RESUMO
Trichophyton rubrum and Trichophyton mentagrophytes spp. are two of the most frequently isolated dermatophytes causing dermatophytosis worldwide. Since the incidence of resistance to antifungal agents is increasing, antifungal susceptibility tests are needed to successfully treat dermatophytoses. Most of the methods currently available are complicated, time-consuming and lack of reference procedures. The aim of this work was to establish a simple protocol to test the susceptibility of dermatophytes isolated from clinical samples against five antifungal drugs using E-test and disk diffusion methods. We used the E-test on non-supplemented Mueller-Hinton agar plates to determine the minimum inhibitory concentrations (MICs) of fluconazole, itraconazole, voriconazole and amphotericin B, and disk diffusion method to determine the interpretive MIC of terbinafine. Fifty dermatophytes-10 T. rubrum and 40 T. mentagrophytes spp.-were assessed after only 96 h of colony growth. Terbinafine was the most active antifungal agent with an inhibition diameter greater than 70 mm (sensitivity > 20 mm), followed by voriconazole, itraconazole and amphotericin B with MICs ranging from 0.032 to 0.38 µg/mL, from 0.006 to 0.125 µg/mL and from 0.5 to 1.5 µg/mL, respectively. All isolates were resistant to fluconazole. Collectively, the less laborious E-test and disk diffusion method were shown to be suitable and reliable to determine antifungal sensitivity of dermatophytes. This simple standard protocol could be employed in the routine of clinical laboratories.
Assuntos
Antifúngicos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Trichophyton/efeitos dos fármacos , Ágar , Meios de Cultura , Humanos , Tinha/microbiologia , Trichophyton/crescimento & desenvolvimentoRESUMO
BACKGROUND: There are a limited number of studies investigating the type of serum proteins capable of differentiating intraductal papillary mucinous neoplasms from benign or malignant diseases of the pancreas. AIMS: To select proteins able to differentiate intraductal papillary mucinous neoplasms from benign and malignant pancreatic disease using semiquantitative proteomics. METHODS: Serum samples were obtained from 74 patients (19 with type II intraductal papillary mucinous neoplasms, 8 with type I/III intraductal papillary mucinous neoplasms, 24 with chronic pancreatitis, 23 with pancreatic ductal adenocarcinomas) and 21 healthy subjects. Small proteins and peptides were assayed by matrix-assisted laser desorption/ionization for the detection of differentially abundant species possibly related to tumor onset. Serum pancreatic amylase, lipase, carcinoembryonic antigen and carbohydrate antigen 19-9 (CA 19-9) were also assayed. RESULTS: Twenty-six of 84 peaks detected were dysregulated (7 more abundant and 19 less abundant in the type II intraductal papillary mucinous neoplasms, p < 0.05). Of the differentially abundant peaks, 17 were commonly dysregulated (3 peaks more abundant and 13 less abundant in type II intraductal papillary mucinous neoplasms, and one at m/z = 9961 at variance), indicating a protein fingerprint shared by types I/III and type II intraductal papillary mucinous neoplasms and pancreatic ductal adenocarcinomas. CONCLUSIONS: These results suggest that our approach can be used to differentiate type II intraductal papillary mucinous neoplasms from type I/III neoplasms, and type II intraductal papillary mucinous neoplasms from pancreatic ductal adenocarcinomas.
Assuntos
Adenocarcinoma/diagnóstico , Carcinoma Ductal Pancreático/diagnóstico , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/diagnóstico , Pancreatite Crônica/sangue , Pancreatite Crônica/diagnóstico , Adenocarcinoma/sangue , Idoso , Biomarcadores/sangue , Carcinoma Ductal Pancreático/sangue , Estudos de Casos e Controles , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
NEU3 sialidase has been shown to be a key player in many physio- and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking.
