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1.
Internist (Berl) ; 61(10): 1094-1105, 2020 Oct.
Artigo em Alemão | MEDLINE | ID: mdl-32897404

RESUMO

BACKGROUND: The early detection and treatment of diabetic nephropathy (DN) is of crucial importance as patients with diabetes mellitus represent the largest proportion of patients on dialysis, with the highest morbidity and mortality. Currently, the first clinical sign of incipient DN is microalbuminuria, but its precision is not optimal. Many studies now report that proteins and peptides are new biomarkers in urine that primarily depict the pathophysiology of DN and thus allow for improved diagnosis of DN. OBJECTIVES: The presentation of new concepts for the early detection and treatment of DN for better patient management. MATERIAL AND METHODS: A systematic literature search was carried out. RESULTS: Many potential markers have been described in the search for new biomarkers to diagnose DN by urinary proteome analysis. However, many of these studies were not meaningful due to the small number of samples. This limitation led to inadequate validation of proteins that could not be confirmed as markers. However, the diagnostic benefit of CKD 273, a multimarker of 273 protein fragments, was sustainably demonstrated for the early diagnosis of DN. This multi-marker shows significant advantages in the precision of diagnosis and prognosis compared to albuminuria. Furthermore, many of its peptide markers map the molecular pathophysiology of DN. CONCLUSIONS: Clinical urinary proteome analysis shows great benefits and is already an appropriate tool for the early detection of incipient DN.


Assuntos
Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/urina , Proteoma/análise , Proteômica/métodos , Albuminúria/diagnóstico , Albuminúria/urina , Biomarcadores/sangue , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/urina , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/urina , Diagnóstico Precoce , Humanos
2.
Nat Commun ; 11(1): 3793, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732981

RESUMO

Reproducible research is the bedrock of experimental science. To enable the deployment of large-scale proteomics, we assess the reproducibility of mass spectrometry (MS) over time and across instruments and develop computational methods for improving quantitative accuracy. We perform 1560 data independent acquisition (DIA)-MS runs of eight samples containing known proportions of ovarian and prostate cancer tissue and yeast, or control HEK293T cells. Replicates are run on six mass spectrometers operating continuously with varying maintenance schedules over four months, interspersed with ~5000 other runs. We utilise negative controls and replicates to remove unwanted variation and enhance biological signal, outperforming existing methods. We also design a method for reducing missing values. Integrating these computational modules into a pipeline (ProNorM), we mitigate variation among instruments over time and accurately predict tissue proportions. We demonstrate how to improve the quantitative analysis of large-scale DIA-MS data, providing a pathway toward clinical proteomics.


Assuntos
Espectrometria de Massas/métodos , Proteoma/análise , Proteômica/métodos , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Masculino , Neoplasias Ovarianas , Neoplasias da Próstata , Reprodutibilidade dos Testes , Saccharomyces cerevisiae
3.
Nat Commun ; 11(1): 3903, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764543

RESUMO

Top-down mass spectrometry (MS)-based proteomics provides a comprehensive analysis of proteoforms to achieve a proteome-wide understanding of protein functions. However, the MS detection of low-abundance proteins from blood remains an unsolved challenge due to the extraordinary dynamic range of the blood proteome. Here, we develop an integrated nanoproteomics method coupling peptide-functionalized superparamagnetic nanoparticles (NPs) with top-down MS for the enrichment and comprehensive analysis of cardiac troponin I (cTnI), a gold-standard cardiac biomarker, directly from serum. These NPs enable the sensitive enrichment of cTnI (<1 ng/mL) with high specificity and reproducibility, while simultaneously depleting highly abundant proteins such as human serum albumin (>1010 more abundant than cTnI). We demonstrate that top-down nanoproteomics can provide high-resolution proteoform-resolved molecular fingerprints of diverse cTnI proteoforms to establish proteoform-pathophysiology relationships. This scalable and reproducible antibody-free strategy can generally enable the proteoform-resolved analysis of low-abundance proteins directly from serum to reveal previously unachievable molecular details.


