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The increased risk for acquiring secondary illnesses in people living with HIV (PLWH) has been associated with immune dysfunction. We have previously found that circulating monocytes from PLWH display a trained phenotype. Here, we evaluated the metabolic profile of these cells and found increased mitochondrial respiration and glycolysis of monocyte-derived macrophages (MDMs) from PLWH. We additionally found that cART shifted the energy metabolism of MDMs from controls toward increased utilization of mitochondrial respiration. Importantly, both downregulation of IKAROS expression and inhibition of the mTOR pathway reversed the metabolic profile of MDMs from PLWH and cART-treated control-MDMs. Altogether, this study reveals a very specific metabolic adaptation of MDMs from PLWH, which involves an IKAROS/mTOR-dependent increase of mitochondrial respiration and glycolysis. We propose that this metabolic adaptation decreases the ability of these cells to respond to environmental cues by "locking" PLWH monocytes in a pro-inflammatory and activated phenotype.
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Infecções por HIV , Humanos , Macrófagos , Monócitos , Fenótipo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Glioblastomas are highly aggressive brain tumors for which therapeutic options are very limited. In a quest for new anti-glioblastoma drugs, we focused on specific structural modifications to the benzoyl-phenoxy-acetamide (BPA) structure present in a common lipid-lowering drug, fenofibrate, and in our first prototype glioblastoma drug, PP1. Here, we propose extensive computational analyses to improve the selection of the most effective glioblastoma drug candidates. Initially, over 100 structural BPA variations were analyzed and their physicochemical properties, such as water solubility (- logS), calculated partition coefficient (ClogP), probability for BBB crossing (BBB_SCORE), probability for CNS penetration (CNS-MPO) and calculated cardiotoxicity (hERG), were evaluated. This integrated approach allowed us to select pyridine variants of BPA that show improved BBB penetration, water solubility, and low cardiotoxicity. Herein the top 24 compounds were synthesized and analyzed in cell culture. Six of them demonstrated glioblastoma toxicity with IC50 ranging from 0.59 to 3.24 µM. Importantly, one of the compounds, HR68, accumulated in the brain tumor tissue at 3.7 ± 0.5 µM, which exceeds its glioblastoma IC50 (1.17 µM) by over threefold.
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Neoplasias Encefálicas , Glioblastoma , Humanos , Barreira Hematoencefálica , Cardiotoxicidade , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Neoplasias Encefálicas/tratamento farmacológico , Simulação por Computador , Acetamidas/farmacologia , Piridinas/farmacologia , Água/farmacologia , Linhagem Celular TumoralRESUMO
Mild Traumatic Brain Injury (mild TBI)/concussion is a common sports injury, especially common in football players. Repeated concussions are thought to lead to long-term brain damage including chronic traumatic encephalopathy (CTE). With the worldwide growing interest in studying sport-related concussion the search for biomarkers for early diagnosis and progression of neuronal injury has also became priority. MicroRNAs are short, non-coding RNAs that regulate gene expression post-transcriptionally. Due to their high stability in biological fluids, microRNAs can serve as biomarkers in a variety of diseases including pathologies of the nervous system. In this exploratory study, we have evaluated changes in the expression of selected serum miRNAs in collegiate football players obtained during a full practice and game season. We found a miRNA signature that can distinguish with good specificity and sensitivity players with concussions from non-concussed players. Furthermore, we found miRNAs associated with the acute phase (let-7c-5p, miR-16-5p, miR-181c-5p, miR-146a-5p, miR-154-5p, miR-431-5p, miR-151a-5p, miR-181d-5p, miR-487b-3p, miR-377-3p, miR-17-5p, miR-22-3p, and miR-126-5p) and those whose changes persist up to 4 months after concussion (miR-17-5p and miR-22-3p).
