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1.
Nucleic Acids Res ; 50(4): e23, 2022 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-34850942

RESUMO

Most RNA footprinting approaches that require ribonuclease cleavage generate RNA fragments bearing a phosphate or cyclic phosphate group at their 3' end. Unfortunately, current library preparation protocols rely only on a 3' hydroxyl group for adaptor ligation or poly-A tailing. Here, we developed circAID-p-seq, a PCR-free library preparation for selective 3' phospho-RNA sequencing. As a proof of concept, we applied circAID-p-seq to ribosome profiling, which is based on sequencing of RNA fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast and highly efficient sequencing of phospho-RNA fragments from eukaryotic cells and tissues. We used circAID-p-seq to portray ribosome occupancy in transcripts, providing a versatile and PCR-free strategy to possibly unravel any endogenous 3'-phospho RNA molecules.


Assuntos
RNA , Ribossomos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fosfatos , RNA/genética , Ribossomos/genética , Análise de Sequência de RNA/métodos
2.
PLoS Comput Biol ; 14(8): e1006169, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30102689

RESUMO

Ribosome profiling is a powerful technique used to study translation at the genome-wide level, generating unique information concerning ribosome positions along RNAs. Optimal localization of ribosomes requires the proper identification of the ribosome P-site in each ribosome protected fragment, a crucial step to determine the trinucleotide periodicity of translating ribosomes, and draw correct conclusions concerning where ribosomes are located. To determine the P-site within ribosome footprints at nucleotide resolution, the precise estimation of its offset with respect to the protected fragment is necessary. Here we present riboWaltz, an R package for calculation of optimal P-site offsets, diagnostic analysis and visual inspection of ribosome profiling data. Compared to existing tools, riboWaltz shows improved accuracies for P-site estimation and neat ribosome positioning in multiple case studies. riboWaltz was implemented in R and is available as an R package at https://github.com/LabTranslationalArchitectomics/RiboWaltz.


Assuntos
Biologia Computacional/métodos , Ribossomos/fisiologia , Análise de Sequência de RNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Software
3.
Catheter Cardiovasc Interv ; 89(1): E13-E25, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27404797

RESUMO

OBJECTIVES: The aim of the present study was to define the feasibility and clinical impact of complying with national consensus recommendations on perioperative management of antiplatelet therapy in patients with coronary stents undergoing cardiac and noncardiac surgery. BACKGROUND: There are limited evidence-based recommendations on the perioperative management of antiplatelet therapy in stented patients undergoing surgery. METHODS: The recommendations provided by the national consensus document were applied in a multicenter, prospective registry of consecutive patients with prior coronary stenting undergoing any type of surgery at 19 hospitals in Italy. The primary end-point was in-hospital net adverse clinical events (NACE) represented by the composite of all-cause death, myocardial infarction, probable/definite stent thrombosis and Bleeding Academic Research Consortium (BARC) grade ≥3 bleeding. Patients were followed for 30 days. RESULTS: A total of 1,082 patients were enrolled. Adherence to consensus recommendations occurred in 85% of the cases. Perioperative aspirin and dual antiplatelet therapy were maintained in 69.7 and 10.5% of the cases, respectively. In-hospital NACE rate was 12.7%, being significantly higher in patients undergoing cardiac surgery (36.3% vs. 7.3%, P < 0.01), mainly due to BARC 3 bleeding events (32.3%). At 30 days, MACE rates were similar (3.5% vs. 3.5%, P = NS) in patients undergoing cardiac and noncardiac surgery, whereas BARC ≥3 bleeding events were significantly higher with cardiac surgery (36.3% vs. 5.6%, P < 0.01). CONCLUSIONS: The results of this registry demonstrate the safety and feasibility of applying a national consensus document on the perioperative management of antiplatelet therapy in stented patients undergoing surgery. © 2016 Wiley Periodicals, Inc.


