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mTOR inhibitors (mTOR-Is) may induce proteinuria in kidney transplant recipients through podocyte damage. However, the mechanism has only been partially defined. Total cell lysates and supernatants of immortalized human podocytes treated with different doses of everolimus (EVE) (10, 100, 200, and 500 nM) for 24 h were subjected to mass spectrometry-based proteomics. Support vector machine and partial least squares discriminant analysis were used for data analysis. The results were validated in urine samples from 28 kidney transplant recipients receiving EVE as part of their immunosuppressive therapy. We identified more than 7000 differentially expressed proteins involved in several pathways, including kinases, cell cycle regulation, epithelial-mesenchymal transition, and protein synthesis, according to gene ontology. Among these, after statistical analysis, 65 showed an expression level significantly and directly correlated with EVE dosage. Polo-Like Kinase 1 (PLK1) content was increased, whereas osteopontin (SPP1) content was reduced in podocytes and supernatants in a dose-dependent manner and significantly correlated with EVE dose (p < 0.0001, FDR < 5%). Similar results were obtained in the urine of kidney transplant patients. This study analyzed the impact of different doses of mTOR-Is on podocytes, helping to understand not only the biological basis of their therapeutic effects but also the possible mechanisms underlying proteinuria.
Assuntos
Everolimo , Imunossupressores , Podócitos , Proteômica , Humanos , Podócitos/metabolismo , Podócitos/efeitos dos fármacos , Everolimo/farmacologia , Proteômica/métodos , Imunossupressores/farmacologia , Transplante de Rim , Quinase 1 Polo-Like , Proteoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Proto-Oncogênicas/metabolismo , Feminino , Proteinúria , Masculino , OsteopontinaRESUMO
The nervous system displays high energy consumption, apparently not fulfilled by mitochondria, which are underrepresented therein. The oxidative phosphorylation (OxPhos) activity, a mitochondrial process that aerobically provides ATP, has also been reported also in the myelin sheath and the rod outer segment (OS) disks. Thus, commonalities and differences between the extra-mitochondrial and mitochondrial aerobic metabolism were evaluated in bovine isolated myelin (IM), rod OS, and mitochondria-enriched fractions (MIT). The subcellular fraction quality and the absence of contamination fractions have been estimated by western blot analysis. Oxygen consumption and ATP synthesis were stimulated by conventional (pyruvate + malate or succinate) and unconventional (NADH) substrates, observing that oxygen consumption and ATP synthesis by IM and rod OS are more efficient than by MIT, in the presence of both kinds of respiratory substrates. Mitochondria did not utilize NADH as a respiring substrate. When ATP synthesis by either sample was assayed in the presence of 10-100 µM ATP in the assay medium, only in IM and OS it was not inhibited, suggesting that the ATP exportation by the mitochondria is limited by extravesicular ATP concentration. Interestingly, IM and OS but not mitochondria appear able to synthesize ATP at a later time with respect to exposure to respiratory substrates, supporting the hypothesis that the proton gradient produced by the electron transport chain is buffered by membrane phospholipids. The putative transfer mode of the OxPhos molecular machinery from mitochondria to the extra-mitochondrial structures is also discussed, opening new perspectives in the field of neurophysiology.
Assuntos
Trifosfato de Adenosina/biossíntese , Membrana Celular/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Prosencéfalo/metabolismo , Retina/metabolismo , Trifosfato de Adenosina/administração & dosagem , Animais , Bovinos , Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Prosencéfalo/efeitos dos fármacos , Retina/efeitos dos fármacosRESUMO
Hirschsprung (HSCR) Associated Enterocolitis (HAEC) is a common life-threatening complication in HSCR. HAEC is suggested to be due to a loss of gut homeostasis caused by impairment of immune system, barrier defense, and microbiome, likely related to genetic causes. No gene has been claimed to contribute to HAEC occurrence, yet. Genetic investigation of HAEC by Whole-Exome Sequencing (WES) on 24 HSCR patients affected (HAEC) or not affected (HSCR-only) by enterocolitis and replication of results on a larger panel of patients allowed the identification of the HAEC susceptibility variant p.H187Q in the Oncostatin-M receptor (OSMR) gene (14.6% in HAEC and 5.1% in HSCR-only, p = 0.0024). Proteomic analysis on the lymphoblastoid cell lines from one HAEC patient homozygote for this variant and one HAEC patient not carrying the variant revealed two well distinct clusters of proteins significantly up or downregulated upon OSM stimulation. A marked enrichment in immune response pathways (q < 0.0001) was shown in the HAEC H187 cell line, while proteins upregulated in the HAEC Q187 lymphoblasts sustained pathways likely involved in pathogen infection and inflammation. In conclusion, OSMR p.H187Q is an HAEC susceptibility variant and perturbates the downstream signaling cascade necessary for the gut immune response and homeostasis maintenance.
