RESUMO
Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.
Assuntos
Benzotiazóis/química , Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Diaminas/química , Substâncias Intercalantes/química , Quinolinas/química , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/normas , DNA/análise , DNA/biossíntese , Primers do DNA/química , Primers do DNA/metabolismo , Humanos , Nasofaringe/virologia , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Dipterans exhibit a remarkable diversity of chromosome end structures in contrast to the conserved system defined by telomerase and short repeats. Within dipteran families, structure of chromosome termini is usually conserved within genera. With the aim to assess whether or not the evolutionary distance between genera implies chromosome end diversification, this report exploits two representatives of Sciaridae, Rhynchosciara americana, and Trichomegalosphyspubescens. METHODS: Probes and plasmid microlibraries obtained by chromosome end microdissection, in situ hybridization, cloning, and sequencing are among the methodological approaches employed in this work. RESULTS: The data argue for the existence of either specific terminal DNA sequences for each chromosome tip in T. pubescens, or sequences common to all chromosome ends but their extension does not allow detection by in situ hybridization. Both sciarid species share terminal sequences that are significantly underrepresented in chromosome ends of T. pubescens. CONCLUSIONS: The data suggest an unusual terminal structure in T. pubescens chromosomes compared to other dipterans investigated. A putative, evolutionary process of repetitive DNA expansion that acted differentially to shape chromosome ends of the two flies is also discussed.
Assuntos
Cromossomos de Insetos/genética , Dípteros/genética , Animais , Sequência de Bases , DNA/biossíntese , Biblioteca Gênica , Microdissecção , Plasmídeos/genética , Cromossomos Politênicos/genéticaRESUMO
We aimed to investigate the mechanisms underlying muscle growth after 12 weeks of resistance training performed with blood flow restriction (RT-BFR) and high-intensity resistance training (HRT) in older individuals. Participants were allocated into the following groups: HRT, RT-BFR, or a control group. High-throughput transcriptome sequencing was performed by the Illumina HiSeq 2500 platform. HRT and RT-BFR presented similar increases in the quadriceps femoris cross-sectional area, and few genes were differently expressed between interventions. The small differences in gene expression between interventions suggest that similar mechanisms may underpin training-induced muscle growth.
Assuntos
Envelhecimento/fisiologia , Músculo Esquelético/metabolismo , Educação Física e Treinamento , Fluxo Sanguíneo Regional/fisiologia , Treinamento Resistido , Transcriptoma/fisiologia , Idoso , DNA/biossíntese , DNA/genética , Dieta , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Perna (Membro)/anatomia & histologia , Perna (Membro)/fisiologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/irrigação sanguínea , Músculo Quadríceps/fisiologia , RNA/biossíntese , RNA/genéticaRESUMO
Endoreplication is a cell cycle program in which cells replicate their genomes without undergoing mitosis and cytokinesis. For the normal development of many organisms (from fungi to humans) and the formation of their organs, endoreplication is indispensable. The aim of the present study was to explore whether endoreplication and DNA synthesis are relevant processes during the induction of trained innate immunity in human monocytes and in the Anopheles albimanus mosquito cell line. During the induction of trained immunity in both models, endoreplication markers were overexpressed and we observed an increase in DNA synthesis with an augmented copy number of genes essential for trained immunity. Blocking DNA synthesis prevented trained immunity from being established. Overall, these findings suggest that DNA synthesis and endoreplication are important mechanisms involved in inducing innate immune memory. They have probably been conserved throughout evolution from invertebrates to humans.