Assuntos
Procedimentos Cirúrgicos Cardíacos , Intervenção Coronária Percutânea/instrumentação , Inibidores da Agregação Plaquetária/uso terapêutico , Padrões de Prática Médica , Stents , Idoso , Idoso de 80 Anos ou mais , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Procedimentos Cirúrgicos Cardíacos/normas , Consenso , Trombose Coronária/etiologia , Quimioterapia Combinada , Estudos de Viabilidade , Feminino , Fidelidade a Diretrizes , Hemorragia/induzido quimicamente , Mortalidade Hospitalar , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/etiologia , Intervenção Coronária Percutânea/efeitos adversos , Intervenção Coronária Percutânea/mortalidade , Intervenção Coronária Percutânea/normas , Inibidores da Agregação Plaquetária/efeitos adversos , Guias de Prática Clínica como Assunto , Padrões de Prática Médica/normas , Estudos Prospectivos , Sistema de Registros , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento
4.
Biochem J ; 472(1): 83-95, 2015 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-26371376

RESUMO

Genome-wide analyses of translation can provide major contributions in our understanding of the complex interplay between virulent factors and host cells. So far, the activation of host translational control mechanisms by bacterial toxins, owing to specific recruitment of mRNAs, RNA-binding proteins (RBPs) and ncRNAs (non-coding RNAs), are far from being understood. In the present study, we characterize for the first time the changes experienced by the translational control system of host cells in response to the well-known Staphylococcus aureus α-haemolysin (AHL) under both sublytic and lytic conditions. By comparing variations occurring in the cellular transcriptome and translatome, we give evidence that global gene expression is primarily rewired at the translational level, with the contribution of the RBP ELAVL1 (HuR) in the sublytic response. These results reveal the importance of translational control during host-pathogen interaction, opening new approaches for AHL-induced diseases.


Assuntos
Toxinas Bacterianas/farmacologia , Variação Genética/efeitos dos fármacos , Proteínas Hemolisinas/farmacologia , Biossíntese de Proteínas/genética , Transcriptoma/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Perfilação da Expressão Gênica/métodos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Humanos , Immunoblotting , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Arch Insect Biochem Physiol ; 83(1): 1-14, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23404797

RESUMO

Laboratory experiments on the cold stenothermal midge Diamesa cinerella (Diptera, Chironomidae) were performed to study the relationship between increasing temperature and heat shock proteins (HSP70) expression at translational level (Western blotting). Thermotolerance of IV instar larvae collected in nature at 1.5-4.3°C during seasons was analyzed through short-term (1 h at ten different temperatures from 26°C to 35°C) and long-term (1-14 h at 26°C and 1-4 h at 32°C) heat shocks. A high thermotolerance was detected (LT50=30.9-32.8°C and LT100=34.0-37.8°C). However, survival decreased consistently with increasing exposure time, especially at higher temperature (LTime50=7.64 h at 26°C and LTime50=1.73 h at 32°C). The relationship between such heat resistance and HSP70 expression appeared evident because a relationship between HSP70 level and larval survival rate was generally found. A heat shock response (HSR) was consistent only in the summer larvae. The absence of HSR in the other populations coupled with even higher amounts of HSP70 than in summer, led us to hypothesize that other macromolecules and other adaptive mechanisms, apart from biochemical ones, are involved in the response of D. cinerella larvae to high temperature. Altogether these results stressed how in this midge the HSP70 protein family confers resistance against cold, being detected under natural conditions in control larvae collected in all seasons, but also against warm under experimental heat shocks. These results give new insights into possible responses to climate changes in freshwater insects within the context of global warming.


Assuntos
Chironomidae/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Temperatura Alta , Animais , Mudança Climática , Larva/metabolismo , Estresse Fisiológico , Fatores de Tempo
6.
Microbiology (Reading) ; 158(Pt 8): 2089-2098, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22556359

