Assuntos
Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Feminino , Humanos , Masculino , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Reação em Cadeia da Polimerase/métodosAssuntos
Antineoplásicos/administração & dosagem , Mesilato de Imatinib/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Carga Tumoral/efeitos dos fármacos , Suspensão de Tratamento/estatística & dados numéricos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Adulto JovemRESUMO
Imatinib is effective for the treatment of chronic myeloid leukemia (CML). However even undetectable BCR-ABL1 by Q-RT-PCR does not equate to eradication of the disease. Digital-PCR (dPCR), able to detect 1 BCR-ABL1 positive cell out of 10(7) , has been recently developed. The ISAV study is a multicentre trial aimed at validating dPCR to predict relapses after imatinib discontinuation in CML patients with undetectable Q-RT-PCR. CML patients under imatinib therapy since more than 2 years and with undetectable PCR for at least 18 months were eligible. Patients were monitored by standard Q-RT-PCR for 36 months. Patients losing molecular remission (two consecutive positive Q-RT-PCR with at least 1 BCR-ABL1/ABL1 value above 0.1%) resumed imatinib. The study enrolled 112 patients, with a median follow-up of 21.6 months. Fifty-two of the 108 evaluable patients (48.1%), relapsed; 73.1% relapsed in the first 9 months but 14 late relapses were observed between 10 and 22 months. Among the 56 not-relapsed patients, 40 (37.0% of total) regained Q-RT-PCR positivity but never lost MMR. dPCR results showed a significant negative predictive value ratio of 1.115 [95% CI: 1.013-1.227]. An inverse relationship between patients age and risk of relapse was evident: 95% of patients <45 years relapsed versus 42% in the class ≥45 to <65 years and 33% of patients ≥65 years [P(χ(2) ) < 0.0001]. Relapse rates ranged between 100% (<45 years, dPCR+) and 36% (>45 years, dPCR-). Imatinib can be safely discontinued in the setting of continued PCR negativity; age and dPCR results can predict relapse.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Mesilato de Imatinib/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto , Fatores Etários , Idoso , Feminino , Seguimentos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/epidemiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , RecidivaRESUMO
Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity.
Assuntos
Nucléolo Celular/metabolismo , Inibidores de Proteassoma , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Proteína SUMO-1/metabolismo , Animais , Nucléolo Celular/química , Inibidores de Cisteína Proteinase/metabolismo , Células HeLa , Humanos , Marcação por Isótopo , Leupeptinas/metabolismo , Dados de Sequência Molecular , Proteína SUMO-1/genéticaRESUMO
BACKGROUND: The COVID-19 pandemic is challenging advanced health systems, which are dealing with an overwhelming number of patients in need of intensive care for respiratory failure, often requiring intubation. Prone positioning in intubated patients is known to reduce mortality in moderate-to-severe acute respiratory distress syndrome. We aimed to investigate feasibility and effect on gas exchange of prone positioning in awake, non-intubated patients with COVID-19-related pneumonia. METHODS: In this prospective, feasibility, cohort study, patients aged 18-75 years with a confirmed diagnosis of COVID-19-related pneumonia receiving supplemental oxygen or non-invasive continuous positive airway pressure were recruited from San Gerardo Hospital, Monza, Italy. We collected baseline data on demographics, anthropometrics, arterial blood gas, and ventilation parameters. After baseline data collection, patients were helped into the prone position, which was maintained for a minimum duration of 3 h. Clinical data were re-collected 10 min after prone positioning and 1 h after returning to the supine position. The main study outcome was the variation in oxygenation (partial pressure of oxygen [PaO2]/fractional concentration of oxygen in inspired air [FiO2]) between baseline and resupination, as an index of pulmonary recruitment. This study is registered on ClinicalTrials.gov, NCT04365959, and is now complete. FINDINGS: Between March 20 and April 9, 2020, we enrolled 56 patients, of whom 44 (79%) were male; the mean age was 57·4 years (SD 7·4) and the mean BMI was 27·5 kg/m2 (3·7). Prone positioning was feasible (ie, maintained for at least 3 h) in 47 patients (83·9% [95% CI 71·7 to 92·4]). Oxygenation substantially improved from supine to prone positioning (PaO2/FiO2 ratio 180·5 mm Hg [SD 76·6] in supine position vs 285·5 mm Hg [112·9] in prone position; p<0·0001). After resupination, improved oxygenation was maintained in 23 patients (50·0% [95% CI 34·9-65·1]; ie, responders); however, this improvement was on average not significant compared with before prone positioning (PaO2/FiO2 ratio 192·9 mm Hg [100·9] 1 h after resupination; p=0·29). Patients who maintained increased oxygenation had increased levels of inflammatory markers (C-reactive protein: 12·7 mg/L [SD 6·9] in responders vs 8·4 mg/L [6·2] in non-responders; and platelets: 241·1â×â103/µL [101·9] vs 319·8â×â103/µL [120·6]) and shorter time between admission to hospital and prone positioning (2·7 days [SD 2·1] in responders vs 4·6 days [3·7] in non-responders) than did those for whom improved oxygenation was not maintained. 13 (28%) of 46 patients were eventually intubated, seven (30%) of 23 responders and six (26%) of 23 non-responders (p=0·74). Five patients died during follow-up due to underlying disease, unrelated to study procedure. INTERPRETATION: Prone positioning was feasible and effective in rapidly ameliorating blood oxygenation in awake patients with COVID-19-related pneumonia requiring oxygen supplementation. The effect was maintained after resupination in half of the patients. Further studies are warranted to ascertain the potential benefit of this technique in improving final respiratory and global outcomes. FUNDING: University of Milan-Bicocca.
Assuntos
Betacoronavirus , Infecções por Coronavirus/fisiopatologia , Pneumonia Viral/fisiopatologia , Decúbito Ventral , Troca Gasosa Pulmonar/fisiologia , Síndrome do Desconforto Respiratório/fisiopatologia , Adolescente , Adulto , Idoso , COVID-19 , Infecções por Coronavirus/terapia , Cuidados Críticos/métodos , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/terapia , Estudos Prospectivos , Síndrome do Desconforto Respiratório/terapia , Síndrome do Desconforto Respiratório/virologia , Mecânica Respiratória/fisiologia , SARS-CoV-2 , Adulto JovemRESUMO
Pharmacological investigations have suggested the involvement of the cAMP transduction pathway in the action of antidepressant drugs and in the pathophysiology of mood disorders. We have extended these studies to determine the time-related effects of two selective serotonin reuptake inhibitors, fluvoxamine (15 mg/kg) and paroxetine (5 mg/kg), on the cAMP-binding in rat cerebral cortex, after short and long-term treatments. Photoaffinity labelling experiments with 8-N(3)-[(32)P]cAMP were carried out in cerebrocortical soluble (S1 or S2) and microtubule fractions. In our conditions, both SSRIs administered for 5 days were unable to affect the cAMP-binding in S1, S2, and in microtubule fractions. After 12 days of treatment, paroxetine and fluvoxamine significantly enhanced the cAMP-binding to the 54 kDa protein, corresponding to the type II regulatory subunit of PKA (RII), in the S1 and microtubule fractions. Any modification in respect to controls was observed in S2, the soluble fraction devoid of microtubules. After 21 days of treatment no changes were observed in the soluble S1 fraction and in microtubules, but the cAMP-binding to the RII subunit was found to be significantly higher in the S2 fraction. The high concentration of RII, demonstrated first in microtubules (12 days) and then in the cytosol (21 days), could be the result of a time-related effect of SSRIs on PKA and its translocation from microtubule compartment to the cytosol. The present findings seem to demonstrate the capacity of SSRIs to modulate the subcellular distribution of PKA and support the involvement of the cAMP pathway in the mechanism of action of these drugs.
RESUMO
BACKGROUND: Abnormal levels of protein kinase A (PKA) were found in patients with bipolar disorder (BD). Since altered levels are generally accompanied by functional modifications, the purpose of this study was to investigate PKA activity in patients with BD. METHODS: PKA activity was assessed in platelets from 20 drug-free bipolar patients and 19 controls. RESULTS: The cAMP-stimulated PKA activity was significantly increased in bipolar patients compared with controls. LIMITATIONS: This study made use of platelets, which may not fully represent changes occurring in specific brain regions. CONCLUSION: This study adds to the growing evidence suggesting that abnormalities of PKA are associated with BD.
Assuntos
Transtorno Bipolar/fisiopatologia , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Adulto , Transtorno Bipolar/complicações , Plaquetas/enzimologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de SinaisRESUMO
MYBBP1A is a predominantly nucleolar transcriptional regulator involved in rDNA synthesis and p53 activation via acetylation. However little further information is available as to its function. Here we report that MYBBP1A is developmentally essential in the mouse prior to blastocyst formation. In cell culture, down-regulation of MYBBP1A decreases the growth rate of wild type mouse embryonic stem cells, mouse embryo fibroblasts (MEFs) and of human HeLa cells, where it also promotes apoptosis. HeLa cells either arrest at G2/M or undergo delayed and anomalous mitosis. At mitosis, MYBBP1A is localized to a parachromosomal region and gene-expression profiling shows that its down-regulation affects genes controlling chromosomal segregation and cell cycle. However, MYBBP1A down-regulation increases the growth rate of the immortalized NIH3T3 cells. Such Mybbp1a down-regulated NIH3T3 cells are more susceptible to Ras-induced transformation and cause more potent Ras-driven tumors. We conclude that MYBBP1A is an essential gene with novel roles at the pre-mitotic level and potential tumor suppressor activity.
Assuntos
Proteínas de Transporte/genética , Ciclo Celular/genética , Embrião de Mamíferos/metabolismo , Mitose/genética , Proteínas Nucleares/genética , Animais , Apoptose/genética , Proteínas de Transporte/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/genética , Células Cultivadas , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Células-Tronco Embrionárias/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Gravidez , Interferência de RNA , Proteínas de Ligação a RNA , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
We have examined glucose homeostasis in mice hypomorphic for the homeotic transcription factor gene Prep1. Prep1-hypomorphic (Prep1(i/i)) mice exhibit an absolute reduction in circulating insulin levels but normal glucose tolerance. In addition, these mice exhibit protection from streptozotocin-induced diabetes and enhanced insulin sensitivity with improved glucose uptake and insulin-dependent glucose disposal by skeletal muscle. This muscle phenotype does not depend on reduced expression of the known Prep1 transcription partner, Pbx1. Instead, in Prep1(i/i) muscle, we find normal Pbx1 but reduced levels of the recently identified novel Prep1 interactor p160. Consistent with this reduction, we find a muscle-selective increase in mRNA and protein levels of PGC-1alpha, accompanied by enhanced expression of the GLUT4 transporter, responsible for insulin-stimulated glucose uptake in muscle. Indeed, using L6 skeletal muscle cells, we induced the opposite effects by overexpressing Prep1 or p160, but not Pbx1. In vivo skeletal muscle delivery of p160 cDNA in Prep1(i/i) mice also reverses the molecular phenotype. Finally, we show that Prep1 controls the stability of the p160 protein. We conclude that Prep1 controls insulin sensitivity through the p160-GLUT4 pathway.
Assuntos
Glicemia/metabolismo , Proteínas de Transporte/metabolismo , Diabetes Mellitus Experimental , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Ligação a DNA , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/prevenção & controle , Feminino , Glucagon/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Proteínas de Homeodomínio/genética , Homeostase , Humanos , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Proteínas Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fenótipo , Fator de Transcrição 1 de Leucemia de Células Pré-B , Proteínas de Ligação a RNA , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The analysis of short tandem repeats (STRs) plays an important role in forensic science, human identification, genetic mapping, and disease diagnostics. Traditional STR analysis utilizes gel- or column-based approaches to analyze DNA repeats. Individual STR alleles are separated and distinguished according to fragment length; thus the assay is generally hampered by its low multiplex capacity. However, use of DNA microarray would employ a simple hybridization and detection for field forensics and biology. Here we demonstrate a rapid, highly sensitive method for STR analysis that utilizes DNA microarray technology. We describe two adaptations to accomplish this: the use of competitive hybridization to remove unpaired ssDNA from an array and the use of neural network classification to automate the analysis. The competitive displacement technique mimics the branch migration process that occurs during DNA recombination. Our technique will facilitate the rapid deduction of identity, length, and number of repeats for the multiple STRs in an unknown DNA sample.
Assuntos
DNA/química , Genética Forense/métodos , Repetições de Microssatélites/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Redes Neurais de Computação , Hibridização de Ácido Nucleico/métodos , OligonucleotídeosRESUMO
Prep1 is known to interact in vivo with Pbx1 to regulate development and organogenesis. We have identified a novel Prep1-interacting protein, p160 c-Myb binding protein (p160). p160 and Pbx1 compete for Prep1 in vitro, and p160 inhibits Prep1-dependent HoxB2 expression in retinoic acid-treated NT2-D1 cells. The N-terminal physiologically truncated form of p160, p67, binds the sequence 63LFPLL67 in the HR1 domain of Prep1. Mutation of both L63 and L66 impairs the binding of Prep1 to both p160/p67 and Pbx1. The sequences required to bind Prep1 are mainly located in residues 51 to 151. Immunofluorescence colocalization and coimmunoprecipitation of endogenous p160 and Prep1 are induced by ActD, which translocates p160 from the nucleolus to the nucleoplasm. These data therefore show that p160 is a novel regulator of Prep1-Pbx1 transcriptional activity.