Assuntos
Neuraminidase/metabolismo , Membrana Celular/enzimologia , Chaperona BiP do Retículo Endoplasmático , Endossomos/enzimologia , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Neuraminidase/química , Neuraminidase/genética , Dobramento de Proteína , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , Regulação para CimaRESUMO
In the skeletal muscle, the ageing process is characterized by a loss of muscle mass and strength, coupled with a decline of mitochondrial function and a decrease of satellite cells. This profile is more pronounced in hindlimb than in forelimb muscles, both in humans and in rodents. Utilizing light and electron microscopy, myosin heavy chain isoform distribution, proteomic analysis by 2D-DIGE, MALDI-TOF MS and quantitative immunoblotting, this study analyzes the protein levels and the nuclear localization of specific molecules, which can contribute to a preferential muscle loss. Our results identify the molecular changes in the hindlimb (gastrocnemius) and forelimb (triceps) muscles during ageing in rats (3- and 22-month-old). Specifically, the oxidative metabolism contributes to tissue homeostasis in triceps, whereas respiratory chain disruption and oxidative-stress-induced damage imbalance the homeostasis in gastrocnemius muscle. High levels of dihydrolipoyllysine-residue acetyltransferase (Dlat) and ATP synthase subunit alpha (Atp5a1) are detected in triceps and gastrocnemius, respectively. Interestingly, in triceps, both molecules are increased in the nucleus in aged rats and are associated to an increased protein acetylation and myoglobin availability. Furthermore, autophagy is retained in triceps whereas an enhanced fusion, decrement of mitophagy and of regenerative potential is observed in aged gastrocnemius muscle.
Assuntos
Envelhecimento , Proteínas Musculares/análise , Músculo Esquelético/patologia , Doenças Musculares/patologia , Animais , Autofagia , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Doenças Musculares/metabolismo , Cadeias Pesadas de Miosina/análise , Cadeias Pesadas de Miosina/metabolismo , Proteômica , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletroforese em Gel Diferencial BidimensionalRESUMO
Sphingolipids have hydrophilic and hydrophobic properties, different saturation and combination of the oligosaccharide chains and mass homology of species located in a narrow m/z region hampering their recognition. To target sphingolipids for diagnostic purposes, standardized methods for lipid extraction, quali- and quantitative assessments are required. In this study, HPTLC-MALDI MS was adopted to establish sphingolipid and glycosphingolipid profiles in muscle, brain and serum to create a database of molecules to be searched in the preclinical and clinical investigations. Specific protocols for lipid extraction were set up based on the characteristics of the tissue or/and fluids; this approach maximizes the HPTLC-MALDI MS analytical throughput both for lipids extracted in organic and aqueous phase. This study indicates that alkaline hydrolysis is necessary for the detection of low abundant species such as Gb3Cer and ceramides in serum and Gb4Cer, CerP and HexCer in muscle tissue. The high hydrophobicity of ceramides has been overcome by the development of HPTLC plate in chloroform:methanol/50:3.5, which increases the number and the intensity of low abundant Cer species. MS/MS analysis has been conducted directly on HPTLC plate allowing the molecular recognition; furthermore a dataset of spectra was acquired to create a database for future profiling of these molecules.
Assuntos
Biomarcadores/metabolismo , Cromatografia em Camada Fina/métodos , Glicoesfingolipídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biomarcadores/sangue , Encéfalo/metabolismo , Glicoesfingolipídeos/sangue , Hidrólise , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismoRESUMO
Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.
Assuntos
Biomarcadores/metabolismo , Diferenciação Celular , Gangliosídeos/metabolismo , Células-Tronco/metabolismo , Fosfatase Alcalina/genética , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Derme/citologia , Relação Dose-Resposta a Droga , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Gangliosídeos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Osteopontina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingolipídeos/metabolismo , Células-Tronco/citologiaRESUMO
Glycosphingolipids (GSLs) are a class of ubiquitous lipids characterized by a wide structural repertoire and a variety of functional implications. Importantly, altered levels have been correlated with different diseases, suggesting their crucial role in health. Conventional methods for the characterization and quantification are based on high-performance TLC (HPTLC) separation and comparison with the migration distance of standard samples or on MS. We set up and herein report the application of an ImagePrep method for glycosphingolipids qualitative and quantitative profiling through direct HPTLC-MALDI with particular application to wild-type and NEU3 sialidase-overexpressing C2C12 myoblasts. Lipids were analyzed by HPTLC, coupled with MALDI-TOF, and the resulting GSLs profiles were compared to the [³H]sphingolipids HPTLC patterns obtained after metabolic radiolabeling. GSLs detection by HPTLC-MALDI was optimized by testing different methods for matrix delivery and by performing quantitative analyses using serial dilutions of GSLs standards. Through this approach an accurate analysis of each variant of neutral and acidic GSLs, including the detection of different fatty-acid chain variants for each GSL, was provided and these results demonstrated that HPTLC-MALDI is an easy and high-throughput analytical method for GSLs profiling, suggesting its use for an early detection of markers in different diseases, including cancer and heart ischemia.