RESUMO

Pseudomonas aeruginosa is a ubiquitous environmental bacterium responsible for a variety of infections in humans, as well as in animal hosts. While the evolution of virulence in P. aeruginosa strains isolated from chronic lung infection in cystic fibrosis (CF) patients has been extensively studied, the virulence phenotype of P. aeruginosa isolated from other infection types or from the environment is currently not well characterized. Here we report an extensive analysis of the virulence of P. aeruginosa strains isolated from acute infections compared with population structure. Virulence profiles of individual strains were also compared with the expression levels of the rhlR gene, the transcriptional regulator of the rhl quorum-sensing system, and the gene encoding Crc, a global regulator controlling catabolite repression and carbon metabolism. Additionally, the presence/absence of the two mutually exclusive genes, exoU and exoS, encoding effectors of the type III secretion system, was assessed. In order to capture the widest range of genetic variability, a collection of 120 clinical strains was initially characterized by repetitive element-based PCR genotyping, and a selection of 27 strains belonging to different clonal lineages was subsequently tested using three different virulence assays, including two Dictyostelium discoideum assays on different growth media, and a Caenorhabditis elegans fast-killing assay. We show that the parallel application of virulence assays can be used to quantitatively assess this complex, multifactorial phenotypic trait. We observed a wide spectrum of virulence phenotypes ranging from weakly to highly aggressive, indicating that clinical strains isolated from acute infections can present a reduced or altered virulence phenotype. Genotypic associations only partially correlated with virulence profiles and virulence gene expression, whereas the presence of either exoU or exoS was not significantly correlated with virulence. Interestingly, the expression of rhlR showed a significant and positive correlation with the virulence profiles obtained with the three assays, while the expression of crc was either negatively or not correlated with virulence, depending on the assay.


Assuntos
Proteínas de Bactérias/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência/genética , Doença Aguda , Animais , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans , Dictyostelium , Expressão Gênica , Humanos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Virulência , Fatores de Virulência/metabolismo
7.
Elife ; 102021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33594971

RESUMO

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.


Assuntos
RNA Longo não Codificante/metabolismo , Ribossomos/metabolismo , Animais , Camundongos , Células-Tronco Embrionárias Murinas , Peptídeos/metabolismo , Biossíntese de Proteínas , Proteômica , RNA Longo não Codificante/genética , Ribossomos/genética
8.
Circulation ; 119(13): 1703-10, 2009 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-19307481

RESUMO

BACKGROUND: The prognostic significance of syncope has not been investigated systematically in hypertrophic cardiomyopathy, and treatment strategies have been based largely on intuition and experience. METHODS AND RESULTS: We assessed the relationship between syncope and sudden death in 1511 consecutive patients with hypertrophic cardiomyopathy. Unexplained (n=153) or neurally mediated (n=52) syncope occurred in 205 patients (14%). Over a 5.6+/-5.2-year follow-up, 74 patients died suddenly. Relative risk of sudden death was 1.78 (95% confidence interval 0.88 to 3.51, P=0.08) in patients with unexplained syncope and 0.91 (95% confidence interval 0.00 to 3.83, P=1.0) in those with neurally mediated syncope compared with patients without syncope. In multivariable analysis, the temporal proximity of unexplained syncope to initial patient evaluation was independently associated with risk of sudden death (P=0.006). Patients with unexplained syncope within 6 months before the initial evaluation showed a 5-fold increase in risk compared with patients without syncope (adjusted hazard ratio 4.89, 95% confidence interval 2.19 to 10.94), a relationship that was maintained throughout all age groups (<18, 18 to 39, and > or =40 years). Older patients (> or =40 years of age) with remote episodes of syncope (>5 years before initial evaluation) did not show an increased risk of sudden death (adjusted hazard ratio 0.38, 95% confidence interval 0.05 to 2.74). CONCLUSIONS: In the present large cohort of patients with hypertrophic cardiomyopathy, unexplained syncope was a risk factor for sudden death. Patients with syncopal events that occurred in close temporal proximity to the initial evaluation showed a substantially higher risk of sudden death than patients without syncope. Older patients with remote syncopal events did not show an increased risk.


Assuntos
Cardiomiopatia Hipertrófica/mortalidade , Morte Súbita Cardíaca/epidemiologia , Síncope/mortalidade , Adolescente , Adulto , Distribuição por Idade , Idoso , Cardiomiopatia Hipertrófica/terapia , Desfibriladores Implantáveis , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Risco , Síncope/terapia , Adulto Jovem
9.
PLoS One ; 15(4): e0227172, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32240200

RESUMO

Cold stenothermal insects living in glacier-fed streams are stressed by temperature variations resulting from glacial retreat during global warming. The molecular aspects of insect response to environmental stresses remain largely unexplored. The aim of this study was to expand our knowledge of how a cold stenothermal organism controls gene expression at the transcriptional, translational, and protein level under warming conditions. Using the chironomid Diamesa tonsa as target species and a combination of RACE, qPCR, polysomal profiling, western blotting, and bioinformatics techniques, we discovered a new molecular pathway leading to previously overlooked adaptive strategies to stress. We obtained and characterized the complete cDNA sequences of three heat shock inducible 70 (hsp70) and two members of heat-shock cognate 70 (hsc70). Strikingly, we showed that a novel pseudo-hsp70 gene encoding a putative long noncoding RNA (lncRNA) which is transcribed during thermal stress, acting as a ribosome sponge to provide post-transcriptional control of HSP70 protein levels. The expression of the pseudo-hsp70 gene and its function suggest the existence of a new and unexpected mechanism to cope with thermal stress: lowering the pace of protein production to save energy and optimize resources for recovery.


