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Background: The high COVID-19 dissemination rate demands active surveillance to identify asymptomatic, presymptomatic, and oligosymptomatic (APO) SARS-CoV-2-infected individuals. This is of special importance in communities inhabiting closed or semi-closed institutions such as residential care homes, prisons, neuropsychiatric hospitals, etc., where risk people are in close contact. Thus, a pooling approach-where samples are mixed and tested as single pools-is an attractive strategy to rapidly detect APO-infected in these epidemiological scenarios. Materials and Methods: This study was done at different pandemic periods between May 28 and August 31 2020 in 153 closed or semi-closed institutions in the Province of Buenos Aires (Argentina). We setup pooling strategy in two stages: first a pool-testing followed by selective individual-testing according to pool results. Samples included in negative pools were presumed as negative, while samples from positive pools were re-tested individually for positives identification. Results: Sensitivity in 5-sample or 10-sample pools was adequate since only 2 Ct values were increased with regard to single tests on average. Concordance between 5-sample or 10-sample pools and individual-testing was 100% in the Ct ≤ 36. We tested 4,936 APO clinical samples in 822 pools, requiring 86-50% fewer tests in low-to-moderate prevalence settings compared to individual testing. Conclusions: By this strategy we detected three COVID-19 outbreaks at early stages in these institutions, helping to their containment and increasing the likelihood of saving lives in such places where risk groups are concentrated.
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RATIONALE: Cocaine base paste (CBP) is an illegal drug of abuse usually consumed by adolescents in a socio-economically vulnerable situation. Repeated drug use targets key brain circuits disrupting the processes that underlie emotions and cognition. At the basis of such neuroadaptations lie changes in the expression of immediate-early genes (IEGs). Nevertheless, changes in transcriptional regulation associated with CBP consumption remain unknown. OBJECTIVES: We aimed to describe behavioral phenotype related to locomotion, anxiety-like behavior, and memory of CBP-injected mice and to study IEGs expression after an abstinence period. METHODS: Five-week-old female CF-1 mice were i.p. injected daily with vehicle or CBP (40 mg/kg) for 10 days and subjected to a 10-day period of abstinence. Open field and novel object recognition tests were used to evaluate locomotion and anxiety-like behaviors and recognition memory, respectively, during chronic administration and after abstinence. After abstinence, prefrontal cortex (mPFC) and nucleus accumbens (NAc) were isolated and gene expression analysis performed through real-time PCR. RESULTS: We found an increase in locomotion and anxiety-like behavior during CBP administration and after the abstinence period. Furthermore, the CBP group showed impaired recognition memory after abstinence. Egr1, FosB, ΔFosB, Arc, Bdnf, and TrkB expression was upregulated in CBP-injected mice in NAc and FosB, ΔFosB, Arc, and Npas4 expression was downregulated in mPFC. We generated an anxiety score and found positive and negative correlations with IEGs expression in NAc and mPFC, respectively. CONCLUSION: Our results suggest that chronic CBP exposure induced alterations in anxiety-like behavior and recognition memory. These changes were accompanied by altered IEGs expression.
Assuntos
Ansiedade/induzido quimicamente , Ansiedade/metabolismo , Cocaína/administração & dosagem , Genes Precoces/fisiologia , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Animais , Ansiedade/psicologia , Cocaína/toxicidade , Inibidores da Captação de Dopamina/administração & dosagem , Inibidores da Captação de Dopamina/toxicidade , Feminino , Regulação da Expressão Gênica , Genes Precoces/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Camundongos , Núcleo Accumbens/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacosRESUMO
E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.
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Dano ao DNA , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F2/metabolismo , Instabilidade Genômica , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cicloeximida/toxicidade , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Dactinomicina/toxicidade , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F2/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Células HEK293 , Histonas/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , MAP Quinase Quinase Quinases/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Inibidores da Síntese de Proteínas/toxicidade , Rad51 Recombinase/metabolismo , Raios Ultravioleta , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.