Assuntos
Glicoesfingolipídeos Acídicos/análise , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia em Camada Fina/métodos , Neuraminidase/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Glicoesfingolipídeos Acídicos/metabolismo , Animais , Área Sob a Curva , Linhagem Celular , Modelos Lineares , Camundongos , MioblastosRESUMO
Context: Insulin resistance, glucose alterations, arterial hypertension (HTN), and the renin-angiotensin-aldosterone system (RAAS) are related in adult obesity. This crosstalk is still unexplored in childhood. Objective: Characterize the relationships of fasting and postload glucose and insulin levels with new American Academy of Pediatrics classification of HTN and RAAS in pediatric obesity. Methods: This was a retrospective observational study; 799 pediatric outpatients (11.4 ± 3.1 years) at a tertiary center who were overweight or obese and not yet on diet were included. The main outcome measures were mean and correlations among parameters of a complete clinical and metabolic screening (body mass index, blood pressure, and glucose and insulin levels during an oral glucose tolerance test, and renin and aldosterone levels and their ratio). Results: 774 subjects had all the parameters, of whom 87.6% had HTN (5% elevated blood pressure, 29.2% stage I HTN, and 53.4% stage II HTN). Eighty subjects had 1 or more glucose alterations, and more frequently presented HTN. Blood pressure levels were higher in subjects with glucose alterations than in those with normal glucose levels. Fasting and stimulated glucose and insulin levels were directly related to the HTN stages, and insulin sensitivity was lower in HTN than in normal blood pressure. Aldosterone, renin, and aldosterone-renin ratio (ARR) were similar in sexes, whereas aldosterone was higher in prepubertal individuals. Subjects with impaired glucose tolerance (IGT) had higher renin and lower ARR. Renin was positively correlated with postload glucose, and ARR was negatively correlated with the Homeostatic Model Assessment for Insulin Resistance index. Conclusion: A close relationship exists among insulin resistance, glucose alterations, HTN, and renin in childhood obesity. Specific categories of risk could provide indicators for strict clinical surveillance.
RESUMO
The synthetic purine reversine has been shown to possess a dual activity as it promotes the de-differentiation of adult cells, including fibroblasts, into stem-cell-like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti-cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced.
Assuntos
Antineoplásicos/farmacologia , Morfolinas/farmacologia , Purinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Benzotiazóis/farmacologia , Western Blotting , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular , Morte Celular , Desdiferenciação Celular , Proliferação de Células , Forma Celular/efeitos dos fármacos , Sobrevivência Celular , Endorreduplicação , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Citometria de Fluxo , Inativação Gênica , Células HeLa , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1α), energy balance (AMPK), mitochondrial biogenesis (PGC-1α), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2-D DIGE, mass spectrometry and antigen-antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF-1α stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up-regulation of adenosylhomocysteinase and acetyl-CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC-1α, a master regulator of mitochondrial biogenesis, was down-regulated. CH was characterized by the up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a general down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic molecules increased accompained by down-regulation of specific target proteins.