Assuntos
Chironomidae/genética , Proteínas de Choque Térmico HSP70/genética , Processamento Pós-Transcricional do RNA/genética , RNA Longo não Codificante/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Resposta ao Choque Térmico/genética , Polirribossomos/genética , Estresse Fisiológico/genética
10.
Nat Cell Biol ; 22(10): 1239-1251, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32958857

RESUMO

The contribution of ribosome heterogeneity and ribosome-associated proteins to the molecular control of proteomes in health and disease remains unclear. Here, we demonstrate that survival motor neuron (SMN) protein-the loss of which causes the neuromuscular disease spinal muscular atrophy (SMA)-binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs that are enriched for translational enhancer sequences in the 5' untranslated region (UTR) and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins that are involved in motor neuron function and stability, including acetylcholinesterase. Thus, SMN plays a crucial role in the regulation of ribosome fluxes along mRNAs encoding proteins that are relevant to SMA pathogenesis.


Assuntos
Neurônios Motores/patologia , Atrofia Muscular Espinal/patologia , Biossíntese de Proteínas , Proteoma/análise , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Camundongos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , RNA Mensageiro/genética , Ribossomos/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Transcriptoma
11.
Clin Cardiol ; 42(11): 1113-1120, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31471975

RESUMO

BACKGROUND: Current guidelines suggest to consider dual antiplatelet therapy (DAPT) continuation for longer than 12 months in selected patients with myocardial infarction (MI). HYPOTHESIS: We sought to assess the criteria used by cardiologists in daily practice to select patients with a history of MI eligible for DAPT continuation beyond 1 year. METHODS: We analyzed data from the EYESHOT Post-MI, a prospective, observational, nationwide study aimed to evaluate the management of patients presenting to cardiologists 1 to 3 years from the last MI event. RESULTS: Out of the 1633 post-MI patients enrolled in the study between March and December 2017, 557 (34.1%) were on DAPT at the time of enrolment, and 450 (27.6%) were prescribed DAPT after cardiologist assessment. At multivariate analyses, a percutaneous coronary intervention (PCI) with multiple stents and the presence of peripheral artery disease (PAD) resulted as independent predictors of DAPT continuation, while atrial fibrillation was the only independent predictor of DAPT interruption for patients both at the second and the third year from MI at enrolment and the time of discharge/end of the visit. CONCLUSIONS: Risk scores recommended by current guidelines for guiding decisions on DAPT duration are underused and misused in clinical practice. A PCI with multiple stents and a history of PAD resulted as the clinical variables more frequently associated with DAPT continuation beyond 1 year from the index MI.


Assuntos
Aspirina/administração & dosagem , Cardiologistas , Clopidogrel/administração & dosagem , Terapia Antiplaquetária Dupla/métodos , Infarto do Miocárdio/tratamento farmacológico , Seleção de Pacientes , Sistema de Registros , Idoso , Esquema de Medicação , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/administração & dosagem , Estudos Prospectivos , Fatores de Tempo , Resultado do Tratamento
12.
J Insect Physiol ; 54(9): 1337-42, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18680747

RESUMO

The effects of temperature and oxygen saturation on the respiration rate of two cold stenothermal chironomids, Diamesa insignipes and Pseudodiamesa branickii were investigated. Fourth instar larvae were collected in winter in a glacio-rhithral stream (1300 m a.s.l., Alps, NE-Italy) and their respiration rate was measured with a Clark's electrode in the range 0-14 degrees C. The respiration rate was significantly higher in D. insignipes than in P. branickii at low temperatures (

Assuntos
Chironomidae/metabolismo , Temperatura Baixa , Oxigênio/metabolismo , Animais , Respiração Celular , Larva/metabolismo , Rios
13.
Cell Rep ; 25(4): 1097-1108.e5, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30355487