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Inibidor de Quinase Dependente de Ciclina p19/metabolismo , Dano ao DNA , Fator de Transcrição E2F1/metabolismo , Ativação Transcricional/efeitos da radiação , Raios Ultravioleta , Animais , Ciclo Celular , Cricetinae , Inibidor de Quinase Dependente de Ciclina p19/genética , DNA/farmacologia , Reparo do DNA , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/fisiologia , Células HEK293 , Humanos , Elementos de Resposta , Transcrição Gênica/efeitos da radiaçãoRESUMO
INTRODUCTION: The role of apoptotic secretory epithelium as a pro-inflammatory triggering factor of exocrine dysfunction is currently explored in Sjogren's syndrome patients and in the nonobese diabetic (NOD) mouse model. Vasoactive intestinal peptide (VIP) has anti-inflammatory effects in various models of chronic inflammation. Our goal was to analyse the effect of TNF-alpha on apoptotic mediators in isolated acinar cells from NOD submandibular gland and their modulation by VIP. METHODS: Acinar cells were isolated from submandibular glands of 16-week-old NOD females with salivary flow decline. Age-matched BALB/c females or eight-week-old NOD females were used as controls. Apoptotic mediators and TNF-alpha receptor expression were assessed by immunoblotting and RT-PCR, caspase 3 activity was assessed by optical density at 405 nm with Ac-DEVD-pNA as a substrate and chromatin condensation by Hoechst stain. They were evaluated in resting conditions and after a 3.5 or 6 hours of culture with TNF-alpha. VIP effects in acinar cells were assessed at 100 nM in TNF-alpha-treated cultures and VIP receptor functional assays by radio immunoassay (cAMP) or enzymatic detection (amylase). RESULTS: NOD acinar cells at 16 weeks present an increased expression of TNF-alpha receptor1 together with increased Bax, tumour protein 53-induced nuclear protein1alpha (TP53INP1alpha), caspase 3 activity and chromatin condensation. Acini from NOD mice were more sensitive to TNF-alpha-induced increases of apoptotic mediators than control cells. VIP inhibited TNF-alpha-induced apoptotic events through functional VPAC1 receptors coupled to the protein kinase A (PKA) signalling pathway. CONCLUSIONS: Our results indicate that acinar cells isolated from submandibular glands of NOD mice with salivary dysfunction are more sensitive to apoptosis induced by TNF-alpha which could be prevented by VIP through a PKA-mediated pathway.
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Apoptose/fisiologia , Síndrome de Sjogren/metabolismo , Glândula Submandibular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Western Blotting , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Síndrome de Sjogren/patologia , Glândula Submandibular/patologiaRESUMO
The growth factor erythropoietin (Epo) has shown neuronal protective action in addition to its well known proerythroid activity. Furthermore, Epo has dealt with cellular inflammation by inhibiting the expression of several proinflammatory cytokines, such as IL-1 and TNF-alpha. The action of TNF can have both apoptotic and antiapoptotic consequences due to altered balance between different cell signalling pathways. This work has focused on the apoptotic effects of this cytokine and the potential protective action of Epo. The model we used was neuroblastoma SH-SY5Y cells cultured in the presence of 25 ng/ml TNF-alpha or pretreated with 25 U/ml Epo for 12 h before the addition of TNF-alpha. Apoptosis was evaluated by differential cell count after Hoechst staining, analysis of DNA ladder pattern, and measurement of caspase activity. Despite its ability to induce NF-kappaB nuclear translocation, TNF-alpha induced cell death, which was found to be associated to upregulation of TNF Receptor 1 expression. On the other hand, cells activated by Epo became resistant to cell death. Prevention of death receptor upregulation and caspase activation may explain this antiapoptotic effect of Epo, which may be also favoured by the induction of a higher expression of protective factors, such as Bcl-2 and NF-kappaB, through mechanisms involving Jak/STAT and PI3K signalling pathways.
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Apoptose , Eritropoetina/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular Tumoral , Citoplasma/metabolismo , Humanos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Recombinantes , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
BACKGROUND/AIMS: Since the reversible phosphorylation of tyrosyl residues is a critical event in cellular signaling pathways activated by erythropoietin (Epo), attention has been focused on protein tyrosine phosphatases (PTPs) and their coordinated action with protein tyrosine kinases. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines, and hormones, such as the downregulation of erythropoietin and insulin receptors. However, little is known about which factor modulates the activity of this enzyme. METHODS: The effect of Epo on PTP1B expression was studied in the UT-7 Epo-dependent cell line. PTP1B expression was analyzed under different conditions by Real-Time PCR and Western blot, while PTP1B phosphatase activity was determined by a p-nitrophenylphosphate hydrolysis assay. RESULTS: Epo rapidly induced an increased expression of PTP1B which was associated with higher PTP1B tyrosine phosphorylation and phosphatase activity. The action of Epo on PTP1B induction involved Janus Kinase 2 (JAK2) and Phosphatidylinositol-3 kinase (PI3K). CONCLUSION: The results allow us to suggest for the first time that, besides modulating Epo/Epo receptor signaling, PTP1B undergoes feedback regulation by Epo.