Assuntos
Análise Química do Sangue/métodos , Proteínas Sanguíneas/análise , Espectrometria de Massas/métodos , Proteômica/métodos , Troponina I/sangue , Biomarcadores/sangue , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestrutura , Nanotecnologia , Processamento de Proteína Pós-Traducional , Proteoma/análise , Reprodutibilidade dos Testes , Albumina Sérica Humana/análise
4.
Nat Commun ; 11(1): 4332, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859902

RESUMO

The group of enteroviruses contains many important pathogens for humans, including poliovirus, coxsackievirus, rhinovirus, as well as newly emerging global health threats such as EV-A71 and EV-D68. Here, we describe an unbiased, system-wide and time-resolved analysis of the proteome and phosphoproteome of human cells infected with coxsackievirus B3. Of the ~3,200 proteins quantified throughout the time course, a large amount (~25%) shows a significant change, with the majority being downregulated. We find ~85% of the detected phosphosites to be significantly regulated, implying that most changes occur at the post-translational level. Kinase-motif analysis reveals temporal activation patterns of certain protein kinases, with several CDKs/MAPKs immediately active upon the infection, and basophilic kinases, ATM, and ATR engaging later. Through bioinformatics analysis and dedicated experiments, we identify mTORC1 signalling as a major regulation network during enterovirus infection. We demonstrate that inhibition of mTORC1 activates TFEB, which increases expression of lysosomal and autophagosomal genes, and that TFEB activation facilitates the release of virions in extracellular vesicles via secretory autophagy. Our study provides a rich framework for a system-level understanding of enterovirus-induced perturbations at the protein and signalling pathway levels, forming a base for the development of pharmacological inhibitors to treat enterovirus infections.


Assuntos
Infecções por Coxsackievirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Proteoma/análise , Animais , Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Sobrevivência Celular , Enterovirus/fisiologia , Enterovirus Humano B/fisiologia , Técnicas de Inativação de Genes , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosforilação , Transdução de Sinais , Proteínas Virais/metabolismo
5.
Parasitol Res ; 119(9): 2837-2850, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32757109

RESUMO

Fasciola hepatica (F. hepatica) is a well-known zoonotic parasite that is crucial for economic and public health worldwide. Quantitative proteomics studies have been performed on proteins expressed by F. hepatica to investigate the differential expression of proteomes in different growth phases. And the screening of several marker proteins for use as early diagnostic antigens is essential. In this study, high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted to analyze the differences in the expression of F. hepatica somatic proteins in different growth phases. Furthermore, gene ontology (GO) functional annotation, KEGG metabolic pathway, and clustering analyses were also performed. LC-MS/MS identified 629, 2286, 2254, and 2192 proteins in metacercariae, juvenile flukes 28dpi, immature flukes 59dpi, and adult phases, respectively. GO analysis revealed that differentially expressed proteins (DEPs) were mainly involved in transport, localization, metabolism, enzyme regulation, protein folding and binding, and nucleoside and nucleotide binding. The DEPs were enriched in cells, intracellular components, organelles, cytoplasm, vesicles, and membranes. KEGG pathway annotation results showed that the DEPs were involved in metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and other processes. These findings provide a theoretical basis for vaccine development and establishing early diagnostic methods in the future.


Assuntos
Fasciola hepatica/crescimento & desenvolvimento , Fasciola hepatica/genética , Proteoma/análise , Animais , Cromatografia Líquida , Análise por Conglomerados , Biologia Computacional , Fasciolíase/parasitologia , Perfilação da Expressão Gênica , Proteoma/genética , Proteômica/métodos , Espectrometria de Massas em Tandem
6.
PLoS One ; 15(7): e0237045, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32735576