RESUMO

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ribossomos/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Microesferas , Puromicina/análogos & derivados , Puromicina/síntese química , Puromicina/química , RNA Mensageiro/metabolismo
14.
Insect Sci ; 24(2): 314-324, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26463003

RESUMO

The threats posed by climate change make it important to expand knowledge concerning cold and heat tolerance in stenothermal species from habitats potentially threatened by temperature changes. Thermal limits and basal metabolism variations were investigated in Pseudodiamesa branickii (Diptera: Chironomidae) under thermal stress between -20 and 37 °C. Supercooling point (SCP), lower (LLTs) and upper lethal temperatures (ULTs), and oxygen consumption rate were measured in overwintering young (1st and 2nd instar) and mature (3rd and 4th instar) larvae from an Alpine glacier-fed stream. Both young and mature larvae were freezing tolerant (SCPs = -7.1 °C and -6.4 °C, respectively; LLT100 -20 °C) and thermotolerant (ULT50 = 31.7 ± 0.4, 32.5 ± 0.3, respectively). However, ontogenetic differences in acute tolerance were observed. The LLT50 calculated for the young larvae (= -7.4 °C) was almost equal to their SCP (= -7.1 °C) and the overlapping of the proportion of mortality curve with the CPIF curve highlighted that the young larvae are borderline between freezing tolerance and freezing avoidance. Furthermore, a lower ULT100 in the young larvae (of ca. 1 °C), suggests that they are less thermotolerant than mature larvae. Finally, young larvae exhibit a higher oxygen consumption rate (mgO2 /gAFDM/h) at any temperature tested and are overall less resistant to oxygen depletion compared to mature larvae at ≥10 °C. These findings suggest that mature larvae enter into a dormant state by lowering their basal metabolism until environmental conditions improve in order to save energy for life cycle completion during stressful conditions.


Assuntos
Chironomidae/crescimento & desenvolvimento , Adaptação Fisiológica , Animais , Metabolismo Basal , Chironomidae/anatomia & histologia , Chironomidae/metabolismo , Congelamento , Itália , Larva/anatomia & histologia , Larva/metabolismo , Consumo de Oxigênio , Temperatura
15.
Chemosphere ; 173: 235-244, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28110013

RESUMO

Copper is one of the predominant components of pesticides employed in agriculture and known to be highly toxic once it reaches aquatic organisms. The impact of sublethal concentrations of this metal on wild insects is not yet completely understood. Studies addressing alterations in different levels of gene expression are still lacking. We previously demonstrated that in a wild population of Chironomus riparius, HSP and CYP families of genes were up-regulated at the transcriptional level after copper exposure. Here, we analyse the impact of copper at the genomic, translational and protein functional level, obtaining a comprehensive picture of the molecular reply to this metal. We studied genotoxicity in C. riparius larvae by Comet Assay, the translational response by polysomal profiling and the detoxification capacity by the CYP450 enzymes activity. Fourth-instar larvae from a mountain stream polluted by agricultural land run-off (NE-Italy) were exposed for 3 h copper concentrations ≤ LC50. We report DNA damage induced by copper, even at sublethal levels, as demonstrated by significant increases in all the comet parameters at concentrations ≥1 mg L-1. By estimating the transcript-specific translational efficiency, we observe a specific up-regulation of CYP4G. Furthermore, the enzymatic activity of CYP450 enzymes is increased at all sublethal copper concentrations, confirming the role of this protein family in the detoxification processes. Surprisingly, the HSP transcripts are up-regulated at the transcriptional level, but these changes are buffered at the translational level suggesting the existence of still unknown post-transcriptional controls that may be connected to survival processes.


