from human brain tumours and in a pilot experiment using a rat glioma model. We found that roscovitine is a potent inhibitor of ongoing DNA synthesis in the developing rat cerebral cortex as well as in human gliomas but showed little or no effect in adult normal tissue. Moreover, roscovitine inhibited preferentially DNA synthesis connected with replicative processes rather than DNA synthesis connected with DNA repair. In addition, some in vitro studies of redox regulation of topoisomerases and the effects of thiol reacting drugs on this enzyme are presented