Assuntos
Hipóxia/metabolismo , Miocárdio/metabolismo , Proteoma/metabolismo , Animais , Apoptose , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Immunoblotting , Camundongos , Proteoma/genética , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletroforese em Gel Diferencial BidimensionalRESUMO
The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology although the serum high dynamic range imposes a major limitation hampering the identification of less abundant species decreasing the quality of MALDI profiling. A setup to improve these parameters has been performed for recombinant human erythropoietin (rhEPO) monitoring in serum, analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α-cyano-4-hydroxycinnamic acid and 2',4'-dihydroxyacetophenone) for peak quality improvement. The immunodepletion capability of both columns was determined by 2-D DIGE, which precisely revealed the efficacy of Hu14 in protein removal and the serum dynamic range decrement. In addition, the type of matrix, the sample dilution, and the efficacy of optimized parameters were used for serum profiling of ten healthy subjects before and after rhEPO treatment. The principal component analysis indicates that a combination of Hu14 column and 2',4'-dihydroxyacetophenone matrix increases data quality allowing the discrimination between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhEPO.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Eritropoetina/sangue , Técnicas de Imunoadsorção/instrumentação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Monitoramento de Medicamentos , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , Análise de Componente Principal , Proteínas RecombinantesRESUMO
The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2-D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (â¼50:50%) head of the quadriceps and in the calf soleus (SOL), a predominantly slow (â¼35:65%) twitch muscle. Two separate studies were performed on six subjects without (BR) and six with resistive vibration exercise (RVE) countermeasures, respectively. Both VL and SOL underwent in BR decrements of â¼15% in cross-sectional area and of â¼22% in maximal torque that were prevented by RVE. Myosin heavy chain distribution showed increased type I and decreased type IIA in BR both in VL and in SOL, the opposite with RVE. A substantial downregulation of proteins involved in aerobic metabolism characterized both in SOL and VL in BR. RVE reversed the pattern more in VL than in SOL, whereas proteins involved in anaerobic glycolysis were upregulated. Proteins from the Z-disk region and from costamers were differently dysregulated during bed rest (both BR and RVE), particularly in VL.
Assuntos
Repouso em Cama/efeitos adversos , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Cadeias Leves de Miosina/metabolismo , Proteômica/métodos , Vibração/uso terapêutico , Adulto , Eletroforese em Gel Bidimensional , Terapia por Exercício/métodos , Humanos , Immunoblotting , Masculino , Cadeias Pesadas de Miosina/química , Cadeias Leves de Miosina/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Músculo Quadríceps/metabolismo , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
Ageing induces a progressive morphological change and functional decline in muscles and in nerves. Light and electron microscopy, 2-D DIGE and MS, were applied to profile the qualitative and quantitative differences in the proteome and morphology of rat gastrocnemius muscle and sciatic nerve, in healthy 22-month-old rats. At muscle level, morphological changes are associated to fibre atrophy accompanied by myofibrillar loss and degeneration, disappearance of sarcomeres and sarcoplasmic reticulum dilatation, internal migration of nuclei, longitudinal fibre splitting, increment of subsarcolemmal mitochondria aggregates and increment of lipofuscin granules. Sciatic nerve shows myelin abnormalities like enfoldings, invaginations, onion bulbs, breakdowns and side axonal atrophy. Proteomic analysis identified changes correlated to morphological abnormalities in metabolic, contractile and cytoskeletal proteins, deregulation of iron homeostasis, change of Ca(2+) balance and stress response proteins, accompanied by a deregulation of myelin membrane adhesion protein and proteins regulating the neuronal caliber. By comparing proteomic results from the two tissues, 16 protein isoforms showed the same up and down regulation trend suggesting that there are changes implying a general process which may act as a signal event of degeneration. Only beta enolase and tropomyosin 1alpha were differentially expressed in the tissues.
Assuntos
Envelhecimento/metabolismo , Perfilação da Expressão Gênica/métodos , Expressão Gênica , Músculo Esquelético , Nervo Isquiático , Animais , Proteínas de Transporte/metabolismo , Proteínas Contráteis/metabolismo , Proteínas do Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Masculino , Espectrometria de Massas , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Cadeias Pesadas de Miosina/metabolismo , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Nervo Isquiático/metabolismo , Nervo Isquiático/ultraestrutura , Transdução de SinaisRESUMO
Recent advances in stem cell biology have demonstrated that terminally differentiated adult cells can be induced to de-differentiate into progenitor cells (induced stem cells) upon proper stimuli. This has been achieved by the induced expression of key regulatory genes by retro- or lenti-viral systems. On the other hand, synthetic "small molecules" can also induce de-differentiation and may represent a potentially safer approach as compared with genetic manipulation. Along this line, a synthetic purine called "reversine" has been shown to induce the de-differentiation of fibroblasts into mesenchymal stem-cell-like progenitors, which can be successively induced to differentiate into skeletal muscle, smooth muscle and bone cells. The mechanism whereby reversine is able to achieve de-differentiation has yet to be clarified. In this context, we defined the protein changes induced by reversine treatment in murine fibroblasts by 2-D difference gel electrophoresis, coupled with MS. Proteins involved in cytoskeletal and cell shape remodeling, RNA export, degradation, folding, stress control and ATP production were found to be remarkably changed after reversine treatment. Ingenuity pathway analysis (IPA) predicted that these protein pattern changes enabled to propose that about 40 proteins might be associated to several biological functional networks, including cellular assembly, cell signaling and cell death. Altogether our data confirm the intrinsic complexity of the de-differentiation process induced by reversine and suggest more selected approaches to investigate the action mechanism of this small molecule.