Assuntos
Suscetibilidade a Doenças , Enterocolite/etiologia , Enterocolite/metabolismo , Doença de Hirschsprung/complicações , Doença de Hirschsprung/genética , Subunidade beta de Receptor de Oncostatina M/genética , Transdução de Sinais , Alelos , Enterocolite/patologia , Expressão Gênica , Frequência do Gene , Variação Genética , Genótipo , Doença de Hirschsprung/diagnóstico , Humanos , Modelos Moleculares , Subunidade beta de Receptor de Oncostatina M/química , Subunidade beta de Receptor de Oncostatina M/metabolismo , Conformação Proteica , Proteômica/métodos , Relação Estrutura-Atividade , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
Liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is currently the method of choice for untargeted metabolomic analysis. The availability of established protocols to achieve a high confidence identification of metabolites is crucial. The aim of this work is to describe the workflow that we have applied to build an Accurate Mass Retention Time (AMRT) database using a commercial metabolite library of standards. LC-HRMS analysis was carried out using a Vanquish Horizon UHPLC system coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Milan, Italy). The fragmentation spectra, obtained with 12 collision energies, were acquired for each metabolite, in both polarities, through flow injection analysis. Several chromatographic conditions were tested to obtain a protocol that yielded stable retention times. The adopted chromatographic protocol included a gradient separation using a reversed phase (Waters Acquity BEH C18) and a HILIC (Waters Acquity BEH Amide) column. An AMRT database of 518 compounds was obtained and tested on real plasma and urine samples analyzed in data-dependent acquisition mode. Our AMRT library allowed a level 1 identification, according to the Metabolomics Standards Initiative, of 132 and 124 metabolites in human pediatric plasma and urine samples, respectively. This library represents a starting point for future metabolomic studies in pediatric settings.
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Metabolômica/métodos , Plasma/química , Urina/química , Adolescente , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Humanos , Masculino , Espectrometria de Massas/métodos , Urinálise/métodosRESUMO
BACKGROUND INFORMATION: Energy demand in human platelets is very high, to carry out their functions. As for most human cells, the aerobic metabolism represents the primary energy source in platelets, even though mitochondria are negligibly represented. Following the hypothesis that other structures could be involved in chemical energy production, in this work, we have investigated the functional expression of an extramitochondrial aerobic metabolism in platelets. RESULTS: Oximetric and luminometric analyses showed that platelets consume large amounts of oxygen and produce ATP in the presence of common respiring substrates, such as pyruvate + malate or succinate, although morphological electron microscopy analysis showed that these contain few mitochondria. However, evaluation of the anaerobic glycolytic metabolism showed that only 13% of consumed glucose was converted to lactate. Interestingly, the highest OXPHOS activity was observed in the presence of NADH, not a readily permeant respiring substrate for mitochondria. Also, oxygen consumption and ATP synthesis fuelled by NADH were not affected by atractyloside, an inhibitor of the adenine nucleotide translocase, suggesting that these processes may not be ascribed to mitochondria. Functional data were confirmed by immunofluorescence microscopy and Western blot analyses, showing a consistent expression of the ß subunit of F1 Fo -ATP synthase and COXII, a subunit of Complex IV, but a low signal of translocase of the inner mitochondrial membrane (a protein not involved in OXPHOS metabolism). Interestingly, the NADH-stimulated oxygen consumption and ATP synthesis increased in the presence of the physiological platelets agonists, thrombin or collagen. CONCLUSIONS: Data suggest that in platelets, aerobic energy production is mainly driven by an extramitochondrial OXPHOS machinery, originated inside the megakaryocyte, and that this metabolism plays a pivotal role in platelet activation. SIGNIFICANCE: This work represents a further example of the existence of an extramitochondrial aerobic metabolism, which can contribute to the cellular energy balance.