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Eritropoetina/farmacologia , Proteínas Tirosina Fosfatases/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Janus Quinase 2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/genética , Tirfostinas/farmacologiaRESUMO
To study the effects of aluminium (Al) on glutathione (GSH) metabolism in the small intestine, adult male Wistar rats were orally treated with AlCl3.6H2O at doses of 30, 60, 120 and 200 mg/kg body weight (b.w.) per day, during seven days. Controls received deionized water. At doses above 120 mg/kg b.w., Al produced both a significant reduction of GSH content and an increase of oxidized/reduced glutathione ratio (P < 0.05). The index of oxidative stress of the intestine mucosa in terms of lipid peroxidation evaluated by thiobarbituric acid reactive substances was significantly increased (52%) at higher Al dose used. The duodenal expression of the multidrug resistance-associated protein 2 in brush border membranes, determined by Western blot technique, was increased 2.7-fold in rats treated with 200mg AlCl3/kg b.w (P < 0.01). Intestine activities of both GSH-synthase (from 60 mg/kg b.w.) and GSSG-reductase (from 120 mg/kg b.w.) were significantly reduced (26% and 31%, respectively) while glutathione-S-transferase showed to be slightly modified in the Al-treated groups. Conversely, gamma-glutamyltranspeptidase activity was significantly increased (P < 0.05) due to the Al treatment. Al reduced in vitro mucosa-to-lumen GSH efflux (P < 0.05). A positive linear correlation between the intestine GSH depletion and reduction of in situ 45Ca intestinal absorption, both produced by Al, was found (r = 0.923, P = 0.038). Taking as a whole, these results show that Al would alter GSH metabolism in small intestine by decreasing its turnover, leading to an unbalance of redox state in the epithelial cells, thus contributing to deteriorate GSH-dependent absorptive functions.
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Alumínio/toxicidade , Glutationa/metabolismo , Intestinos/efeitos dos fármacos , Administração Oral , Animais , Glutationa Sintase/metabolismo , Glutationa Transferase/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/enzimologia , Peroxidação de Lipídeos , Masculino , Proteínas Mitocondriais/metabolismo , Ratos , Ratos Wistar , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Aluminum (Al) and iron (Fe) share several physicochemical characteristics and they both bind to transferrin (Tf), entering the cell via Tf receptors (TfR). Previously, we found similar values of affinity constant for the binding of TfR to Tf carrying either Al or Fe. The competitive interaction between both metals prevented normal Fe incorporation into K562 cells and triggered the upregulation of Fe transport. In the present work we demonstrated that Al modified Fe uptake without affecting the expression of Tf receptors. Both TfR and TfR2 mRNA levels, evaluated by RT-PCR, and TfR antigenic sites, analyzed by flow cytometry, were found unchanged after Al exposure. In turn, Al did induce upregulation of non-Tf bound Fe (NTBI) uptake. This modulation was not due to intracellular Fe decrease since NTBI transport proved not to be regulated by Fe depletion. Unlike its behavior in the presence of Tf, Al was unable to compete with NTBI uptake, suggesting that both metals do not share the same alternative transport pathway. We propose that Al interference with TfR-mediated Fe incorporation might trigger the upregulation of NTBI uptake, an adaptation aimed at incorporating the essential metal required for cellular metabolism without allowing the simultaneous access of a potentially toxic metal.
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Alumínio/farmacologia , Ferro/metabolismo , Transferrina/metabolismo , Transporte Biológico/efeitos dos fármacos , Butiratos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , RNA Mensageiro/genética , Receptores da Transferrina/genética , Transferrina/genéticaRESUMO
Erythropoietin (Epo) promotes the development of erythroid progenitors by triggering intracellular signals through the binding to its specific receptor (EpoR). Previous results related to the action of aluminum (Al) on erythropoiesis let us suggest that the metal affects Epo interaction with its target cells. In order to investigate this effect on cell activation by the Epo-EpoR complex, two human cell lines with different dependence on Epo were subjected to Al exposure. In the Epo-independent K562 cells, Al inhibited Epo antiapoptotic action and triggered a simultaneous decrease in protein and mRNA EpoR levels. On the other hand, proliferation of the strongly Epo-dependent UT-7 cells was enhanced by long-term Al treatment, in agreement with the upregulation of EpoR expression during Epo starvation. Results provide some clues to the way by which Epo supports cell survival and growth, and demonstrate that not all the intracellular factors needed to guarantee the different signaling pathways of Epo-cell activation are available or activated in cells expressing EpoR. This study then suggests that at least one of the mechanisms by which Al interfere with erythropoiesis might involve EpoR modulation.