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BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of estrogen receptor, progesterone receptor, and HER2. Our lab previously characterized miR-3189-3p as a microRNA with potent anti-cancer activity against glioblastoma. Here, we hypothesized a similar activity in TNBC cells. As miR-3189-3p is predicted to target a variety of RNA binding proteins, we further hypothesized an inhibitory effect of this miRNA on protein synthesis. METHODS: MDA-MB-231 and MDA-MB-468 cells were used to investigate the effect of miR-3189-3p on cell proliferation, migration, and invasion. TGCA database was used to analyze the expression of miR-3189-3p, c-MYC, 4EPB1, and eIF4E in breast cancer. Western blotting and RT-qPCR assays were used to assess the expression of selected proteins and RNAs after transfections. RESULTS: Although c-MYC is not a predicted gene target for miR-3189-3p, we discovered that c-MYC protein is downregulated in miRNA-treated TNBC cells. We found that the downregulation of c-MYC by miR-3189-3p occurs in both normal growth conditions and in the absence of serum. The mechanism involved the direct inhibition of eIF4EBP1 by miR-3189-3p. Additionally, we found that miR-3189-3p could negatively affect cap-independent translation mediated by internal ribosome entry sites (IRES) or by m6A. Finally, miR-3189-3p sensitized TNBC cells to doxorubicin. CONCLUSION: Overall, results indicated that miR-3189-3p exerts its anti-tumor activity through targeting translational regulatory proteins leading to an impairment in c-MYC translation, and possibly other oncogenic factors, suggesting that miR-3189-3p, alone or in combination, could be a valuable therapeutic approach against a malignancy with few treatment options.
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Glioblastomas are the most aggressive brain tumors for which therapeutic options are limited. Current therapies against glioblastoma include surgical resection, followed by radiotherapy plus concomitant treatment and maintenance with temozolomide (TMZ), however, these standard therapies are often ineffective, and average survival time for glioblastoma patients is between 12 and 18 months. We have previously reported a strong anti-glioblastoma activity of several metabolic compounds, which were synthetized based compounds, which were synthetized based on the chemical structure of a common lipid-lowering drug, fenofibrate, and share a general molecular skeleton of benzoylphenoxyacetamide (BPA). Extensive computational analyses of phenol and naphthol moieties added to the BPA skeleton were performed in this study with the objective of selecting new BPA variants for subsequent compound preparation and anti-glioblastoma testing. Initially, 81 structural variations were considered and their physical properties such as solubility (logS), blood-brain partitioning (logBB), and probability of entering the CNS calculated by the Central Nervous System-Multiparameter Optimization (MPO-CNS) algorithm were evaluated. From this initial list, 18 compounds were further evaluated for anti-glioblastoma activity in vitro. Nine compounds demonstrated desirable glioblastoma cell toxicity in cell culture, and two of them, HR51, and HR59 demonstrated significantly improved capability of crossing the model blood-brain-barrier (BBB) composed of endothelial cells, astrocytes and pericytes.
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Neoplasias Encefálicas , Glioblastoma , Antineoplásicos Alquilantes/farmacologia , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/patologia , Células Endoteliais/metabolismo , Glioblastoma/patologia , Humanos , Temozolomida/farmacologiaRESUMO
OBJECTIVES: To report and analyse the characteristics and performance of the first cohort of Italian radiologists completing the national mammography self-evaluation online test established by the Italian Society of Medical Radiology (SIRM). METHODS: A specifically-built dataset of 132 mammograms (24 with screen-detected cancers and 108 negative cases) was preliminarily tested on 48 radiologists to define pass thresholds (62% sensitivity and 86% specificity) and subsequently made available online to SIRM members during a 13-month timeframe between 2018 and 2019. Associations between participants' characteristics, pass rates, and diagnostic accuracy were then investigated with descriptive statistics and univariate and multivariable regression analyses. RESULTS: A total of 342 radiologists completed the test, 151/342 (44.2%) with success. All individual variables, except gender, showed a significant correlation with pass rates and diagnostic sensitivity, confirmed by univariate logistic regression, while only involvement in organised screening programs and number of mammograms read per year showed a positive association with specificity at univariate logistic regression. In the multivariable regression analysis, fewer variables remained significant: > 3000 mammograms read per year for success rate; female gender, public practice setting, and higher experience self-judgement for sensitivity; no variables were significantly associated with specificity. CONCLUSIONS: This national self-evaluation test effectively differentiated multiple aspects of mammographic reading experience, but specific breast imaging experience was shown not to strictly guarantee good diagnostic accuracy. Due to its easy use and the validity of obtained results, this test could be extended to all Italian breast radiologists, regardless of their experience, also as a Breast Unit accreditation criterion. KEY POINTS: ⢠This self-evaluation test was found to be able to differentiate various degrees of mammographic interpretation experience. ⢠Breast cancer screening readers should undergo a self-assessment test, since experience parameters alone do not guarantee diagnostic ability.