Assuntos
Plaquetas/fisiologia , Metabolismo Energético , Consumo de Oxigênio , Trifosfato de Adenosina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glucose/metabolismo , Glicólise , Voluntários Saudáveis , Humanos , Ácido Láctico/metabolismo , Mitocôndrias/metabolismo , OxirreduçãoRESUMO
The retinal rod outer segment (OS) is a stack of disks surrounded by the plasma membrane, housing proteins related to phototransduction, as well as mitochondrial proteins involved in oxidative phosphorylation (OxPhos). This prompted us to compare the proteome of bovine OS disks and mitochondria to assess the significant top gene signatures of each sample. The two proteomes, obtained by LTQ-Orbitrap Velos mass spectrometry, were compared by statistical analyses. In total, 4139 proteins were identified, 2045 of which overlapping in the two sets. Nonhierarchical Spearman's correlogram revealed that the groups were clearly discriminated. Partial least square discriminant plus support vector machine analysis identified the major discriminative proteins, implied in phototransduction and lipid metabolism, respectively. Gene Ontology analysis identified top gene signatures of the disk proteome, enriched in vesiculation, glycolysis, and OxPhos proteins. The tricarboxylic acid cycle and the electron transport proteins were similarly enriched in the two samples, but the latter was up regulated in disks. Data suggest that the mitochondrial OxPhos proteins may represent a true OS proteome component, outside the mitochondrion. This knowledge may help the scientific community in the further studies of retinal physiology and pathology.
Assuntos
Proteínas do Olho/isolamento & purificação , Mitocôndrias/genética , Proteínas Mitocondriais/isolamento & purificação , Proteoma/isolamento & purificação , Segmento Externo da Célula Bastonete/metabolismo , Animais , Bovinos , Cromatografia Líquida , Ciclo do Ácido Cítrico/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Ontologia Genética , Glicólise/genética , Análise dos Mínimos Quadrados , Transdução de Sinal Luminoso , Metabolismo dos Lipídeos/genética , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Anotação de Sequência Molecular , Fosforilação Oxidativa , Proteoma/genética , Proteoma/metabolismo , Segmento Externo da Célula Bastonete/ultraestrutura , Máquina de Vetores de Suporte , Espectrometria de Massas em TandemRESUMO
Exosomes are secreted nanovesicles that are able to transfer RNA and proteins to target cells. The emerging role of mesenchymal stem cell (MSC) exosomes as promoters of aerobic ATP synthesis restoration in damaged cells, prompted us to assess whether they contain an extramitochondrial aerobic respiration capacity. Exosomes were isolated from culture medium of human MSCs from umbilical cord of ≥37-wk-old newborns or between 28- to 30-wk-old newborns (i.e.,term or preterm infants). Characterization of samples was conducted by cytofluorometry. Oxidative phosphorylation capacity was assessed by Western blot analysis, oximetry, and luminometric and fluorometric analyses. MSC exosomes express functional respiratory complexes I, IV, and V, consuming oxygen. ATP synthesis was only detectable in exosomes from term newborns, suggestive of a specific mechanism that is not completed at an early gestational age. Activities are outward facing and comparable to those detected in mitochondria isolated from term MSCs. MSC exosomes display an unsuspected aerobic respiratory ability independent of whole mitochondria. This may be relevant for their ability to rescue cell bioenergetics. The differential oxidative metabolism of pretermvs.term exosomes sheds new light on the preterm newborn's clinical vulnerability. A reduced ability to repair damaged tissue and an increased capability to cope with anoxic environment for preterm infants can be envisaged.-Panfoli, I., Ravera, S., Podestà, M., Cossu, C., Santucci, L., Bartolucci, M., Bruschi, M., Calzia, D., Sabatini, F., Bruschettini, M., Ramenghi, L. A., Romantsik, O., Marimpietri, D., Pistoia, V., Ghiggeri, G., Frassoni, F., Candiano, G. Exosomes from human mesenchymal stem cells conduct aerobic metabolism in term and preterm newborn infants.