RESUMO

The dead organs enclosing embryos (DOEEs) emerge as central components of the dispersal unit (DU) capable for long-term storage of active proteins and other substances that affect seed performance and fate. We studied the effect of maternal environment (salt and salt+heat) on progeny DU (dry indehiscent fruit) focusing on pericarp properties of Anastatica hierochuntica. Stressed plants displayed increased seed abortion and low level and rate of germination. Hydrated pericarps released antimicrobial factors and allelopathic substances that inhibit germination of heterologous species. Proteome analysis of dead pericarps revealed hundreds of proteins, among them nucleases, chitinases and proteins involved in reactive oxygen species detoxification and cell wall modification. Salt treatment altered the composition and level of proteins stored in the pericarp. We observed changes in protein profile released from seeds of salt-treated plants with a notable increase in a small anti-fungal protein, defensin. The levels of phytohormones including IAA, ABA and salicylic acid were reduced in dead pericarps of stressed plants. The data presented here highlighted the predominant effects of maternal environment on progeny DUs of the desert plant A. hierochuntica, particularly on pericarp properties, which in turn might affect seed performance and fate, soil fertility and consequently plant biodiversity.


Assuntos
Brassicaceae , Frutas , Brassicaceae/embriologia , Brassicaceae/metabolismo , Defensinas/metabolismo , Frutas/embriologia , Frutas/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Proteoma/análise , Sementes/metabolismo , Estresse Fisiológico
7.
Nat Commun ; 11(1): 3850, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737322

RESUMO

Resolving the distribution of specific proteins at the nanoscale in the ultrastructural context of the cell is a major challenge in fluorescence microscopy. We report the discovery of a new principle for an optical contrast equivalent to electron microscopy (EM) which reveals the ultrastructural context of the cells with a conventional confocal microscope. By decrowding the intracellular space through 13 to 21-fold physical expansion while simultaneously retaining the proteins, bulk (pan) labeling of the proteome resolves local protein densities and reveals the cellular nanoarchitecture by standard light microscopy.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Proteoma/análise , Coloração e Rotulagem/métodos , Acrilamidas/química , Reagentes para Ligações Cruzadas/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Hidrogéis/química , Espaço Intracelular/química , Succinimidas/química , Inclusão do Tecido/métodos
8.
N Engl J Med ; 383(4): 321-333, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32706533

RESUMO

BACKGROUND: Environmental enteric dysfunction (EED) is an enigmatic disorder of the small intestine that is postulated to play a role in childhood undernutrition, a pressing global health problem. Defining the incidence of this disorder, its pathophysiological features, and its contribution to impaired linear and ponderal growth has been hampered by the difficulty in directly sampling the small intestinal mucosa and microbial community (microbiota). METHODS: In this study, among 110 young children (mean age, 18 months) with linear growth stunting who were living in an urban slum in Dhaka, Bangladesh, and had not benefited from a nutritional intervention, we performed endoscopy in 80 children who had biopsy-confirmed EED and available plasma and duodenal samples. We quantified the levels of 4077 plasma proteins and 2619 proteins in duodenal biopsy samples obtained from these children. The levels of bacterial strains in microbiota recovered from duodenal aspirate from each child were determined with the use of culture-independent methods. In addition, we obtained 21 plasma samples and 27 fecal samples from age-matched healthy children living in the same area. Young germ-free mice that had been fed a Bangladeshi diet were colonized with bacterial strains cultured from the duodenal aspirates. RESULTS: Of the bacterial strains that were obtained from the children, the absolute levels of a shared group of 14 taxa (which are not typically classified as enteropathogens) were negatively correlated with linear growth (length-for-age z score, r = -0.49; P = 0.003) and positively correlated with duodenal proteins involved in immunoinflammatory responses. The representation of these 14 duodenal taxa in fecal microbiota was significantly different from that in samples obtained from healthy children (P<0.001 by permutational multivariate analysis of variance). Enteropathy of the small intestine developed in gnotobiotic mice that had been colonized with cultured duodenal strains obtained from children with EED. CONCLUSIONS: These results provide support for a causal relationship between growth stunting and components of the small intestinal microbiota and enteropathy and offer a rationale for developing therapies that target these microbial contributions to EED. (Funded by the Bill and Melinda Gates Foundation and others; ClinicalTrials.gov number, NCT02812615.).