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Neoplasias da Mama , Radiologia , Neoplasias da Mama/diagnóstico por imagem , Autoavaliação Diagnóstica , Feminino , Humanos , Mamografia , Programas de Rastreamento , Autoavaliação (Psicologia) , Sensibilidade e EspecificidadeRESUMO
Persons living with HIV (PLWH) are at higher risk of developing secondary illnesses than their uninfected counterparts, suggestive of a dysfunctional immune system in these individuals. Upon exposure to pathogens, monocytes undergo epigenetic remodeling that results in either a trained or a tolerant phenotype, characterized by hyper-responsiveness or hypo-responsiveness to secondary stimuli, respectively. We utilized CD14+ monocytes from virally suppressed PLWH and healthy controls for in vitro analysis following polarization of these cells toward a pro-inflammatory monocyte-derived macrophage (MDM) phenotype. We found that in PLWH-derived MDMs, pro-inflammatory signals (TNFA, IL6, IL1B, miR-155-5p, and IDO1) dominate over negative feedback signals (NCOR2, GSN, MSC, BIN1, and miR-146a-5p), favoring an abnormally trained phenotype. The mechanism of this reduction in negative feedback involves the attenuated expression of IKZF1, a transcription factor required for de novo synthesis of RELA during LPS-induced inflammatory responses. Furthermore, restoring IKZF1 expression in PLWH-MDMs partially reinstated expression of negative regulators of inflammation and lowered the expression of pro-inflammatory cytokines. Overall, this mechanism may provide a link between dysfunctional immune responses and susceptibility to co-morbidities in PLWH with low or undetectable viral load.
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Suscetibilidade a Doenças/imunologia , Infecções por HIV/imunologia , Fator de Transcrição Ikaros/metabolismo , Macrófagos/imunologia , Fator de Transcrição RelA/metabolismo , Fármacos Anti-HIV/administração & dosagem , Estudos de Casos e Controles , Citocinas/metabolismo , Retroalimentação Fisiológica , Feminino , Regulação da Expressão Gênica/imunologia , HIV/imunologia , HIV/isolamento & purificação , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Voluntários Saudáveis , Humanos , Inflamação/sangue , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fator de Transcrição RelA/genética , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
Neurocognitive disorders associated with HIV-1 infection affect more than half of persons living with HIV (PLWH) under retroviral therapy. Understanding the molecular mechanisms and the complex cellular network communication underlying neurological dysfunction is critical for the development of an effective therapy. As with other neurological disorders, challenges to studying HIV infection of the brain include limited access to clinical samples and proper reproducibility of the complexity of brain networks in cellular and animal models. This review focuses on cellular models used to investigate various aspects of neurological dysfunction associated with HIV infection.
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Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.
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Separação Celular/métodos , Monócitos/citologia , Transfecção , Animais , Polaridade Celular , Sobrevivência Celular , Células Cultivadas , Regulação para Baixo , Humanos , Ativação de Macrófagos , Macrófagos/citologia , MicroRNAs/genética , MicroRNAs/metabolismo , Fenótipo , RNA Interferente Pequeno/metabolismoRESUMO
Insulin receptor substrate 1 (IRS-1) is a common cytosolic adaptor molecule involved in signal transduction from insulin and insulin-like growth factor I (IGF-I) receptors. IRS-1 can also be found in the nucleus. We report here a new finding of unique IRS-1 nuclear structures, which we observed initially in glioblastoma biopsy specimens and glioblastoma xenografts. These nuclear structures can be reproduced in vitro by the ectopic expression of IRS-1 cDNA cloned in frame with the nuclear localization signal (NLS-IRS-1). In these structures, IRS-1 localizes at the periphery, while the center harbors a key autophagy protein, LC3. These new nuclear structures are highly dynamic, rapidly exchange IRS-1 molecules with the surrounding nucleoplasm, disassemble during mitosis, and require a growth stimulus for their reassembly and maintenance. In tumor cells engineered to express NLS-IRS-1, the IRS-1/LC3 nuclear structures repress autophagy induced by either amino acid starvation or rapamycin treatment. In this process, IRS-1 nuclear structures sequester LC3 inside the nucleus, possibly preventing its cytosolic translocation and the formation of new autophagosomes. This novel mechanism provides a quick and reversible way of inhibiting autophagy, which could counteract autophagy-induced cancer cell death under severe stress, including anticancer therapies.