Assuntos
Metabolismo Energético , Exossomos/metabolismo , Recém-Nascido Prematuro/metabolismo , Células-Tronco Mesenquimais/metabolismo , Nascimento a Termo/metabolismo , Trifosfato de Adenosina/biossíntese , Western Blotting , Células Cultivadas , Complexo I de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Recém-Nascido , Recém-Nascido Prematuro/sangue , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação Oxidativa , Oximetria , Consumo de Oxigênio , Nascimento a Termo/sangueRESUMO
Exosomes are nanovesicles, derived from the endocytic pathway, released by most cell types and found in many body fluids, including urine. A variety of exosomal functions have been reported, including transfer of RNA, cell communication, control of apoptosis and protein lifespan. Exosomes from mesenchymal stem cells can rescue bioenergetics of injured cells. Here the urinary exosome proteome, non-urinary exosome proteome and urinome are compared. A consistent number of identified proteins cluster to metabolic functions. Cytoscape software analysis based on biological processes gene ontology database shows that metabolic pathways such as aerobic glycolysis and oxidative phosphorylation have a high probability (p ≤ 0.05) of being expressed and therefore functional. A metabolic function appears to be associated with human urinary exosomes, whose relevance experimental studies can assess.
Assuntos
Exossomos/metabolismo , Urina , HumanosRESUMO
Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and ß subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/biossíntese , Bainha de Mielina/metabolismo , Nervo Óptico/metabolismo , ATPases Translocadoras de Prótons/biossíntese , Trifosfato de Adenosina/biossíntese , Animais , Axônios/metabolismo , Bovinos , Masculino , Camundongos , Mitocôndrias/metabolismo , NADH Desidrogenase/biossíntese , Neuroglia/metabolismo , Fosforilação OxidativaRESUMO
Systemic autoimmune diseases are complex conditions characterized by an immune system dysregulation and an aberrant activation against self-antigens, leading to tissue and organ damage. Even though genetic predisposition plays a role, it cannot fully explain the onset of these diseases, highlighting the significant impact of non-heritable influences such as environment, hormones and infections. The exposome represents all those factors, ranging from chemical pollutants and dietary components to psychological stressors and infectious agents. Epigenetics, which studies changes in gene expression without altering the DNA sequence, is a crucial link between exposome and the development of autoimmune diseases. Key epigenetic mechanisms include DNA methylation, histone modifications, and non-coding RNAs. These epigenetic modifications could provide a potential piece of the puzzle in understanding systemic autoimmune diseases and their connection with the exposome. In this work we have collected the most important and recent evidence in epigenetic changes linked to systemic autoimmune diseases (systemic lupus erythematosus, idiopathic inflammatory myopathies, ANCA-associated vasculitis, and rheumatoid arthritis), emphasizing the roles these changes may play in disease pathogenesis, their potential as diagnostic biomarkers and their prospective in the development of targeted therapies.
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Down syndrome (DS) is the most common genetic cause of cognitive disability. However, it is largely unclear how triplication of a small gene subset may impinge on diverse aspects of DS brain physiopathology. Here, we took a multi-omic approach and simultaneously analyzed by RNA-seq and proteomics the expression signatures of two diverse regions of human postmortem DS brains. We found that the overexpression of triplicated genes triggered global expression dysregulation, differentially affecting transcripts, miRNAs, and proteins involved in both known and novel biological candidate pathways. Among the latter, we observed an alteration in RNA splicing, specifically modulating the expression of genes involved in cytoskeleton and axonal dynamics in DS brains. Accordingly, we found an alteration in axonal polarization in neurons from DS human iPSCs and mice. Thus, our study provides an integrated multilayer expression database capable of identifying new potential targets to aid in designing future clinical interventions for DS.
Assuntos
Encéfalo , Síndrome de Down , Síndrome de Down/metabolismo , Síndrome de Down/genética , Síndrome de Down/patologia , Humanos , Encéfalo/metabolismo , Encéfalo/patologia , Animais , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteômica/métodos , Masculino , Neurônios/metabolismo , Axônios/metabolismo , Axônios/patologia , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , Splicing de RNA , MultiômicaRESUMO
Pediatric pilocytic astrocytoma (PA) is the most common brain tumor in children. Complete resection provides a favorable prognosis, except for unresectable PA forms. There is an incomplete understanding of the molecular and cellular pathogenesis of PA. Potential biomarkers for PA patients, especially the non-BRAF-mutated ones are needed. Cerebrospinal fluid (CSF) is a valuable source of brain tumor biomarkers. Extracellular vesicles (EVs), circulating in CSF, express valuable disease targets. These can be isolated from CSF from waste extraventricular drainage (EVD). We analyzed the proteome of EVD CSF from PA, congenital hydrocephalus (CH, non-tumor control), or medulloblastoma (MB, unrelated tumoral control) patients. A total of 3072 proteins were identified, 47.1%, 65.6%, and 86.2% of which were expressed in the unprocessed total and in its large-EV (LEV), and small-EV (SEV) fractions. Bioinformatics identified 50 statistically significant proteins in the comparison between PA and HC, and PA and MB patients, in the same fractions. Kinase enrichment analysis predicted five enriched kinases involved in signaling. Among these, only Cyclin-dependent kinase 2 (CDK2) kinase was overexpressed in PA samples. PLS-DA highlighted the inactive carboxypeptidase-like protein X2 (CPXM2) and aquaporin-4 (AQP4) as statistically significant in all the comparisons, with CPXM2 being overexpressed (validated by ELISA and Western blot) and AQP4 downregulated in PA. These proteins were considered the most promising potential biomarkers for discriminating among pilocytic astrocytoma and unrelated tumoral (MB) or non-tumoral conditions in all the fractions examined, and are proposed to be prospectively validated in the plasma for translational medicine applications.