Assuntos
Duodeno/microbiologia , Microbioma Gastrointestinal , Transtornos do Crescimento/microbiologia , Transtornos da Nutrição do Lactente/complicações , Animais , Bactérias/isolamento & purificação , Bangladesh , Duodenoscopia , Duodeno/patologia , Doença Ambiental/complicações , Fezes/microbiologia , Feminino , Vida Livre de Germes , Crescimento , Transtornos do Crescimento/etiologia , Humanos , Lactente , Doenças Inflamatórias Intestinais/complicações , Fator de Crescimento Insulin-Like I/análise , Enteropatias/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise Multivariada , Proteínas Associadas a Pancreatite/análise , Proteoma/análise
9.
PLoS One ; 15(7): e0230400, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639965

RESUMO

Alterations in the cortico-cerebellar-thalamic-cortical circuit might underlie the diversity of symptoms in schizophrenia. However, molecular changes in cerebellar neuronal circuits, part of this network, have not yet been fully determined. Using LC-MS/MS, we screened altered candidates in pooled grey matter of cerebellum from schizophrenia subjects who committed suicide (n = 4) and healthy individuals (n = 4). Further validation by immunoblotting of three selected candidates was performed in two cohorts comprising schizophrenia (n = 20), non-schizophrenia suicide (n = 6) and healthy controls (n = 21). We found 99 significantly altered proteins, 31 of them previously reported in other brain areas by proteomic studies. Transport function was the most enriched category, while cell communication was the most prevalent function. For validation, we selected the vacuolar proton pump subunit 1 (VPP1), from transport, and two EF-hand calcium-binding proteins, calmodulin and parvalbumin, from cell communication. All candidates showed significant changes in schizophrenia (n = 7) compared to controls (n = 7). VPP1 was altered in the non-schizophrenia suicide group and increased levels of parvalbumin were linked to antipsychotics. Further validation in an independent cohort of non-suicidal chronic schizophrenia subjects (n = 13) and non-psychiatric controls (n = 14) showed that parvalbumin was increased, while calmodulin was decreased in schizophrenia. Our findings provide evidence of calcium-binding protein dysregulation in the cerebellum in schizophrenia, suggesting an impact on normal calcium-dependent synaptic functioning of cerebellar circuits. Our study also links VPP1 to suicide behaviours, suggesting a possible impairment in vesicle neurotransmitter refilling and release in these phenotypes.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cerebelo/metabolismo , Esquizofrenia/patologia , Adulto , Calmodulina/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Parvalbuminas/metabolismo , Proteoma/análise , Esquizofrenia/metabolismo , Tentativa de Suicídio , Espectrometria de Massas em Tandem , Regulação para Cima
10.
Nat Commun ; 11(1): 3581, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665645

RESUMO

We still know very little about how the human immune system responds to SARS-CoV-2. Here we construct a SARS-CoV-2 proteome microarray containing 18 out of the 28 predicted proteins and apply it to the characterization of the IgG and IgM antibodies responses in the sera from 29 convalescent patients. We find that all these patients had IgG and IgM antibodies that specifically bind SARS-CoV-2 proteins, particularly the N protein and S1 protein. Besides these proteins, significant antibody responses to ORF9b and NSP5 are also identified. We show that the S1 specific IgG signal positively correlates with age and the level of lactate dehydrogenase (LDH) and negatively correlates with lymphocyte percentage. Overall, this study presents a systemic view of the SARS-CoV-2 specific IgG and IgM responses and provides insights to aid the development of effective diagnostic, therapeutic and vaccination strategies.