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Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Autofagia/fisiologia , Núcleo Celular/fisiologia , Sobrevivência Celular/genética , Glioblastoma/metabolismo , Células HeLa , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/ultraestrutura , Fator de Crescimento Insulin-Like I/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Neoplasias , Fosfoproteínas , Receptor IGF Tipo 1/fisiologia , Transdução de SinaisRESUMO
Recent evidence indicates synaptic dysfunction as an early mechanism affected in neuroinflammatory diseases, such as multiple sclerosis, which are characterized by chronic microglia activation. However, the mode(s) of action of reactive microglia in causing synaptic defects are not fully understood. In this study, we show that inflammatory microglia produce extracellular vesicles (EVs) which are enriched in a set of miRNAs that regulate the expression of key synaptic proteins. Among them, miR-146a-5p, a microglia-specific miRNA not present in hippocampal neurons, controls the expression of presynaptic synaptotagmin1 (Syt1) and postsynaptic neuroligin1 (Nlg1), an adhesion protein which play a crucial role in dendritic spine formation and synaptic stability. Using a Renilla-based sensor, we provide formal proof that inflammatory EVs transfer their miR-146a-5p cargo to neuron. By western blot and immunofluorescence analysis we show that vesicular miR-146a-5p suppresses Syt1 and Nlg1 expression in receiving neurons. Microglia-to-neuron miR-146a-5p transfer and Syt1 and Nlg1 downregulation do not occur when EV-neuron contact is inhibited by cloaking vesicular phosphatidylserine residues and when neurons are exposed to EVs either depleted of miR-146a-5p, produced by pro-regenerative microglia, or storing inactive miR-146a-5p, produced by cells transfected with an anti-miR-146a-5p. Morphological analysis reveals that prolonged exposure to inflammatory EVs leads to significant decrease in dendritic spine density in hippocampal neurons in vivo and in primary culture, which is rescued in vitro by transfection of a miR-insensitive Nlg1 form. Dendritic spine loss is accompanied by a decrease in the density and strength of excitatory synapses, as indicated by reduced mEPSC frequency and amplitude. These findings link inflammatory microglia and enhanced EV production to loss of excitatory synapses, uncovering a previously unrecognized role for microglia-enriched miRNAs, released in association to EVs, in silencing of key synaptic genes.
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Vesículas Extracelulares/imunologia , Inflamação/metabolismo , MicroRNAs/metabolismo , Neuroglia/imunologia , Neurônios/imunologia , Sinapses/imunologia , Animais , Células Cultivadas , Líquido Cefalorraquidiano/metabolismo , Técnicas de Cocultura , Vesículas Extracelulares/patologia , Feminino , Hipocampo/imunologia , Hipocampo/patologia , Humanos , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Neuroglia/patologia , Plasticidade Neuronal/fisiologia , Neurônios/patologia , Cultura Primária de Células , Ratos Sprague-Dawley , Sinapses/patologiaRESUMO
HIV-associated neurocognitive disorders (HAND) affects more than half of persons living with HIV-1/AIDS (PLWHA). Identification of biomarkers representing the cognitive status of PLWHA is a critical step for implementation of successful cognitive, behavioral and pharmacological strategies to prevent onset and progression of HAND. However, the presence of co-morbidity factors in PLWHA, the most common being substance abuse, can prevent the identification of such biomarkers. We have optimized a protocol to profile plasma miRNAs using quantitative RT-qPCR and found a miRNA signature with very good discriminatory ability to distinguish PLWHA with cognitive impairment from those without cognitive impairment. Here, we have evaluated this miRNA signature in PLWHA with alcohol use disorder (AUD) at LSU Health Sciences Center (LSUHSC). The results show that AUD is a potential confounding factor for the miRNAs associated with cognitive impairment in PLWHA. Furthermore, we have investigated the miRNA signature associated with cognitive impairment in an independent cohort of PLWHA using plasma samples from the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) program. Despite differences between the two cohorts in socioeconomic status, AUD, and likely misuse of illicit or prescription drugs, we validated a miRNA signature for cognitive deficits found at LSUHSC in the CHARTER samples.