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Glioblastoma multiforme (GBM) is the most aggressive brain cancer, characterized by a rapid and drug-resistant progression. GBM "builds" around its primary core a genetically heterogeneous tumor-microenvironment (TME), recruiting surrounding healthy brain cells by releasing various intercellular signals. Glioma-associated microglia (GAM) represent the largest population of collaborating cells, which, in the TME, usually exhibit the anti-inflammatory M2 phenotype, thus promoting an immunosuppressing environment that helps tumor growth. Conversely, "classically activated" M1 microglia could provide proinflammatory and antitumorigenic activity, expected to exert a beneficial effect in defeating glioblastoma. In this work, an immunotherapy approach based on proinflammatory modulation of the GAM phenotype is proposed, through a controlled and localized electrical stimulation. The developed strategy relies on the wireless ultrasonic excitation of polymeric piezoelectric nanoparticles coated with GBM cell membrane extracts, to exploit homotypic targeting in antiglioma applications. Such camouflaged nanotransducers locally generate electrical cues on GAM membranes, activating their M1 phenotype and ultimately triggering a promising anticancer activity. Collected findings open new perspectives in the modulation of immune cell activities through "smart" nanomaterials and, more specifically, provide an innovative auspicious tool in glioma immunotherapy.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Microglia , Nanopartículas , Glioblastoma/patologia , Glioblastoma/metabolismo , Microglia/metabolismo , Microglia/efeitos dos fármacos , Humanos , Nanopartículas/química , Linhagem Celular Tumoral , Neoplasias Encefálicas/patologia , Microambiente Tumoral/efeitos dos fármacos , Imunoterapia/métodos , Ondas Ultrassônicas , AnimaisRESUMO
Besides recent advances in neonatal care, preterm newborns still develop sex-biased behavioral alterations. Preterms fail to receive placental insulin-like growth factor-1 (IGF-1), a major fetal growth hormone in utero, and low IGF-1 serum levels correlate with preterm poor neurodevelopmental outcomes. Here, we mimicked IGF-1 deficiency of preterm newborns in mice by perinatal administration of an IGF-1 receptor antagonist. This resulted in sex-biased brain microstructural, functional, and behavioral alterations, resembling those of ex-preterm children, which we characterized performing parallel mouse/human behavioral tests. Pharmacological enhancement of GABAergic tonic inhibition by the U.S. Food and Drug Administration-approved drug ganaxolone rescued functional/behavioral alterations in mice. Establishing an unprecedented mouse model of prematurity, our work dissects the mechanisms at the core of abnormal behaviors and identifies a readily translatable therapeutic strategy for preterm brain disorders.
Assuntos
Encefalopatias , Fator de Crescimento Insulin-Like I , Estados Unidos , Criança , Humanos , Recém-Nascido , Gravidez , Feminino , Animais , Camundongos , Receptor IGF Tipo 1 , Placenta , Recém-Nascido Prematuro , Encefalopatias/tratamento farmacológicoRESUMO
Polydopamine nanoparticles (PDA NPs) are proposed as an anti-cancer tool against hepatocellular carcinoma through the combination of near-infrared (NIR)-mediated hyperthermia and loading with a chemotherapeutic drug, sorafenib (SRF). Cell membranes isolated from a liver cancer cell line (HepG2) have been exploited for the coating of the nanoparticles (thus obtaining CM-SRF-PDA NPs), to promote homotypic targeting toward cancer cells. The selective targeting ability and the combined photothermal and chemotherapeutic activity of the CM-SRF-PDA NPs following NIR irradiation have been evaluated on cell cultures in static and dynamic conditions, besides three-dimensional culture models. Eventually, the therapeutic effectiveness of the proposed approach has also been tested ex ovo on HepG2 spheroid-grafted quail embryos. This comprehensive investigation, supported by proteomic analysis, showed the effectiveness of the proposed nanoplatform and strongly suggests further pre-clinical testing in the treatment of liver cancer.