Assuntos
Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Análise Serial de Proteínas/métodos , Adulto , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , L-Lactato Desidrogenase/sangue , Masculino , Pessoa de Meia-Idade , Proteínas do Nucleocapsídeo/imunologia , Pandemias , Pneumonia Viral/imunologia , Proteoma/análise , Proteoma/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas não Estruturais Virais/imunologia
11.
PLoS One ; 15(7): e0235904, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32663208

RESUMO

Pancreatic ductal adenocarcinoma is one of the most aggressive types of cancer. Certain proteins in the tumor microenvironment have attracted considerable attention owing to their association with tumor invasion and metastasis. Here, we used proteomics to identify proteins associated with lymph-node metastasis, which is one of the prognostic factors. We selected lymph node metastasis-positive and -negative patients (n = 5 each) who underwent pancreatectomy between 2005 and 2015 and subjected to comprehensive proteomic profiling of tumor stroma. A total of 490 proteins were detected by mass spectrometry. Software analysis revealed that nine of these proteins were differentially expressed between the two patient groups. We focused on hemopexin and ferritin light chain based on immunohistochemistry results. We assessed the clinicopathological data of 163 patients and found that hemopexin expression was associated with UICC N2 (p = 0.0399), lymph node ratio (p = 0.0252), venous invasion (p = 0.0096), and lymphatic invasion (p = 0.0232). Notably, in vitro assays showed that hemopexin promotes invasion of the pancreatic cancer cells. Our findings suggest that hemopexin is a lymph node metastasis-associated protein that could potentially serve as a useful therapeutic target or biomarker of pancreatic ductal adenocarcinoma.


Assuntos
Carcinoma Ductal Pancreático/patologia , Hemopexina/metabolismo , Neoplasias Pancreáticas/patologia , Idoso , Apoferritinas/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Neoplasias Pancreáticas/metabolismo , Prognóstico , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem
12.
Proc Natl Acad Sci U S A ; 117(29): 17094-17103, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611817

RESUMO

Declining ejaculate performance with male age is taxonomically widespread and has broad fitness consequences. Ejaculate success requires fully functional germline (sperm) and soma (seminal fluid) components. However, some aging theories predict that resources should be preferentially diverted to the germline at the expense of the soma, suggesting differential impacts of aging on sperm and seminal fluid and trade-offs between them or, more broadly, between reproduction and lifespan. While harmful effects of male age on sperm are well known, we do not know how much seminal fluid deteriorates in comparison. Moreover, given the predicted trade-offs, it remains unclear whether systemic lifespan-extending interventions could ameliorate the declining performance of the ejaculate as a whole. Here, we address these problems using Drosophila melanogaster. We demonstrate that seminal fluid deterioration contributes to male reproductive decline via mating-dependent mechanisms that include posttranslational modifications to seminal proteins and altered seminal proteome composition and transfer. Additionally, we find that sperm production declines chronologically with age, invariant to mating activity such that older multiply mated males become infertile principally via reduced sperm transfer and viability. Our data, therefore, support the idea that both germline and soma components of the ejaculate contribute to male reproductive aging but reveal a mismatch in their aging patterns. Our data do not generally support the idea that the germline is prioritized over soma, at least, within the ejaculate. Moreover, we find that lifespan-extending systemic down-regulation of insulin signaling results in improved late-life ejaculate performance, indicating simultaneous amelioration of both somatic and reproductive aging.


Assuntos
Envelhecimento , Drosophila melanogaster , Proteínas de Plasma Seminal , Espermatozoides , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Feminino , Fertilidade/genética , Fertilidade/fisiologia , Infertilidade Masculina/genética , Infertilidade Masculina/fisiopatologia , Masculino , Proteoma/análise , Proteoma/genética , Proteoma/fisiologia , Proteínas de Plasma Seminal/análise , Proteínas de Plasma Seminal/fisiologia , Comportamento Sexual Animal/fisiologia , Espermatozoides/química , Espermatozoides/fisiologia
13.
PLoS One ; 15(7): e0236148, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32692761