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Alcohol use disorders (AUD) exacerbate neurocognitive dysfunction in Human Immunodeficiency Virus (HIV+) patients. We have shown that chronic binge alcohol (CBA) administration (13-14 g EtOH/kg/wk) prior to and during simian immunodeficiency virus (SIV) infection in rhesus macaques unmasks learning deficits in operant learning and memory tasks. The underlying mechanisms of neurocognitive alterations due to alcohol and SIV are not known. This exploratory study examined the CBA-induced differential expression of hippocampal genes in SIV-infected (CBA/SIV+; n = 2) macaques in contrast to those of sucrose administered, SIV-infected (SUC/SIV+; n = 2) macaques. Transcriptomes of hippocampal samples dissected from brains obtained at necropsy (16 months post-SIV inoculation) were analyzed to determine differentially expressed genes. MetaCore from Thomson Reuters revealed enrichment of genes involved in inflammation, immune responses, and neurodevelopment. Functional relevance of these alterations was examined in vitro by exposing murine neural progenitor cells (NPCs) to ethanol (EtOH) and HIV trans-activator of transcription (Tat) protein. EtOH impaired NPC differentiation as indicated by decreased ßIII tubulin expression. These findings suggest a role for neuroinflammation and neurogenesis in CBA/SIV neuropathogenesis and warrant further investigation of their potential contribution to CBA-mediated neurobehavioral deficits.
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Consumo Excessivo de Bebidas Alcoólicas/complicações , Perfilação da Expressão Gênica/métodos , Hipocampo/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Animais , Consumo Excessivo de Bebidas Alcoólicas/genética , Consumo Excessivo de Bebidas Alcoólicas/imunologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Etanol/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Macaca mulatta/virologia , Camundongos , Neurogênese/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/imunologiaRESUMO
Human Immunodeficiency Virus (HIV)-infected individuals are at increased risk for developing neurocognitive disorders and depression. These conditions collectively affect more than 50% of people living with HIV/AIDS and adversely impact adherence to HIV therapy. Thus, identification of early markers of neurocognitive impairment could lead to interventions that improve psychosocial functioning and slow or reverse disease progression through improved treatment adherence. Evidence has accumulated for the role and function of microRNAs in normal and pathological conditions. We have optimized a protocol to profile microRNAs in body fluids. Using this methodology, we have profiled plasma microRNA expression for 30 age-matched, HIV-infected (HIV(+) ) patients and identified highly sensitive and specific microRNA signatures distinguishing HIV(+) patients with cognitive impairment from those without cognitive impairment. These results justify follow-on studies to determine whether plasma microRNA signatures can be used as a screening or prognostic tool for HIV(+) patients with neurocognitive impairment. J. Cell. Physiol. 231: 829-836, 2016. © 2015 Wiley Periodicals, Inc.
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Disfunção Cognitiva/sangue , Disfunção Cognitiva/complicações , Infecções por HIV/sangue , Infecções por HIV/complicações , MicroRNAs/sangue , Adulto , Disfunção Cognitiva/genética , Demografia , Infecções por HIV/genética , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
The HIV-1 transactivator protein Tat is implicated in the neuronal damage that contributes to neurocognitive impairment affecting people living with HIV/AIDS. Aberrant splicing of TAU exon 10 results in tauopathies characterized by alterations in the proportion of TAU isoforms containing three (3R) or four (4R) microtubule-binding repeats. The splicing factor SC35/SRSF2 binds to nuclear RNA and facilitates the incorporation of exon 10 in the TAU molecule. Here, we utilized clinical samples, an animal model, and neuronal cell cultures and found that Tat promotes TAU 3R up-regulation through increased levels of phosphorylated SC35, which is retained in nuclear speckles. This mechanism involved Tat-mediated increased expression of DYRK1A and was prevented by DYRK1A silencing. In addition, we found that Tat associates with TAU RNA, further demonstrating that Tat interferes with host RNA metabolism in the absence of viral infection. Altogether, our data unravel a novel mechanism of Tat-mediated neuronal toxicity through dysregulation of the SC35-dependent alternative splicing of TAU exon 10. Furthermore, the increased immunostaining of DYRK1A in HIV+ brains without pathology points at dysregulation of DYRK1A as an early event in the neuronal complications of HIV infection.