Assuntos
Antineoplásicos , Indóis , Neoplasias Hepáticas , Nanopartículas , Terapia Fototérmica , Polímeros , Indóis/química , Indóis/farmacologia , Polímeros/química , Polímeros/farmacologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patologia , Nanopartículas/química , Nanopartículas/uso terapêutico , Células Hep G2 , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Sorafenibe/química , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Sobrevivência Celular/efeitos dos fármacosRESUMO
Myelin sheath is the proteolipid membrane wrapping the axons of CNS and PNS. We have shown data suggesting that CNS myelin conducts oxidative phosphorylation (OXPHOS), challenging its role in limiting the axonal energy expenditure. Here, we focused on PNS myelin. Samples were: (i) isolated myelin vesicles (IMV) from sciatic nerves, (ii) mitochondria from primary Schwann cell cultures, and (iii) sciatic nerve sections, from wild type or Charcot-Marie-Tooth type 1A (CMT1A) rats. The latter used as a model of dys-demyelination. O2 consumption and activity of OXPHOS proteins from wild type (Wt) or CMT1A sciatic nerves showed some differences. In particular, O2 consumption by IMV from Wt and CMT1A 1-month-old rats was comparable, while it was severely impaired in IMV from adult affected animals. Mitochondria extracted from CMT1A Schwann cell did not show any dysfunction. Transmission electron microscopy studies demonstrated an increased mitochondrial density in dys-demyelinated axons, as to compensate for the loss of respiration by myelin. Confocal immunohistochemistry showed the expression of OXPHOS proteins in the myelin sheath, both in Wt and dys-demyelinated nerves. These revealed an abnormal morphology. Taken together these results support the idea that also PNS myelin conducts OXPHOS to sustain axonal function.
Assuntos
Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Fosforilação Oxidativa , Doenças do Sistema Nervoso Periférico/metabolismo , Nervo Isquiático/fisiologia , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Axônios/metabolismo , Western Blotting , Células Cultivadas , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Doenças Desmielinizantes/patologia , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Bainha de Mielina/ultraestrutura , Consumo de Oxigênio/fisiologia , Doenças do Sistema Nervoso Periférico/patologia , ATPases Translocadoras de Prótons/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Células de Schwann/metabolismo , Nervo Isquiático/patologiaRESUMO
Myelin sheath is a lipid-rich membrane, consisting of 70% lipid and 30% proteins, that is involved in physiological and pathological processes. For this reason its protein composition has been often investigated, principally by two-dimensional electrophoresis; however, the consistent lipid content makes it difficult to obtain good proteins separation. To improve the resolution of myelin proteins in a denaturing monodimensional gel electrophoresis, we examined several mixtures for the denaturation of the sample, utilizing different detergents and reducing agents. The definition of the protein pattern was analyzed by both "Blue Silver" Coomassie staining and Western Blot analysis against myelin basic protein, one of the most represented myelin proteins. The best resolution is observed when the sample was incubated with a mixture containing 1.25% dithiothreitol, 4 M urea, and 1% dodecyl maltoside or 1 % 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, prior to addition of denaturing agents. In conclusion, this work describes a novel method to improve the separation of myelin proteins in a monodimensional gel electrophoresis. It may be also useful for investigating other lipid-rich samples.