Assuntos
Anopheles , DNA , Imunidade Inata , Memória Imunológica , Modelos Imunológicos , Monócitos , Animais , Anopheles/imunologia , Anopheles/metabolismo , DNA/biossíntese , DNA/imunologia , Humanos , Monócitos/imunologia , Monócitos/metabolismoRESUMO
Ruthenium-based compounds have gained great interest due to their potent cytotoxicity in cancer cells; however, much of their potential applications remain unexplored. In this paper, we report the synthesis of a novel ruthenium complex with xanthoxylin (RCX) and the investigation of its cellular and molecular action in human hepatocellular carcinoma HepG2 cells. We found that RCX exhibited a potent cytotoxic effect in a panel of cancer cell lines in monolayer cultures and in a 3D model of multicellular cancer spheroids formed from HepG2 cells. This compound is detected at a high concentration in the cell nuclei, induces DNA intercalation and inhibits DNA synthesis, arresting the cell cycle in the S-phase, which is followed by the activation of the caspase-mediated apoptosis pathway in HepG2 cells. Gene expression analysis revealed changes in the expression of genes related to cell cycle control, apoptosis and the MAPK pathway. In addition, RCX induced the phosphorylation of ERK1/2, and pretreatment with U-0126, an MEK inhibitor known to inhibit the activation of ERK1/2, prevented RCX-induced apoptosis. In contrast, pretreatment with a p53 inhibitor (cyclic pifithrin-α) did not prevent RCX-induced apoptosis, indicating the activation of a p53-independent apoptosis pathway. RCX also presented a potent in vivo antitumor effect in C.B-17 SCID mice engrafted with HepG2 cells. Altogether, these results indicate that RCX is a novel anticancer drug candidate.
Assuntos
Acetofenonas/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Rutênio/farmacologia , Fase S/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Acetofenonas/síntese química , Acetofenonas/química , Animais , Antineoplásicos/farmacologia , Inibidores de Caspase/farmacologia , Caspases/metabolismo , DNA/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Substâncias Intercalantes/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos SCID , Modelos Biológicos , Inibidores de Proteínas Quinases/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The 3'-azido-3'-deoxythymidine or Zidovudine (AZT) was the first antiretroviral drug used in the treatment of HIV patients, which has good effectiveness but also hepatotoxic side effects that include cell cycle arrest and oxidative/nitrative mitochondrial damage. Whether such an oxidative damage may affect the proliferative-regenerative capacity of liver remains to be clearly specified at doses commonly used in the clinical practice. In this study, we described the oxidative-proliferative effect of AZT administered at a common clinical dose in rat liver submitted to 70% partial hepatectomy (PH). The results indicate that AZT significantly decreased DNA synthesis and the number of mitosis in liver subjected to PH in a synchronized way with the promotion of organelle-selective lipid peroxidation events (especially those observed in plasma membrane and cytosolic fractions) and with liver enzyme release to the bloodstream. Then at the dose used in clinical practice AZT decreased liver regeneration but stimulates oxidative events involved during the proliferation process in a way that each membrane system inside the cell preserves its integrity in order to maintain the cell proliferative process. Here, the induction of large amounts of free ammonia in the systemic circulation could become a factor capable of mediating the deleterious effects of AZT on PH-induced rat liver regeneration.
Assuntos
DNA/biossíntese , Regeneração Hepática/efeitos dos fármacos , Fígado/metabolismo , Mitose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Zidovudina/farmacologia , Animais , Hepatectomia , Fígado/cirurgia , Masculino , Ratos , Ratos WistarRESUMO
Genome lesions trigger biological responses that help cells manage damaged DNA, improving cell survival. Pol eta is a translesion synthesis (TLS) polymerase that bypasses lesions that block replicative polymerases, avoiding continued stalling of replication forks, which could lead to cell death. p53 also plays an important role in preventing cell death after ultraviolet (UV) light exposure. Intriguingly, we show that p53 does so by favoring translesion DNA synthesis by pol eta. In fact, the p53-dependent induction of pol eta in normal and DNA repair-deficient XP-C human cells after UV exposure has a protective effect on cell survival after challenging UV exposures, which was absent in p53- and Pol H-silenced cells. Viability increase was associated with improved elongation of nascent DNA, indicating the protective effect was due to more efficient lesion bypass by pol eta. This protection was observed in cells proficient or deficient in nucleotide excision repair, suggesting that, from a cell survival perspective, proper bypass of DNA damage can be as relevant as removal. These results indicate p53 controls the induction of pol eta in DNA damaged human cells, resulting in improved TLS and enhancing cell tolerance to DNA damage, which parallels SOS responses in bacteria.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Sobrevivência Celular , Cromatina/metabolismo , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/genética , Relação Dose-Resposta à Radiação , Fibroblastos/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Raios UltravioletaRESUMO