Assuntos
Comunicação Celular/efeitos dos fármacos , Eletroforese em Gel Bidimensional/métodos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Espectrometria de Massas/métodos , Morfolinas/farmacologia , Purinas/farmacologia , Animais , Anexinas/metabolismo , Ciclo Celular/efeitos dos fármacos , Desdiferenciação Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/citologia , Gluconeogênese/efeitos dos fármacos , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Proteoma/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: The use of phase contrast in urinalysis has been highly recommended. A new system, sediMAX conTRUST PRO, is now available providing simultaneous automated phase contrast and bright field microscopy. This study aimed to evaluate both analytical and diagnostic performance of this new analyzer. METHODS: Results from 504 samples evaluated with the sediMAX conTRUST PRO were compared to those obtained from the same samples by manual microscopy (MM). Analytical and diagnostic performance were assessed according to established guidelines. RESULTS: The concentration of red blood cells (RBCs)and white blood cells (WBCs) at which the LoQ satisfied a CV<â¯25% was 12 particles per µL (p/µL) and 8â¯p/µL, respectively. Within one grade of agreement concordance was quite high, 97.8% for RBCs and 98.0% for WBCs, and above 90% for all other particles. Overall, diagnostic sensitivity and specificity were good (>80%) for the particles considered, although lower sensitivities, 70.6% and 61.8%, were respectively found for hyaline and pathological casts. CONCLUSIONS: The sediMAX conTRUST PRO provides very good performance in terms of RBC and WBC recognition and enumeration, and quite good performance for all other particles. Hyaline cast and pathological cast identification is fine and comparable to other automated systems, but could use further improvement.
Assuntos
Microscopia de Contraste de Fase , Urinálise/instrumentação , Automação , Humanos , Limite de Detecção , Modelos LinearesRESUMO
INTRODUCTION: Chest pain and its clinical manifestations are the most common reasons for presentation to the emergency department (ED). Given that the prevalence of chest pain due to acute myocardial infarction (AMI) in the ED is modest, clinicians should use cardiac troponins to safely and rapidly rule out AMI, avoiding the delayed release of low risk patients. The study aims to develop and validate an algorithm to early rule-out of non-ST elevation myocardial infarction (NSTEMI) in subjects admitted to the ED with symptoms of myocardial infarction. METHODS: High sensitivity cardiac Troponin T (hs-cTnT) serial measurements (baseline, T0; after 1â¯h, T1; after 3â¯h, T3) were used to develop and validate the algorithm, respectively, in 6403 and 773 consecutive admissions suggestive of AMI. RESULTS: Patients were classified as having or not having NSTEMI according to clinical assessment, diagnostic imaging, and serial measurements ofhs-cTnT; ROC curve analysis allowed to find changes in consecutive hs-cTnT associated with diagnostic sensitivity close to 100%. Only patients with hs-cTnTat T0 lower than 14â¯ng/L resultedto be eligible for the safe rule-out of NSTEMI. CONCLUSIONS: Although some points remain to be improved, the results obtained indicate that algorithms for fast NSTEMI rule-out are feasible and safe.
Assuntos
Algoritmos , Análise Química do Sangue/métodos , Serviço Hospitalar de Emergência , Limite de Detecção , Infarto do Miocárdio sem Supradesnível do Segmento ST/sangue , Infarto do Miocárdio sem Supradesnível do Segmento ST/diagnóstico , Troponina T/sangue , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Admissão do Paciente , Curva ROC , Fatores de TempoRESUMO
After hind limb suspension, a remodeling of postural muscle phenotype is observed. This remodeling results in a shift of muscle profile from slow-oxidative to fast-glycolytic. These metabolic changes and fiber type shift increase muscle fatigability. Acetyl-L-carnitine (ALCAR) influences the skeletal muscle phenotype of soleus muscle suggesting a positive role of dietary supplementation of ALCAR during unloading. In the present study, we applied a 2-D DIGE, mass spectrometry and biochemical assays, to assess qualitative and quantitative differences in the proteome of rat slow-twitch soleus muscle subjected to disuse. Meanwhile, the effects of ALCAR administration on muscle proteomic profile in both unloading and normal-loading conditions were evaluated. The results indicate a modulation of troponin I and tropomyosin complex to regulate fiber type transition. Associated, or induced, metabolic changes with an increment of glycolytic enzymes and a decreased capacity of fat oxidation are observed. These metabolic changes appear to be counteracted by ALCAR treatment, which restores the mitochondrial mass and decreases the glycolytic enzyme expression, suggesting a normalization of the metabolic shift observed in unloaded animals. This normalization is accompanied by a maintenance of body weight and seems to prevent a switch of fiber type.