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Eletroforese em Gel de Poliacrilamida/métodos , Proteínas da Mielina/isolamento & purificação , Bainha de Mielina/química , Animais , Western Blotting , Bovinos , Ácidos Cólicos/química , Detergentes/química , Ditiotreitol/química , Glucosídeos/química , Desnaturação Proteica , Substâncias Redutoras/química , Ureia/químicaRESUMO
Neuroblastoma (NB) is the most common extracranial solid tumor during infancy, causing up to 10% of mortality in children; thus, identifying novel early and accurate diagnostic and prognostic biomarkers is mandatory. NB-derived exosomes carry proteins (Exo-prots) reflecting the status of the tumor cell of origin. The purpose of this study was to characterize, for the first time, the Exo-prots specifically expressed in NB patients associated with tumor phenotype and disease stage. We isolated exosomes from plasma specimens of 24 HR-NB patients and 24 low-risk (LR-NB) patients at diagnosis and of 24 age-matched healthy controls (CTRL). Exo-prot expression was measured by liquid chromatography-mass spectrometry. The data are available via ProteomeXchange (PXD042422). The NB patients had a different Exo-prot expression profile compared to the CTRL. The deregulated Exo-prots in the NB specimens acted mainly in the tumor-associated pathways. The HR-NB patients showed a different Exo-prot expression profile compared to the LR-NB patients, with the modulation of proteins involved in cell migration, proliferation and metastasis. NCAM, NCL, LUM and VASP demonstrated a diagnostic value in discriminating the NB patients from the CTRL; meanwhile, MYH9, FN1, CALR, AKAP12 and LTBP1 were able to differentiate between the HR-NB and LR-NB patients with high accuracy. Therefore, Exo-prots contribute to NB tumor development and to the aggressive metastatic NB phenotype.
Assuntos
Exossomos , Neuroblastoma , Criança , Humanos , Exossomos/metabolismo , Prognóstico , Neuroblastoma/genética , Fenótipo , Biomarcadores/metabolismoRESUMO
Renal normothermic machine perfusion (NMP) is an organ preservation method based on the circulation of a warm (35-37 °C) perfusion solution through the renal vasculature to deliver oxygen and nutrients. However, its biological effects on marginal kidneys are unclear. We therefore used mass spectrometry to determine the proteomic profile of kidney tissue and urine from eight organs reconditioned for 120 min using a Kidney Assist device. Biopsies were taken during the pre-implantation histological evaluation (T-1), at the start of back table preparation (T0), and after 60 and 120 min of perfusion (T60, T120). Urine samples were collected at T0 (urine produced in the first 15 min after the beginning of normothermic reperfusion), T30, T60 and T120. Multiple algorithms, support vector machine learning and partial least squares discriminant analysis were used to select the most discriminative proteins during NMP. Statistical analysis revealed the upregulation of 169 proteins and the downregulation of 196 during NMP. Machine learning algorithms identified the top 50 most discriminative proteins, five of which were concomitantly upregulated (LXN, ETFB, NUDT3, CYCS and UQCRC1) and six downregulated (CFHR3, C1S, CFI, KNG1, SERPINC1 and F9) in the kidney and urine after NMP. Latexin (LXN), an endogenous carboxypeptidase inhibitor, resulted the most-upregulated protein at T120, and this result was confirmed by ELISA. In addition, functional analysis revealed that the most strongly upregulated proteins were involved in the oxidative phosphorylation system and ATP synthesis, whereas the downregulated proteins represented the complement system and coagulation cascade. Our proteomic analysis demonstrated that even brief periods of NMP induce remarkable metabolic and biochemical changes in marginal organs, which supports the use of this promising technique in the clinic.
Assuntos
Transplante de Rim , Rim/metabolismo , Transplante de Rim/métodos , Perfusão/métodos , Proteômica , Regulação para Cima , Proteínas do Tecido Nervoso/metabolismoRESUMO
Cerebrospinal fluid (CSF) is a biochemical-clinical window into the brain. Unfortunately, its wide dynamic range, low protein concentration, and small sample quantity significantly limit the possibility of using it routinely. Extraventricular drainage (EVD) of CSF allows us to solve quantitative problems and to study the biological role of extracellular vesicles (EVs). In this study, we implemented bioinformatic analysis of our previous data of EVD of CSF and its EVs obtained from congenital hydrocephalus with the aim of identifying a comprehensive list of potential tumor and non-tumor biomarkers of central nervous system diseases. Among all proteins identified, those enriched in EVs are associated with synapses, synaptosomes, and nervous system diseases including gliomas, embryonal tumors, and epilepsy. Among these EV-enriched proteins, given the broad consensus present in the recent scientific literature, we validated syntaxin-binding protein 1 (STXBP1) as a marker of malignancy in EVD of CSF and its EVs from patients with pilocytic astrocytoma and medulloblastoma. Our results show that STXBP1 is negatively enriched in EVs compared to non-tumor diseases and its downregulation correlates with adverse outcomes. Further experiments are needed to validate this and other EV markers in the blood of pediatric patients for translational medicine applications.