BACKGROUND: The liver has the remarkable capacity to regenerate in order to compensate for lost or damaged hepatic tissue. However, pre-existing pathological abnormalities, such as hepatic steatosis (HS), inhibits the endogenous regenerative process, becoming an obstacle for liver surgery and living donor transplantation. Recent evidence indicates that multipotent mesenchymal stromal cells (MSCs) administration can improve hepatic function and increase the potential for liver regeneration in patients with liver damage. Since HS is the most common form of chronic hepatic illness, in this study we evaluated the role of MSCs in liver regeneration in an animal model of severe HS with impaired liver regeneration. METHODS: C57BL/6 mice were fed with a regular diet (normal mice) or with a high-fat diet (obese mice) to induce HS. After 30 weeks of diet exposure, 70% hepatectomy (Hpx) was performed and normal and obese mice were divided into two groups that received 5 × 105 MSCs or vehicle via the tail vein immediately after Hpx. RESULTS: We confirmed a significant inhibition of hepatic regeneration when liver steatosis was present, while the hepatic regenerative response was promoted by infusion of MSCs. Specifically, MSC administration improved the hepatocyte proliferative response, PCNA-labeling index, DNA synthesis, liver function, and also reduced the number of apoptotic hepatocytes. These effects may be associated to the paracrine secretion of trophic factors by MSCs and the hepatic upregulation of key cytokines and growth factors relevant for cell proliferation, which ultimately improves the survival rate of the mice. CONCLUSIONS: MSCs represent a promising therapeutic strategy to improve liver regeneration in patients with HS as well as for increasing the number of donor organs available for transplantation.
Assuntos
Fígado Gorduroso/terapia , Regeneração Hepática/fisiologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Obesidade/terapia , Animais , Apoptose , Biomarcadores/metabolismo , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , DNA/biossíntese , Dieta Hiperlipídica , Fígado Gorduroso/etiologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Expressão Gênica , Hepatectomia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/metabolismo , Obesidade/etiologia , Obesidade/genética , Obesidade/patologia , Comunicação Parácrina , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transplante HomólogoRESUMO
Congenital transmission of Trypanosoma cruzi (T. cruzi) is partially responsible for the progressive globalization of Chagas disease. During congenital transmission the parasite must cross the placental barrier where the trophoblast, a continuous renewing epithelium, is the first tissue in contact with the parasite. The trophoblast turnover implies cellular proliferation, differentiation and apoptotic cell death. The epithelial turnover is considered part of innate immunity. We previously demonstrated that T. cruzi induces cellular differentiation and apoptosis in this tissue. Here we demonstrate that T. cruzi induces cellular proliferation in a trophoblastic cell line. We analyzed the cellular proliferation in BeWo cells by determining DNA synthesis by BrdU incorporation assays, mitotic index, cell cycle analysis by flow cytometry, as well as quantification of nucleolus organizer regions by histochemistry and expression of the proliferation markers PCNA and Ki67 by Western blotting and/or immunofluorescence. Additionally, we determined the ERK1/2 MAPK pathway activation by the parasite by Western blotting.
Assuntos
Proliferação de Células , Trofoblastos/citologia , Trofoblastos/parasitologia , Trypanosoma cruzi/fisiologia , Animais , Divisão Celular , Linhagem Celular Tumoral , DNA/biossíntese , Citometria de Fluxo , Fase G2 , Antígeno Ki-67/metabolismo , Sistema de Sinalização das MAP Quinases , Índice Mitótico , Região Organizadora do Nucléolo/ultraestrutura , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fase S , Trofoblastos/metabolismoRESUMO
The levels of the cyclin-dependent kinase (CDK) inhibitor p21 are low in S phase and insufficient to inhibit CDKs. We show here that endogenous p21, instead of being residual, it is functional and necessary to preserve the genomic stability of unstressed cells. p21depletion slows down nascent DNA elongation, triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions, namely common fragile sites (CFS). The p21's PCNA interacting region (PIR), and not its CDK binding domain, is needed to prevent the replication defects and the genomic instability caused by p21 depletion. The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa. Hence, in CDK-independent manner, endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis.