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Human CD4(+)CD25(hi)Foxp3(+)CD127(-) Treg and CD4(+)CD25(-)Foxp3(-) Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells.
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Ácidos Graxos/metabolismo , Glucose/metabolismo , Glicólise , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Antígenos CD4/metabolismo , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Oxirredução , Proteômica , TranscriptomaRESUMO
Eukaryotic DNA is organized in nucleosomes, which package DNA and regulate its accessibility to transcription, replication, recombination and repair. Here, we show that in living cells nucleosomes protect DNA from high-energy radiation and reactive oxygen species. We combined sequence-based methods (ATAC-seq and BLISS) to determine the position of both nucleosomes and double strand breaks (DSBs) in the genome of nucleosome-rich malignant mesothelioma cells, and of the same cells partially depleted of nucleosomes. The results were replicated in the human MCF-7 breast carcinoma cell line. We found that, for each genomic sequence, the probability of DSB formation is directly proportional to the fraction of time it is nucleosome-free; DSBs accumulate distal from the nucleosome dyad axis. Nucleosome free regions and promoters of actively transcribed genes are more sensitive to DSB formation, and consequently to mutation. We argue that this may be true for a variety of chemical and physical DNA damaging agents.
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Quebras de DNA de Cadeia Dupla/efeitos da radiação , DNA/efeitos da radiação , Nucleossomos/metabolismo , Animais , Linhagem Celular , Humanos , Células MCF-7 , CamundongosRESUMO
Amyloidosis is a relatively rare human disease caused by the deposition of abnormal protein fibres in the extracellular space of various tissues, impairing their normal function. Proteomic analysis of patients' biopsies, developed by Dogan and colleagues at the Mayo Clinic, has become crucial for clinical diagnosis and for identifying the amyloid type. Currently, the proteomic approach is routinely used at National Amyloidosis Centre (NAC, London, UK) and Istituto di Tecnologie Biomediche-Consiglio Nazionale delle Ricerche (ITB-CNR, Milan, Italy). Both centres are members of the European Proteomics Amyloid Network (EPAN), which was established with the aim of sharing and discussing best practice in the application of amyloid proteomics. One of the EPAN's activities was to evaluate the quality and the confidence of the results achieved using different software and algorithms for protein identification. In this paper, we report the comparison of proteomics results obtained by sharing NAC proteomics data with the ITB-CNR centre. Mass spectrometric raw data were analysed using different software platforms including Mascot, Scaffold, Proteome Discoverer, Sequest and bespoke algorithms developed for an accurate and immediate amyloid protein identification. Our study showed a high concordance of the obtained results, suggesting a good accuracy of the different bioinformatics tools used in the respective centres. In conclusion, inter-centre data exchange is a worthwhile approach for testing and validating the performance of software platforms and the accuracy of results, and is particularly important where the proteomics data contribute to a clinical diagnosis.
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Amiloidose/diagnóstico , Biologia Computacional , Disseminação de Informação , Proteômica/métodos , Software , Algoritmos , Proteínas Amiloidogênicas/metabolismo , Amiloidose/metabolismo , Humanos , Itália , Reino UnidoRESUMO
Objective- Vascular calcification (VC) is age dependent and a risk factor for cardiovascular and all-cause mortality. VC involves the senescence-induced transdifferentiation of vascular smooth muscle cells (SMCs) toward an osteochondrogenic lineage resulting in arterial wall mineralization. miR-34a increases with age in aortas and induces vascular SMC senescence through the modulation of its target SIRT1 (sirtuin 1). In this study, we aimed to investigate whether miR-34a regulates VC. Approach and Results- We found that miR-34a and Runx2 (Runt-related transcription factor 2) expression correlates in young and old mice. Mir34a+/+ and Mir34a-/- mice were treated with vitamin D, and calcium quantification revealed that Mir34a deficiency reduces soft tissue and aorta medial calcification and the upregulation of the VC Sox9 (SRY [sex-determining region Y]-box 9) and Runx2 and the senescence p16 and p21 markers. In this model, miR-34a upregulation was transient and preceded aorta mineralization. Mir34a-/- SMCs were less prone to undergo senescence and under osteogenic conditions deposited less calcium compared with Mir34a+/+ cells. Furthermore, unlike in Mir34a+/+ SMC, the known VC inhibitors SIRT1 and Axl (AXL receptor tyrosine kinase) were only partially downregulated in calcifying Mir34a-/- SMC. Strikingly, constitutive miR-34a overexpression to senescence-like levels in human aortic SMCs increased calcium deposition and enhanced Axl and SIRT1 decrease during calcification. Notably, we also showed that miR-34a directly decreased Axl expression in human aortic SMC, and restoration of its levels partially rescued miR-34a-dependent growth arrest. Conclusions- miR-34a promotes VC via vascular SMC mineralization by inhibiting cell proliferation and inducing senescence through direct Axl and SIRT1 downregulation, respectively. This miRNA could be a good therapeutic target for the treatment of VC.
Assuntos
Senescência Celular/fisiologia , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Sirtuína 1/metabolismo , Calcificação Vascular , Adulto , Envelhecimento/patologia , Animais , Aorta/metabolismo , Proliferação de Células , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo , Humanos , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Fatores de Transcrição SOX9/metabolismo , Regulação para Cima , Adulto Jovem , Receptor Tirosina Quinase AxlRESUMO
BACKGROUND: We have demonstrated that intramyocardial delivery of human mesenchymal stem cells preconditioned with a hyaluronan mixed ester of butyric and retinoic acid (MSCp+) is more effective in preventing the decay of regional myocardial contractility in a swine model of myocardial infarction (MI). However, the understanding of the role of MSCp+ in proteomic remodeling of cardiac infarcted tissue is not complete. We therefore sought to perform a comprehensive analysis of the proteome of infarct remote (RZ) and border zone (BZ) of pigs treated with MSCp+ or unconditioned stem cells. METHODS: Heart tissues were analyzed by MudPIT and differentially expressed proteins were selected by a label-free approach based on spectral counting. Protein profiles were evaluated by using PPI networks and their topological analysis. RESULTS: The proteomic remodeling was largely prevented in MSCp+ group. Extracellular proteins involved in fibrosis were down-regulated, while energetic pathways were globally up-regulated. Cardioprotectant pathways involved in the production of keto acid metabolites were also activated. Additionally, we found that new hub proteins support the cardioprotective phenotype characterizing the left ventricular BZ treated with MSCp+. In fact, the up-regulation of angiogenic proteins NCL and RAC1 can be explained by the increase of capillary density induced by MSCp+. CONCLUSIONS: Our results show that angiogenic pathways appear to be uniquely positioned to integrate signaling with energetic pathways involving cardiac repair. GENERAL SIGNIFICANCE: Our findings prompt the use of proteomics-based network analysis to optimize new approaches preventing the post-ischemic proteomic remodeling that may underlie the limited self-repair ability of adult heart.
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Fenômenos Biológicos/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Regulação para Baixo/efeitos dos fármacos , Fibrose/metabolismo , Humanos , Cetoácidos/metabolismo , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Neovascularização Patológica/metabolismo , Proteômica/métodos , Suínos , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Myocardial hibernation (MH) is a well-known feature of human ischaemic cardiomyopathy (ICM), whereas its presence in human idiopathic dilated cardiomyopathy (DCM) is still controversial. We investigated the histological and molecular features of MH in left ventricle (LV) regions of failing DCM or ICM hearts. We examined failing hearts from DCM (n = 11; 41.9 ± 5.45 years; left ventricle-ejection fraction (LV-EF), 18 ± 3.16%) and ICM patients (n = 12; 58.08 ± 1.7 years; LVEF, 21.5 ± 6.08%) undergoing cardiac transplantation, and normal donor hearts (N, n = 8). LV inter-ventricular septum (IVS) and antero-lateral free wall (FW) were transmurally (i.e. sub-epicardial, mesocardial and sub-endocardial layers) analysed. LV glycogen content was shown to be increased in both DCM and ICM as compared with N hearts (P < 0.001), with a U-shaped transmural distribution (lower values in mesocardium). Capillary density was homogenously reduced in both DCM and ICM as compared with N (P < 0.05 versus N), with a lower decrease independent of the extent of fibrosis in sub-endocardial and sub-epicardial layers of DCM as compared with ICM. HIF1-α and nestin, recognized ischaemic molecular hallmarks, were similarly expressed in DCM-LV and ICM-LV myocardium. The proteomic profile was overlapping by ~50% in DCM and ICM groups. Morphological and molecular features of MH were detected in end-stage ICM as well as in end-stage DCM LV, despite epicardial coronary artery patency and lower fibrosis in DCM hearts. Unravelling the presence of MH in the absence of coronary stenosis may be helpful to design a novel approach in the clinical management of DCM.
Assuntos
Cardiomiopatia Dilatada/patologia , Miocárdio Atordoado/patologia , Adulto , Apoptose , Capilares/patologia , Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/diagnóstico por imagem , Cardiomiopatia Dilatada/fisiopatologia , Tamanho Celular , Colágeno Tipo I/metabolismo , Conexina 43/metabolismo , Feminino , Fibronectinas/metabolismo , Insuficiência Cardíaca/patologia , Transplante de Coração , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Hemodinâmica , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/patologia , Miocárdio Atordoado/complicações , Miocárdio Atordoado/metabolismo , Miócitos Cardíacos/patologia , Fenótipo , Proteômica , Ultrassonografia , Vimentina/metabolismoRESUMO
Considering the important advances in treating specific types of systemic amyloidoses, unequivocal typing of amyloid deposits is now essential. Subcutaneous abdominal fat aspiration is the easiest, most common diagnostic procedure. We developed a novel, automated approach, based on Multidimensional Protein Identification Technology, for typing amyloidosis. Fat aspirates were obtained from patients with the most common systemic amyloidoses (ALλ, ALκ, transthyretin, and reactive amyloidosis), with Congo red score more than or equal to 3+, and nonaffected controls. Peptides from extracted and digested proteins were analyzed by Multidimensional Protein Identification Technology. On semiquantitative differential analysis (patients vs controls) of mass spectrometry data, specific proteins up-represented in patients were identified and used as deposit biomarkers. An algorithm was developed to classify patients according to type and abundance of amyloidogenic proteins in samples; in all cases, proteomic characterization was concordant with fibril identification by immunoelectron microscopy and consistent with clinical presentation. Our approach allows reliable amyloid classification using readily available fat aspirates.
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Amiloidose/metabolismo , Proteoma/análise , Proteômica/métodos , Gordura Subcutânea/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Amiloide/análise , Amiloide/química , Amiloidose/classificação , Amiloidose/patologia , Biópsia por Agulha Fina , Vermelho Congo/química , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Gordura Subcutânea/patologiaRESUMO
BACKGROUND: Treatments of eating disorders result too often in partial psychological and physical remission, chronicization, dropout, relapse and death, with no fully known explanations for this failure. In order to clarify this problem, we conducted three studies to identify the biochemical background of cognitive-behavioural psychotherapy (CBT), individual psychology brief psychotherapy (IBPP), and psychotherapy-pharmacotherapy with CBT + olanzapine in anorexics (AN) and bulimics (BN) by measuring the levels of plasma homovanillic acid (HVA) for dopamine secretion, plasma 3-methoxy-4-hydroxy-phenylglycol (MHPG) for noradrenalin secretion, and platelet [3H]-Paroxetin-binding Bmax and Kd for serotonin transporter function. The data were then compared with psychopathological and physical alterations. METHODS: Study 1 investigated the effects of 4 months of CBT on plasma HVA, MHPG and [3H]-Par-binding in 14 AN-restricted, 14 AN-bingeing/purging, and 22 BN inpatients. Study 2 investigated the effects of 4 months of IBPP on plasma HVA in 15 AN and 17 BN outpatients. Study 3 investigated the effect of 3 months of CBT + olanzapine (5 mg/day) in 30 AN outpatients. The data were analyzed using one-way ANOVA for repeated measures for the changes between basal and post-treatment biological and psychological parameters, two-way ANOVA for repeated measures for the differences in the psychobiological data in the 3 groups, Spearman's test for the correlations between basal and final changes in the psychological and biological scores. RESULTS: Study 1 revealed significant amelioration of the psychopathology in the AN and BN patients, no effects on HVA, MHPG or Paroxetin binding Kd, and a significant increase in Par-binding Bmax only in the BN patients. Study 2 revealed a significant effect of IBPP on psychopathology in the AN and BN patients, and a significant increase in HVA only in the BN patients. Study 3 revealed a significant positive effect of CBT + olanzapine therapy on the psychopathology and increased HVA values. No correlations were observed in the 3 groups between biological and psychological effects of the three treatments. CONCLUSIONS: Our data advance suggestions on the mechanism of action of the three therapies; however, the lack of correlations between biochemical and psychological effects casts doubt on their significance. Clinical Trials.gov. Identifier NCT01990755 .
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Anorexia Nervosa/terapia , Antipsicóticos/uso terapêutico , Benzodiazepinas/uso terapêutico , Bulimia Nervosa/terapia , Terapia Cognitivo-Comportamental/métodos , Psicoterapia Breve/métodos , Administração Oral , Adulto , Análise de Variância , Anorexia Nervosa/sangue , Bulimia Nervosa/sangue , Terapia Combinada , Método Duplo-Cego , Feminino , Ácido Homovanílico/metabolismo , Humanos , Masculino , Metoxi-Hidroxifenilglicol/metabolismo , Olanzapina , Paroxetina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Falha de TratamentoRESUMO
Eating disorders (ED) are characterized by alterations in food choice and in the quantity and quality of nutrient intake. In a population of 124 female patients with ED (anorexia nervosa restricting subtype [AN-R, n=37]; AN bingeing-purging subtype [AN-BP, n=18]; bulimia nervosa purging subtype [BN-P, n=40]; and binge eating disorder [BED, n=29]) and healthy age-matched controls ([C], n=20) we compared food choice and macronutrient intake with psychopathologic symptoms of the disorders. Data were collected from the probands' 7-day food diaries and the scores from two assessment scales (Eating Disorder Inventory-2 [EDI-2] and Temperament and Character Inventory-revised [TCI-R]) that measure symptom domains, dimensions of personality and character dimensions, respectively. Multiple regression analysis was applied to the nutritional data and scale scores. When compared to the values for the control group, intake of animal proteins (grams) was significantly lower for all patient groups, intake of lactoproteins was lower for the AN-R and AN-BP than BN-P and BED groups, intake of vegetal proteins was higher for the AN-R, AN-BP, BN-P and BED groups, intake of dietary fats was lower for the AN-R and AN-BP subtype groups, and intake of total carbohydrates and oligosaccharides was lower for the AN-R and AN-BP groups, and oligosaccharides also for the BED, when calculated in grams but not when expressed in percent. When studied as percent values animal proteins were lower in patients than in controls, lactoprotein in BN-P and BED, vegetal proteins higher in all the patients, fat lower in AN-R and AN-BP, while carbohydrates did not differ between patients and controls. Significant correlations emerged between food choice and TCI-R and EDI-2 scale scores. Food choice in ED might depend on alterations in neurotransmitter peptides, neuropeptides, and peripheral peptides, which regulate and are regulated by macronutrient intake and underlie psychological and temperamental alterations.
Assuntos
Anorexia Nervosa/psicologia , Bulimia Nervosa/psicologia , Preferências Alimentares/psicologia , Adulto , Anorexia Nervosa/fisiopatologia , Bulimia Nervosa/fisiopatologia , Estudos de Casos e Controles , Dieta , Feminino , Humanos , Estado Nutricional , Adulto JovemRESUMO
A large body of literature suggests the occurrence of a dysregulation in both central and peripheral modulators of appetite in patients with anorexia nervosa (AN) and bulimia nervosa (BN), but at the moment, the state or trait-dependent nature of those changes is far from being clear. It has been proposed, although not definitively proved, that peptide alterations, even when secondary to malnutrition and/or to aberrant eating behaviours, might contribute to the genesis and the maintenance of some symptomatic aspects of AN and BN, thus affecting the course and the prognosis of these disorders. This review focuses on the most significant literature studies that explored the physiology of those central and peripheral peptides, which have prominent effects on eating behaviour, body weight and energy homeostasis in patients with AN and BN. The relevance of peptide dysfunctions for the pathophysiology of eating disorders is critically discussed.
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Anorexia Nervosa/fisiopatologia , Bulimia Nervosa/fisiopatologia , Comportamento Alimentar/fisiologia , Homeostase/fisiologia , Peptídeos/fisiologia , Apetite/fisiologia , Peso Corporal/fisiologia , HumanosRESUMO
Over the past three decades, cell therapy development has fallen short of expectations, with many cellular sources demonstrating a 'Janus effect' and raising safety concerns. Extracellular vesicles (EVs), supported by advanced technologies, present a promising avenue in regenerative medicine, offering benefits such as immune tolerance and avoidance of negative aspects associated with cell transplants. Our previous research showcased enhanced and organized subcutaneous vascularization using three-dimensional bioprinted patches containing HUVEC-derived EVs in immunodeficient animal models. In this context, stress conditions on the cells of origin further boosted the EVs' neoangiogenic potential. Since neovascularization is the first regenerative target requiring restoration, the present study aims to complement our previous work by employing an injectable gelatin methacrylate (GelMA) hydrogel functionalized with HUVEC-derived EVs in a pathological condition of acute myocardial infarction. This bioactive hydrogel resulted in reduced fibrosis, improved contractility, and promoted angiogenesis, showing promise in countering tissue deterioration and addressing vascular deficits. Moreover, the molecular characterization of EVs through miRNome and proteomic analyses further supports their potential as bio-additives for hydrogel functionalization. This cell-free approach mitigates immune rejection and oncogenic risks, offering innovative therapeutic advantages.
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Vesículas Extracelulares , Células Endoteliais da Veia Umbilical Humana , Hidrogéis , Infarto do Miocárdio , Neovascularização Fisiológica , Humanos , Animais , Infarto do Miocárdio/terapia , Infarto do Miocárdio/patologia , Hidrogéis/química , Neovascularização Fisiológica/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/transplante , Metacrilatos/química , Gelatina/química , Injeções , MasculinoRESUMO
In systemic amyloidosis, accumulation of misfolded proteins as extracellular amyloid fibrils in tissues causes severe organ dysfunction, but the molecular events of tissue damage related to amyloid deposition are still largely unknown. Through the use of the MudPIT proteomic approach, comprehensive protein profiles of human amyloid-affected adipose tissue from patients and its control (non-amyloid-affected) counterpart were acquired. Label-free comparison between patients and controls made it possible to highlight differences related to the presence of amyloid, by describing up- and down-represented proteins, connected into interacting networks. In particular, extracellular matrix (ECM), protein folding, lipid metabolism, and mitochondrial functions were among the most affected structural/functional pathways. The reported results, obtained with no a priori hypotheses, represent a significant step forward in the clarification of the molecular mechanisms involved in amyloidoses at tissue level and are the premise for understanding protein misfolding diseases.
Assuntos
Tecido Adiposo/química , Amiloide/genética , Amiloidose/genética , Matriz Extracelular/genética , Redes Reguladoras de Genes , Proteínas Mitocondriais/genética , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Amiloide/química , Amiloide/metabolismo , Amiloidose/metabolismo , Amiloidose/patologia , Estudos de Casos e Controles , Cromatografia Líquida , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina , Metabolismo dos Lipídeos/genética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Anotação de Sequência Molecular , Dobramento de Proteína , Mapeamento de Interação de Proteínas , Espectrometria de Massas em TandemRESUMO
OBJECTIVE: This article addresses the state of the art concerning the treatment of binge eating disorder (BED). Pharmacological and psychotherapeutic strategies, together with issues concerning the involvement in bariatric surgery are considered. METHOD: A Medline enquiry of published articles was performed using the following keywords: BED, pharmacological treatment, duloxetine, venlafaxine, SSRI, psychotherapy, bariatric surgery; reviews and single-case studies were also analyzed. RESULTS: Psychological interventions that have shown efficacy in the treatment of Bulimia Nervosa have also been tested in BED with positive results, in particular modified cognitive behavioral therapy, interpersonal therapy and dialectical behavior therapy. In addition pharmacotherapy with SSRIs is successful in transiently reducing binge-eating and body weight; the SNRI duloxetine is effective for reducing binge eating, and global severity of illness with a controversial effect on body weight; both topiramate and sibutramine seem promising, but their use is restricted due to labeling and side effect limitations, respectively. Finally, adequate psychological/pharmacological support can help BED patients obtain positive outcomes from bariatric surgery. CONCLUSION: Studies on BED treatment are burdened by several limitations as selection biases (e.g. mostly women and overweight), small samples, high drop-out rates and placebo response.
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Antidepressivos/uso terapêutico , Transtorno da Compulsão Alimentar/terapia , Terapia Cognitivo-Comportamental , Cirurgia Bariátrica , Transtorno da Compulsão Alimentar/tratamento farmacológico , Transtorno da Compulsão Alimentar/psicologia , Humanos , Resultado do TratamentoRESUMO
Cystic formation in human primary brain tumors is a relatively rare event whose incidence varies widely according to the histotype of the tumor. Composition of the cystic fluid has mostly been characterized in samples collected at the time of tumor resection and no indications of the evolution of cystic content are available. We characterized the evolution of the proteome of cystic fluid using a bottom-up proteomic approach on sequential samples obtained from secretory meningioma (SM), cystic schwannoma (CS) and cystic high-grade glioma (CG). We identified 1008 different proteins; 74 of these proteins were found at least once in the cystic fluid of all tumors. The most abundant proteins common to all tumors studied derived from plasma, with the exception of prostaglandin D2 synthase, which is a marker of cerebrospinal fluid origin. Overall, the protein composition of cystic fluid obtained at different times from the same tumor remained stable. After the identification of differentially expressed proteins (DEPs) and the protein-protein interaction network analysis, we identified the presence of tumor-specific pathways that may help to characterize tumor-host interactions. Our results suggest that plasma proteins leaking from local blood-brain barrier disruption are important contributors to cyst fluid formation, but cerebrospinal fluid (CSF) and the tumor itself also contribute to the cystic fluid proteome and, in some cases, as with immunoglobulin G, shows tumor-specific variations that cannot be simply explained by differences in vessel permeability or blood contamination.
RESUMO
Transcription factor dynamics is fundamental to determine the activation of accurate transcriptional programs and yet is heterogeneous at a single-cell level, even within homogeneous populations. We asked how such heterogeneity emerges for the nuclear factor κB (NF-κB). We found that clonal populations of immortalized fibroblasts derived from a single mouse embryo display robustly distinct NF-κB dynamics upon tumor necrosis factor É (TNF-É) stimulation including persistent, oscillatory, and weak activation, giving rise to differences in the transcription of its targets. By combining transcriptomics and simulations we show how less than two-fold differences in the expression levels of genes coding for key proteins of the signaling cascade and feedback system are predictive of the differences of the NF-κB dynamic response of the clones to TNF-É and IL-1ß. We propose that small transcriptional differences in the regulatory circuit of a transcription factor can lead to distinct signaling dynamics in cells within homogeneous cell populations and among different cell types.
RESUMO
AL amyloidosis is caused by the misfolding of immunoglobulin light chains leading to an impaired function of tissues and organs in which they accumulate. Due to the paucity of -omics profiles from undissected samples, few studies have addressed amyloid-related damage system wide. To fill this gap, we evaluated proteome changes in the abdominal subcutaneous adipose tissue of patients affected by the AL isotypes κ and λ. Through our retrospective analysis based on graph theory, we have herein deduced new insights representing a step forward from the pioneering proteomic investigations previously published by our group. ECM/cytoskeleton, oxidative stress and proteostasis were confirmed as leading processes. In this scenario, some proteins, including glutathione peroxidase 1 (GPX1), tubulins and the TRiC complex, were classified as biologically and topologically relevant. These and other results overlap with those already reported for other amyloidoses, supporting the hypothesis that amyloidogenic proteins could induce similar mechanisms independently of the main fibril precursor and of the target tissues/organs. Of course, further studies based on larger patient cohorts and different tissues/organs will be essential, which would be a key point that would allow for a more robust selection of the main molecular players and a more accurate correlation with clinical aspects.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina , Humanos , Proteômica/métodos , Estudos Retrospectivos , Biópsia , Gordura Subcutânea/metabolismoRESUMO
An excess of circulating monoclonal free immunoglobulin light chains (FLC) is common in plasma cell disorders. A subset of FLC, as amyloidogenic ones, possess intrinsic pathogenicity. Because of their complex purification, little is known on the biochemical features of serum FLC, possibly related to their pathogenic spectrum. We developed an immunopurification approach to isolate serum FLC from patients with monoclonal gammopathies, followed by proteomic characterization. Serum monoclonal FLC were detected and quantified by immunofixation and immunonephelometry. Immunoprecipitation was performed by serum incubation with agarose beads covalently linked to polyclonal anti-κ or λ FLC antibodies. Isolated FLC were analyzed by SDS-PAGE, 2D-PAGE, immunoblotting, mass spectrometry (MS). Serum FLC were immunoprecipitated from 15 patients with ALλ amyloidosis (serum λ FLC range: 98-2350mg/L), 5 with ALκ amyloidosis and 1 with κ light chain (LC) myeloma (κ FLC range: 266-2660mg/L), and 3 controls. Monoclonal FLC were the prevalent eluted species in patients. On 2D-PAGE, both λ and κ FLC originated discrete spots with multiple pI isoforms. The nature of eluted FLC and coincidence with the LC sequence from the bone marrow clone was confirmed by MS, which also detected post-translational modifications, including truncation, tryptophan oxidation, cysteinylation, peptide dimerization. Serum FLC were purified in soluble form and adequate amounts for proteomics, which allowed studying primary sequence and detecting post-translational modifications. This method is a novel instrument for studying the molecular bases of FLC pathogenicity, allowing for the first time the punctual biochemical description of the circulating forms.