Assuntos
Chironomidae/efeitos dos fármacos , Cobre/toxicidade , Sistema Enzimático do Citocromo P-450/genética , Dano ao DNA/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Chironomidae/genética , Chironomidae/metabolismo , Ensaio Cometa , Sistema Enzimático do Citocromo P-450/metabolismo , Inativação Metabólica/efeitos dos fármacos , Itália , Larva/metabolismo , Regulação para Cima
16.
Cell Rep ; 21(4): 953-965, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29069603

RESUMO

Genetic alterations impacting ubiquitously expressed proteins involved in RNA metabolism often result in neurodegenerative conditions, with increasing evidence suggesting that translation defects can contribute to disease. Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, whose role in pathogenesis remains unclear. Here, we identified in vivo and in vitro translation defects that are cell autonomous and SMN dependent. By determining in parallel the in vivo transcriptome and translatome in SMA mice, we observed a robust decrease in translation efficiency arising during early stages of disease. We provide a catalogue of RNAs with altered translation efficiency, identifying ribosome biology and translation as central processes affected by SMN depletion. This was further supported by a decrease in the number of ribosomes in SMA motor neurons in vivo. Overall, our findings suggest ribosome biology as an important, yet largely overlooked, factor in motor neuron degeneration.


Assuntos
Atrofia Muscular Espinal/metabolismo , Polirribossomos/metabolismo , Transcriptoma , Animais , Células Cultivadas , Camundongos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Biossíntese de Proteínas , Proteoma/genética , Proteoma/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
17.
J Am Coll Cardiol ; 45(7): 1076-80, 2005 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-15808767

RESUMO

OBJECTIVES: The aim of this study was to investigate whether the prognostic importance of left ventricular (LV) outflow obstruction in hypertrophic cardiomyopathy (HCM) is influenced by other predictors of morbidity and mortality. BACKGROUND: It remains unknown whether the effect of outflow obstruction on clinical outcome in HCM is influenced by other determinants of survival. METHODS: We assessed the impact of severity of symptoms, LV wall thickness, atrial fibrillation, and age on the prognostic importance of outflow obstruction in a large HCM population. RESULTS: Of 526 consecutive HCM patients, 141 (27%) had outflow obstruction. During a follow-up of 4.5 +/- 4.1 years, 34 patients died of HCM. The incidence of cardiovascular death was significantly higher among patients with LV obstruction than among those without obstruction (relative risk [RR] = 2.14; p = 0.02). The prognostic power of the outflow gradient changed in relation to severity of symptoms (p = 0.024). At initial evaluation, LV obstruction was a significant predictor of cardiovascular mortality only in New York Heart Association (NYHA) functional class I to II patients (hazard ratio [HR] = 2.38; p = 0.025). During follow-up, at time of development of severe symptoms, the outflow gradient lost its prognostic significance (HR = 1.18; p = 0.66), whereas NYHA functional class III to IV was associated with an eight-fold increase in risk of cardiovascular mortality (HR = 7.90; p < 0.001). CONCLUSIONS: In patients with HCM, the prognostic importance of LV outflow obstruction varies in relation to the severity of symptoms. In patients with mild or no symptoms, obstruction is an important predictor of cardiovascular death. After development of severe symptoms, NYHA functional class becomes the dominant marker of prognosis independently of the presence of an outflow gradient.


Assuntos
Cardiomiopatia Hipertrófica/mortalidade , Índice de Gravidade de Doença , Obstrução do Fluxo Ventricular Externo/mortalidade , Cardiomiopatia Hipertrófica/complicações , Cardiomiopatia Hipertrófica/diagnóstico por imagem , Cardiomiopatia Hipertrófica/patologia , Feminino , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Análise de Sobrevida , Ultrassonografia , Obstrução do Fluxo Ventricular Externo/complicações , Obstrução do Fluxo Ventricular Externo/diagnóstico por imagem , Obstrução do Fluxo Ventricular Externo/patologia
18.
Sci Total Environ ; 557-558: 183-91, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-26994805