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Éxons , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Tirosina Quinases/biossíntese , Ribonucleoproteínas/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Proteínas tau/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Infecções por HIV/genética , HIV-1/genética , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina , Regulação para Cima , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Proteínas tau/genética , Quinases DyrkRESUMO
Glioblastoma is one of the most aggressive brain tumors. We have previously found up-regulation of growth differentiation factor 15 (GDF15) in glioblastoma cells treated with the anticancer agent fenofibrate. Sequence analysis of GDF15 revealed the presence of a microRNA, miR-3189, in the single intron. We then asked whether miR-3189 was expressed in clinical samples and whether it was functional in glioblastoma cells. We found that expression of miR-3189-3p was down-regulated in astrocytoma and glioblastoma clinical samples compared with control brain tissue. In vitro, the functionality of miR-3189-3p was tested by RNA-binding protein immunoprecipitation, and miR-3189-3p coimmunoprecipitated with Argonaute 2 together with two of its major predicted gene targets, the SF3B2 splicing factor and the guanine nucleotide exchange factor p63RhoGEF. Overexpression of miR-3189-3p resulted in a significant inhibition of cell proliferation and migration through direct targeting of SF3B2 and p63RhoGEF, respectively. Interestingly, miR-3189-3p levels were increased by treatment of glioblastoma cells with fenofibrate, a lipid-lowering drug with multiple anticancer activities. The attenuated expression of miR-3189-3p in clinical samples paralleled the elevated expression of SF3B2, which could contribute to the activation of SF3B2 growth-promoting pathways in these tumors. Finally, miR-3189-3p-mediated inhibition of tumor growth in vivo further supported the function of this microRNA as a tumor suppressor.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , MicroRNAs/genética , Animais , Sequência de Bases , Sítios de Ligação , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Glioblastoma/genética , Glioblastoma/patologia , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Camundongos Nus , Transplante de Neoplasias , Interferência de RNA , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Fatores de Troca de Nucleotídeo Guanina Rho/biossíntese , Fatores de Troca de Nucleotídeo Guanina Rho/genéticaRESUMO
Fenofibrate (FF) is a common lipid-lowering drug and a potent agonist of the peroxisome proliferator-activated receptor alpha (PPARα). FF and several other agonists of PPARα have interesting anticancer properties, and our recent studies demonstrate that FF is very effective against tumor cells of neuroectodermal origin. In spite of these promising anticancer effects, the molecular mechanism(s) of FF-induced tumor cell toxicity remains to be elucidated. Here we report a novel PPARα-independent mechanism explaining FF's cytotoxicity in vitro and in an intracranial mouse model of glioblastoma. The mechanism involves accumulation of FF in the mitochondrial fraction, followed by immediate impairment of mitochondrial respiration at the level of complex I of the electron transport chain. This mitochondrial action sensitizes tested glioblastoma cells to the PPARα-dependent metabolic switch from glycolysis to fatty acid ß-oxidation. As a consequence, prolonged exposure to FF depletes intracellular ATP, activates the AMP-activated protein kinase-mammalian target of rapamycin-autophagy pathway, and results in extensive tumor cell death. Interestingly, autophagy activators attenuate and autophagy inhibitors enhance FF-induced glioblastoma cytotoxicity. Our results explain the molecular basis of FF-induced glioblastoma cytotoxicity and reveal a new supplemental therapeutic approach in which intracranial infusion of FF could selectively trigger metabolic catastrophe in glioblastoma cells.