Assuntos
Cadeias Leves de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/isolamento & purificação , Cadeias lambda de Imunoglobulina/isolamento & purificação , Proteômica/métodos , Adulto , Idoso , Sequência de Aminoácidos , Amiloidose/sangue , Anticorpos Monoclonais/isolamento & purificação , Cromatografia Líquida , Feminino , Humanos , Cadeias Leves de Imunoglobulina/isolamento & purificação , Imunoprecipitação/métodos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Alinhamento de SequênciaRESUMO
Although exosomes are extracellular nanovesicles mainly involved in cardioprotection, it is not known whether plasma exosomes of older patients undergoing different types of on-pump cardiac surgery protect cardiomyocytes from apoptosis. Since different exosomal proteins confer pro-survival effects, we have analyzed the protein cargo of exosomes circulating early after aortic unclamping. Plasma exosomes and serum cardiac troponin I levels were measured in older cardiac surgery patients (NYHA II-III) who underwent first-time on-pump coronary artery bypass graft (CABG; n = 15) or minimally invasive heart valve surgery (mitral valve repair, n = 15; aortic valve replacement, n = 15) at induction of anesthesia (T0, baseline), 3 h (T1) and 72 h (T2) after aortic unclamping. Anti-apoptotic role of exosomes was assessed in HL-1 cardiomyocytes exposed to hypoxia/re-oxygenation (H/R) by TUNEL assay. Protein exosomal cargo was characterized by mass spectrometry approach. Exosome levels increased at T1 (P < 0.01) in accord with troponin values in all groups. In CABG group, plasma exosomes further increased at T2 (P < 0.01) whereas troponin levels decreased. In vitro, all T1-exosomes prevented H/R-induced apoptosis. A total of 340 exosomal proteins were identified in all groups, yet 10% of those proteins were unique for each surgery type. In particular, 22 and 12 pro-survival proteins were detected in T1-exosomes of heart valve surgery and CABG patients, respectively. Our results suggest that endogenous intraoperative cardioprotection in older cardiac surgery patients is early mediated by distinct exosomal proteins regardless of surgery type.
Assuntos
Procedimentos Cirúrgicos Cardíacos , Exossomos , Idoso , Apoptose , Humanos , Miócitos CardíacosRESUMO
In the last decades, it has been demonstrated that the regenerative therapeutic efficacy of mesenchymal stromal cells is primarily due to the secretion of soluble factors and extracellular vesicles, collectively known as secretome. In this context, our work described the preparation and characterization of a freeze-dried secretome (Lyosecretome) from adipose tissue-derived mesenchymal stromal cells for the therapy of equine musculoskeletal disorder. An intraarticular injectable pharmaceutical powder has been formulated, and the technological process has been validated in an authorized facility for veterinary clinical-use medicinal production. Critical parameters for quality control and batch release have been identified regarding (i) physicochemical properties; (ii) extracellular vesicle morphology, size distribution, and surface biomarker; (iii) protein and lipid content; (iv) requirements for injectable pharmaceutical dosage forms such as sterility, bacterial endotoxin, and Mycoplasma; and (v) in vitro potency tests, as anti-elastase activity and proliferative activity on musculoskeletal cell lines (tenocytes and chondrocytes) and mesenchymal stromal cells. Finally, proteins putatively responsible for the biological effects have been identified by Lyosecretome proteomic investigation: IL10RA, MXRA5, RARRES2, and ANXA1 modulate the inflammatory process RARRES2, NOD1, SERPINE1, and SERPINB9 with antibacterial activity. The work provides a proof-of-concept for the manufacturing of clinical-grade equine freeze-dried secretome, and prototypes are now available for safety and efficacy clinical trials in the treatment of equine musculoskeletal diseases.