RESUMO

Copper (Cu) and azadirachtin (AZA-A+B) are pesticides allowed in organic agriculture whose environmental risk and toxicity for aquatic wildlife is only partially known. Reverse Transcription Polymerase Chain Reaction was used to assess the molecular effect of acute and short-term exposure (3, 24h) of Cu (0.01, 0.05, 1, 10, 25mgl(-1)) and AZA-A+B (0.2, 0.3, 0.4, 0.5, 1mgl(-1)) on the expression of five candidate genes (hsp70, hsc70, hsp40, hsp10 and cyP450) in a non-target species, Chironomus riparius. Fourth-instar larvae were collected from a mountain stream polluted by agricultural land run-off. All genes were responsive to both pesticides but each gene had a specific response to the different experimental concentrations and exposure times. A few similarities in transcriptional profiling were observed, such as a linear concentration-dependent response of hsp70 after 24h of exposure (at ≥1mgl(-1) of Cu and ≥0.2mgl(-1) of AZA-A+B) and an up-regulation regardless of the concentration of hsc70 after 24h of exposure (at ≥0mgl(-1) of Cu and ≥0.2mgl(-1) of AZA-A+B and the up-regulation of hsp70 after 3h of exposure at ~LC50 (Cu-LC50=26.1±2.5mgl(-1), AZA-A+B-LC50=1.1±0.2mgl(-1)). According to the results, hsp40, hsp10 and cyP450 may be defined as pesticide-dependent (i.e., hsp40 and hsp10 seemed to responded mainly to AZA-A+B and cyP450 to Cu), while hsc70 as time-dependent regardless of the pesticide (i.e., hsc70 responded only after 24h of treatment with Cu and AZA-A+B). This study gives new insights on the potential role of the C. riparius's hsps and cyP450 genes as sensitive biomarkers for freshwater monitoring.


Assuntos
Chironomidae/fisiologia , Praguicidas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Monitoramento Ambiental , Proteínas de Choque Térmico HSP70/metabolismo , Larva , Agricultura Orgânica , Praguicidas/análise , Poluentes Químicos da Água/análise
19.
J Vis Exp ; (109)2016 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-27023752

RESUMO

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.


Assuntos
Encéfalo/ultraestrutura , Microscopia de Força Atômica/métodos , Polirribossomos/ultraestrutura , Animais , Encéfalo/metabolismo , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Camundongos , Polirribossomos/metabolismo
20.
J Am Coll Cardiol ; 40(10): 1864-9, 2002 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-12446072

RESUMO

OBJECTIVES: We sought to assess mortality and morbidity in pregnant women with hypertrophic cardiomyopathy (HCM). BACKGROUND: The risk associated with pregnancy in women with HCM is an important and increasingly frequent clinical issue for which systematic data are not available and a large measure of uncertainty persists. METHODS: Maternal mortality in 91 consecutively evaluated families with HCM was compared with that reported in the general population. The study cohort included 100 women with HCM with one or more live births, for a total of 199 live births. Morbidity related to HCM during pregnancy was investigated in 40 women evaluated within five years of their pregnancy. RESULTS: Two pregnancy-related deaths occurred, both in patients at a particularly high risk. The maternal mortality rate was 10 per 1,000 live births (95% confidence interval [CI] 1.1 to 36.2/1,000) and was in excess of the expected mortality in the general Italian population (relative risk 17.1, 95% CI 2.0 to 61.8). In the 40 patients evaluated within close proximity of their pregnancy, 1 (4%) of the 28 who were previously asymptomatic and 5 (42%) of the 12 with symptoms progressed to functional class III or IV during pregnancy (p < 0.01). One patient had atrial fibrillation and one had syncope, both of whom had already experienced similar and recurrent events before their pregnancy. CONCLUSIONS: Maternal mortality is increased in patients with HCM compared with the general population. However, absolute maternal mortality is low and appears to be principally confined to women at a particularly high risk. In the presence of a favorable clinical profile, the progression of symptoms, atrial fibrillation, and syncope are also uncommon during pregnancy.


Assuntos
Cardiomiopatia Hipertrófica/complicações , Cardiomiopatia Hipertrófica/epidemiologia , Complicações Cardiovasculares na Gravidez/epidemiologia , Complicações Cardiovasculares na Gravidez/etiologia , Adolescente , Adulto , Cardiomiopatia Hipertrófica/diagnóstico , Ecocardiografia Doppler , Saúde da Família , Feminino , Humanos , Hipertrofia Ventricular Esquerda/complicações , Hipertrofia Ventricular Esquerda/diagnóstico , Hipertrofia Ventricular Esquerda/epidemiologia , Itália , Mortalidade Materna , Bem-Estar Materno , Morbidade , Gravidez , Complicações Cardiovasculares na Gravidez/diagnóstico , Resultado da Gravidez , Fatores de Risco , Índice de Gravidade de Doença , Volume Sistólico/fisiologia , Função Ventricular Esquerda/fisiologia , Obstrução do Fluxo Ventricular Externo/complicações , Obstrução do Fluxo Ventricular Externo/diagnóstico , Obstrução do Fluxo Ventricular Externo/epidemiologia
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