Assuntos
Acetilcarnitina/farmacologia , Elevação dos Membros Posteriores/fisiologia , Proteínas Musculares/efeitos dos fármacos , Músculo Esquelético/fisiologia , Animais , Eletroforese em Gel Bidimensional , Masculino , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
In the diagnosis of Alzheimer's disease (AD) total tau (T-tau), tau phosphorylated at threonine 181 (P-tau181), and the 42 amino acid isoform of alpha ß-amyloid (Aß) are well established surrogate CSF markers. However, there is a constant need for new diagnostic markers to identify the disease at a very early stage. The identification of new molecules for AD diagnosis and monitoring in CSF is hampered by several "confounding" factors including intra- and inter-individual, pre-analytical and analytical variabilities. In an attempt to partially overcome patient's variability and to determine new molecules significantly dysregulated in CSF, we assessed the proteome profile of low molecular weight protein species in CSF and serum of the same patients. CSFs and sera from 36 ADs, 32 iNPHs (idiopathic normal pressure hydrocephalus) and 12 controls were compared by MALDI profiling (non-parametric statistics, CV<20%, AUC>0.750). After protein identification by mass spectrometry, the proteoform composition was assessed by 2-D DIGE/MS. Results indicated that CSF of iNPH can be used as control. Serum and CSF of AD patients shows a specific protein profile compared to iNPH samples. A variation (p<0.01) of Apo A-1 levels in AD, together with a specific dysregulation of Apo A-1 proteoforms was observed. The profiling of CSF and serum of the same patients, suggests that the decrement of total Apo A-1 occurs specifically in CSF. Serum and CSF of AD shows a characteristic Apo A-1 proteoform pattern suggesting it as potential marker which can support the clinical workflow adopted for AD diagnosis and progression.
Assuntos
Doença de Alzheimer/diagnóstico , Apolipoproteína A-I/líquido cefalorraquidiano , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Apolipoproteína A-I/sangue , Apolipoproteína A-I/metabolismo , Área Sob a Curva , Estudos de Casos e Controles , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Hidrocefalia/sangue , Hidrocefalia/líquido cefalorraquidiano , Hidrocefalia/diagnóstico , Masculino , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Fosforilação , Análise de Componente Principal , Curva ROC , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Estatísticas não Paramétricas , Proteínas tau/líquido cefalorraquidianoRESUMO
In mammals, hypoxic stress management is under the control of the Hypoxia Inducible Factors, whose activity depends on the stabilization of their labile α subunit. In particular, the skeletal muscle appears to be able to react to changes in substrates and O2 delivery by tuning its metabolism. The present study provides a comprehensive overview of skeletal muscle metabolic adaptation to hypoxia in mice and in human subjects exposed for 7/9 and 19 days to high altitude levels. The investigation was carried out combining proteomics, qRT-PCR mRNA transcripts analysis, and enzyme activities assessment in rodents, and protein detection by antigen antibody reactions in humans and rodents. Results indicate that the skeletal muscle react to a decreased O2 delivery by rewiring the TCA cycle. The first TCA rewiring occurs in mice in 2-day hypoxia and is mediated by cytosolic malate whereas in 10-day hypoxia the rewiring is mediated by Idh1 and Fasn, supported by glutamine and HIF-2α increments. The combination of these specific anaplerotic steps can support energy demand despite HIFs degradation. These results were confirmed in human subjects, demonstrating that the TCA double rewiring represents an essential factor for the maintenance of muscle homeostasis during adaptation to hypoxia.