RESUMO

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an unexplained chronic, debilitating illness characterized by fatigue, sleep disturbances, cognitive dysfunction, orthostatic intolerance and gastrointestinal problems. Using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), we analyzed the plasma proteomes of 39 ME/CFS patients and 41 healthy controls. Logistic regression models, with both linear and quadratic terms of the protein levels as independent variables, revealed a significant association between ME/CFS and the immunoglobulin heavy variable (IGHV) region 3-23/30. Stratifying the ME/CFS group based on self-reported irritable bowel syndrome (sr-IBS) status revealed a significant quadratic effect of immunoglobulin lambda constant region 7 on its association with ME/CFS with sr-IBS whilst IGHV3-23/30 and immunoglobulin kappa variable region 3-11 were significantly associated with ME/CFS without sr-IBS. In addition, we were able to predict ME/CFS status with a high degree of accuracy (AUC = 0.774-0.838) using a panel of proteins selected by 3 different machine learning algorithms: Lasso, Random Forests, and XGBoost. These algorithms also identified proteomic profiles that predicted the status of ME/CFS patients with sr-IBS (AUC = 0.806-0.846) and ME/CFS without sr-IBS (AUC = 0.754-0.780). Our findings are consistent with a significant association of ME/CFS with immune dysregulation and highlight the potential use of the plasma proteome as a source of biomarkers for disease.


Assuntos
Linfócitos B/imunologia , Biomarcadores/sangue , Síndrome de Fadiga Crônica/sangue , Síndrome de Fadiga Crônica/imunologia , Proteoma/análise , Linfócitos B/patologia , Estudos de Casos e Controles , Síndrome de Fadiga Crônica/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Espectrometria de Massas em Tandem
14.
PLoS Negl Trop Dis ; 14(6): e0008299, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511239

RESUMO

Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites.


Assuntos
Bothrops , Venenos de Crotalídeos/análise , Proteoma/análise , Serina Proteases/análise , Animais , Antivenenos , Brasil , Metaloproteases/análise , Isoformas de Proteínas/análise , Proteômica , Mordeduras de Serpentes/terapia
15.
PLoS One ; 15(6): e0231681, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555742

RESUMO

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.


Assuntos
Mucosa Intestinal/metabolismo , Tecido Parenquimatoso/metabolismo , Platelmintos/metabolismo , Proteoma/análise , Proteômica/métodos , Animais , Carpas/parasitologia , Catepsinas/análise , Catepsinas/metabolismo , Cromatografia Líquida de Alta Pressão , Brânquias/parasitologia , Microdissecção e Captura a Laser , Peptídeo Hidrolases/análise , Peptídeo Hidrolases/metabolismo , Espectrometria de Massas em Tandem
16.
Tumour Biol ; 42(6): 1010428320936410, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32586207

RESUMO

Pancreatic ductal adenocarcinoma is the most common and aggressive type of pancreatic cancer, with a 5-year survival rate that is less than 10%. New biomarkers to aid in predicting the prognosis of pancreatic ductal adenocarcinoma patients are needed. Previous proteomic studies have to a great extent focused on finding proteins of value for the diagnosis of pancreatic ductal adenocarcinoma. There is a lack of studies that have profiled the serum or plasma proteome in order to discover candidates for new prognostic biomarkers. In this study, we have used ultra-performance liquid chromatography-ultra-definition mass spectrometry to analyze the serum samples of 21 pancreatic ductal adenocarcinoma patients with short or long survival. Statistical analysis discovered 31 proteins whose expression differed significantly between pancreatic ductal adenocarcinoma patients with short or long survival. Pathway analysis discovered multiple canonical pathways enriched in this data set, with several pathways having roles in inflammation and lipid metabolism. The serum proteins identified here, which include complement components and several enzymes, could be of value as candidates for new noninvasive prognostic markers.