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Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Fenofibrato/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/farmacologia , Astrócitos/citologia , Neoplasias Encefálicas/metabolismo , Morte Celular , Linhagem Celular Tumoral , Transporte de Elétrons , Feminino , Glioblastoma/metabolismo , Glicólise , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Transplante de Neoplasias , Oxigênio/metabolismo , Consumo de Oxigênio , PPAR alfa/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: The aim of this study was to evaluate the presence of digital lesions in very early diagnosis of SSc (VEDOSS) patients and its possible association with internal organ involvement. METHODS: One hundred and ten VEDOSS patients were investigated for the presence of digital ulcers (DUs), digital pitting scars, calcinosis, necrosis or gangrene, nailfold videocapillaroscopic abnormalities, disease-specific autoantibodies (ACA and anti-topo I) and internal organ involvement. RESULTS: Four patients reported a history of digital pitting scars, while 25 patients presented an active DU or reported a history of DUs. In particular, 16 patients presented with active DUs (14/16 also reporting a history of previous DUs), while the other 9 patients reported a history of DUs only. A statistically significant association between DUs and oesophageal manometry alteration was found in the whole DU population, as well as in the history of DU and the presence of active DU with/without a history of DU subgroups (P < 0.01, P = 0.01 and P < 0.05, respectively). DUs were observed in VEDOSS patients with internal organ involvement but not in those without organ involvement. CONCLUSION: DUs are already present in VEDOSS patients characterized by internal organ involvement, significantly correlating and associating with gastrointestinal involvement. DUs may be a sentinel sign for early organ involvement in VEDOSS patients.
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Dedos , Gastroenteropatias/etiologia , Pneumopatias/etiologia , Escleroderma Sistêmico/complicações , Escleroderma Sistêmico/diagnóstico , Úlcera/diagnóstico , Úlcera/etiologia , Adulto , Calcinose/diagnóstico , Calcinose/patologia , Estudos de Coortes , Diagnóstico Precoce , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Unhas/irrigação sanguínea , Necrose/diagnóstico , Necrose/patologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Fatores de Risco , Úlcera/patologiaRESUMO
MicroRNAs (miRNAs) are small non-coding RNA species that can bind to both untranslated and coding regions of target mRNAs, causing their degradation or post-transcriptional modification. Currently, over 2500 miRNAs have been identified in the human genome. Burgeoning evidence suggests that dysregulation of human miRNAs can play a role in the pathogenesis of a variety of diseases, including cancer. In contrast, only a small subset of human miRNAs has been functionally validated in the pathogenesis of oncogenic viruses, in particular, Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of several human cancers, such as primary effusion lymphoma (PEL) and Kaposi's sarcoma (KS), which are mostly seen in acquired immune deficiency syndrome (AIDS) patients or other immuno-suppressed subpopulation. This review summarizes recent literature outlining mechanisms for KSHV/viral proteins regulation of cellular miRNAs contributing to viral pathogenesis, as well as recent findings about the unique signature of miRNAs induced by KSHV infection or KSHV-related malignancies.
Assuntos
Regulação da Expressão Gênica , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/fisiologia , Interações Hospedeiro-Patógeno , MicroRNAs/metabolismo , Proteínas Virais/metabolismo , Infecções por Herpesviridae/patologia , HumanosRESUMO
OBJECTIVE: Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation and hyperplasia. Tumor necrosis factor-α (TNF-α) plays a pivotal role in RA by interfering with the Fas-Fas ligand (FasL) proapoptotic pathway. We investigated the circulating levels of soluble Fas (sFas) and soluble FasL (sFasL), and their possible correlation with disease activity and improvement after anti-TNF-α treatment in RA. METHODS: Serum levels of sFas and sFasL were measured by quantitative ELISA in 52 patients with RA before and after 3 months of anti-TNF-α treatment (adalimumab, n = 32; infliximab, n = 20). Disease activity measures [Disease Activity Score at 28 joints-erythrocyte sedimentation rate (DAS28-ESR), C-reactive protein (CRP)] were recorded before and after treatment. Forty age-matched and sex-matched healthy subjects served as controls. RESULTS: No significant differences in serum sFas levels were detected between anti-TNF-α-naive patients with RA and controls. After anti-TNF-α treatment, serum sFas levels significantly increased in patients with RA compared to both anti-TNF-α-naive patients and controls. Increased sFas levels inversely correlated with disease activity variables (DAS28-ESR: r = -0.739, CRP: r = -0.636, both p < 0.001). No significant differences in sFasL levels were detected in patients with RA before and after anti-TNF-α treatment. CONCLUSION: In RA, an increase in sFas levels closely correlates with improvement in disease activity induced by TNF-α inhibitors, suggesting their ability to modulate Fas-mediated synoviocyte apoptosis.