Assuntos
Adenocarcinoma/mortalidade , Biomarcadores Tumorais/metabolismo , Proteínas Sanguíneas/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Neoplasias Pancreáticas/mortalidade , Proteoma/metabolismo , Proteômica/métodos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Biomarcadores Tumorais/análise , Proteínas Sanguíneas/análise , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Projetos Piloto , Prognóstico , Mapas de Interação de Proteínas , Proteoma/análise , Taxa de Sobrevida
17.
Ann Clin Lab Sci ; 50(3): 308-313, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32581017

RESUMO

OBJECTIVE: The COVID-19 pandemic has challenged the world economically and medically. Understanding and defining the biology of this specific coronavirus infection may lead to targeted therapies to lessen its virulence and expand the host resistance. This study's objective was to apply morphoproteomics to pulmonary lung sections from a forensic autopsy of an untreated COVID-19 victim, so that we may better define its biology from the perspective of its interaction with the host and provide options for therapeutic targets. DESIGN: Morphoproteomic analysis from a case study of this COVID-19 pulmonary infection included immunohistochemical probes to detect phosphorylated p-STAT3 (Tyr 705), as part of the interleukin (IL)-6 pathway; cyclooxygenase (COX)-2, CD8+ cytotoxic lymphocytes, Programmed Death (PD)-1 receptor+ lymphoid cells, CD56+ NK lymphoid cells, CD163+ (M2 polarized monocytes/macrophages), and programmed death-ligand 1 (PD-L1) expression as part of the host response to interaction with the COVID-19 virus. RESULTS: Representative sections of the COVID-19 victim's lung showed: nuclear expression of p-STAT3 (Tyr 705) in many of the alveolar pneumocytes and in occasional endothelial cells; COX-2 expression in the alveolar pneumocytes; a relative paucity of CD8+ cytotoxic lymphocytes; absence of CD56+ NK lymphoid cells; abundance of intra-alveolar and alveolar interstitial CD163+ macrophages/monocytes; PD-L1 expression on occasional macrophages, focally on collections of alveolar pneumocytes, and on cells in the alveolar interstitium; and rare PD-1+ lymphocytes in similar regions as CD8+ lymphocytes. CONCLUSION: Morphoproteomics and microanatomical features coincide with the etiopathogenic features of pulmonary coronavirus infection and the host response. This suggests that a targeted therapy could address the biology of COVID-19 pneumonia, enhance the host immune response and prevent its progression to a life-threatening, ventilator-dependent clinical situation.


Assuntos
Betacoronavirus/isolamento & purificação , Biomarcadores/metabolismo , Infecções por Coronavirus/complicações , Pneumopatias/metabolismo , Pneumopatias/patologia , Pneumonia Viral/complicações , Proteoma/análise , Biomarcadores/análise , Infecções por Coronavirus/virologia , Evolução Fatal , Humanos , Pneumopatias/etiologia , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/virologia , Proteoma/metabolismo
18.
Anim Sci J ; 91(1): e13388, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32578273

RESUMO

Serum-based biomarkers hold propitious applications for addressing livestock health, and management. However, discovery of protein biomarkers in complex biological fluids like serum is wholly intractable due to the large dynamic range of protein concentrations; that is, ˜10-12 high abundance proteins constitute >90% of the total protein content and effectively mask proteomic detection of low-abundance biomarkers. Toward addressing this limitation, we test a continuous elution size-based fractionation method, and two approaches that use affinity interaction-based separation of proteins in preparing bovine serum, and compare liquid chromatography tandem mass spectrometry protein identification to neat serum. Our results identify the high-abundance proteins in bovine serum, and demonstrate dynamic range compression and improved protein identification with the different enrichment methods. Although these findings indicate the highest protein number identified in bovine serum (445 proteins, all methods combined), and by any single sample processing method (312 proteins) to date, they still remain lower than levels deemed necessary for biomarker discovery. As such, this investigation revealed limitations to resolving the bovine serum proteome, and the need for species-specific tools for immunodepleting high-abundance proteins. In concert, this study represents a step toward advancing sample preparation methods for bovine serum biomarker identification.


Assuntos
Proteínas Sanguíneas/análise , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Proteoma/análise , Soro/química , Espectrometria de Massas em Tandem/métodos , Animais , Biomarcadores/sangue , Bovinos , Feminino , Manejo de Espécimes/métodos
19.
J Vet Sci ; 21(3): e45, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32476319

RESUMO

BACKGROUND: Feline mammary carcinoma is the third most common cancer that affects female cats. OBJECTIVES: The purpose of this study was to screen differential serum proteins in feline and clarify the relationship between them and the occurrence of feline mammary carcinoma. METHODS: Chinese pastoral cats were used as experimental animals. Six serum samples from cats with mammary carcinoma (group T) and six serum samples from healthy cats (group C) were selected. Differential protein analysis was performed using a Label-free technique, while parallel reaction monitoring (PRM) was performed to verify the screened differential proteins. RESULTS: A total of 82 differential proteins were detected between group T and group C, of which 55 proteins were down regulated and 27 proteins were up regulated. Apolipoprotein A-I, Apolipoprotein A-II (ApoA-II), Apolipoprotein B (ApoB), Apolipoprotein C-III (ApoC-III), coagulation factor V, coagulation factor X, C1q, albumen (ALB) were all associated with the occurrence of feline mammary carcinoma. Differential proteins were involved in a total of 40 signaling pathways, among which the metabolic pathways associated with feline mammary carcinoma were the complement and coagulation cascade and cholesterol metabolism. According to the Label-free results, ApoB, ApoC-III, ApoA-II, FN1, an uncharacterized protein, and ALB were selected for PRM target verification. The results were consistent with the trend of the label-free. CONCLUSIONS: This experimen is the first to confirm ApoA-II and ApoB maybe new feline mammary carcinoma biomarkers and to analyze their mechanisms in the development of such carcinoma in feline.


Assuntos
Proteínas Sanguíneas/análise , Carcinoma/veterinária , Doenças do Gato/sangue , Neoplasias Mamárias Animais/sangue , Proteoma/análise , Animais , Carcinoma/sangue , Gatos , Feminino , Proteômica , Soro/química
20.
Arch Biochem Biophys ; 689: 108476, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585310

RESUMO

BACKGROUND: Proteomics is expected to provide novel insights in the underlying pathophysiology of type 2 diabetes mellitus. In the present study, we aimed to identify and biochemically characterize proteins associated with diabetes mellitus in a Qatari population. METHODS: In a diabetes case-control study (175 cases, 164 controls; Arab, South Asian and Philippine ethnicities), we conducted a discovery study to screen 1141 blood protein levels for associations with diabetes mellitus. Additional analyses were done in controls in relation to Hb1Ac, and biochemical characterization of the main findings was performed with metabolomics (501 metabolites). We performed two-sample Mendelian Randomization to provide evidence of potential causality using data from European descent of the DIAGRAM consortium (74,124 cases of diabetes mellitus and 824,006 controls) for the identified proteins for T2D and Hb1Ac. RESULTS: After accounting for multiple testing, 30 protein levels were different (p-values<8.6e-5) between cases and controls. Of these, a higher Hb1Ac in controls was associated with a lower IGFBP-2 level (p-value = 4.1e-6). IGFBP-2 protein level was found lower among cases compared with controls across all ethnicities. In controls, IGFBP-2 was associated with 21 metabolite levels, but specifically connected to the metabolite citrulline in network analyses. We observed no evidence, however, that the association between IGFBP-2 and diabetes mellitus was causal. CONCLUSIONS: We specifically identified IGFBP-2 to be associated with diabetes mellitus, although with no evidence for causality, which was specifically connected to citrulline metabolism.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Adulto , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/metabolismo , Estudos de Casos e Controles , Citrulina/sangue , Citrulina/metabolismo , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Masculino , Metaboloma , Pessoa de Meia-Idade , Proteoma/análise , Proteoma/metabolismo
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