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1.
BMC Bioinformatics ; 21(1): 376, 2020 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-32867673

RESUMO

BACKGROUND: Two-dimensional gel electrophoresis (2-DGE) is a commonly used tool for proteomic analysis. This gel-based technique separates proteins in a sample according to their isoelectric point and molecular weight. 2-DGE images often present anomalies due to the acquisition process, such as: diffuse and overlapping spots, and background noise. This study proposes a joint pre-processing framework that combines the capabilities of nonlinear filtering, background correction and image normalization techniques for pre-processing 2-DGE images. Among the most important, joint nonlinear diffusion filtering, adaptive piecewise histogram equalization and multilevel thresholding were evaluated using both synthetic data and real 2-DGE images. RESULTS: An improvement of up to 46% in spot detection efficiency was achieved for synthetic data using the proposed framework compared to implementing a single technique of either normalization, background correction or filtering. Additionally, the proposed framework increased the detection of low abundance spots by 20% for synthetic data compared to a normalization technique, and increased the background estimation by 67% compared to a background correction technique. In terms of real data, the joint pre-processing framework reduced the false positives up to 93%. CONCLUSIONS: The proposed joint pre-processing framework outperforms results achieved with a single approach. The best structure was obtained with the ordered combination of adaptive piecewise histogram equalization for image normalization, geometric nonlinear diffusion (GNDF) for filtering, and multilevel thresholding for background correction.


Assuntos
Eletroforese em Gel Bidimensional/métodos , Bases de Dados de Proteínas , Humanos , Processamento de Imagem Assistida por Computador , Proteínas/análise , Proteômica/métodos , Razão Sinal-Ruído
2.
Life Sci ; 259: 118193, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32763293

RESUMO

AIMS: Circulating long non-coding RNAs (lncRNAs) have proven to be useful non-invasive tools for diagnosis of various cancers. FAM83H antisense RNA 1 (FAM83H-AS1) and lncRNA activated by TGF ß (lncRNA-ATB) are two lncRNAs that have been shown to play an important role in different cancer types including breast cancer. The primary aim of our study was to investigate the potential role of serum FAM83H-AS1 and lncRNA-ATB as diagnostic/prognostic markers for breast cancer patients. MAIN METHODS: Serum expression levels of FAM83H-AS1 and lncRNA-ATB were analyzed in 90 breast cancer patients and 30 age- and sex-matched healthy controls using RT-qPCR. KEY FINDINGS: We found that FAM83H-AS1 and lncRNA-ATB were significantly overexpressed in sera of breast cancer patients compared to controls (p = 0.000 for both). Analysis of receiver operating characteristic curve demonstrated that lncRNA-ATB had a higher area under curve (AUC) value than the conventional tumor marker cancer antigen 15-3 (CA15-3) (AUC: 0.844, p = 0.000 versus 0.738, p = 0.002) for early diagnosis of breast cancer in patients with stage I-II. On the other hand, FAM83H-AS1 showed a significant correlation with tumor-node metastasis (TNM) stages, large tumor size and lymph node metastasis, suggesting a prognostic rather than diagnostic value. SIGNIFICANCE: This is the first study to demonstrate that serum lncRNA-ATB could be used as a non-invasive diagnostic marker for early stages of breast cancer. Furthermore, serum FAM83H-AS1 has a potential ability for monitoring of progression and staging of breast cancer.


Assuntos
Biomarcadores/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Proteínas/análise , RNA Longo não Codificante/sangue , Adulto , Idoso , Diagnóstico Precoce , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Mucina-1/sangue , Valor Preditivo dos Testes , Prognóstico , RNA Antissenso , Curva ROC , Fator de Crescimento Transformador beta
3.
Opt Express ; 28(12): 18479-18492, 2020 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-32680046

RESUMO

Biomolecule sensing plays an important role in both fundamental biological studies and medical diagnostic applications. Infrared (IR) spectroscopy presents opportunities for sensing biomolecules as it allows their fingerprints to be determined by directly measuring their absorption spectra. However, the detection of biomolecules at low concentrations is difficult with conventional IR spectroscopy due to signal-to-noise considerations. This has led to recent interest on the use of nanostructured surfaces to boost the signals from biomolecules in a method termed surface enhanced infrared spectroscopy. So far, efforts have largely involved the use of metallic nanoantennas (which produce large field enhancement) or graphene nanostructures (which produce strong field confinement and provide electrical tunability). Here, we propose a nanostructured surface that combines the large field enhancement of metallic nanoantennas with the strong field confinement and electrical tunability of graphene plasmons. Our device consists of an array of plasmonic nanoantennas and graphene nanoslits on a resonant substrate. We perform systematic electromagnetic simulations to quantify the sensing performance of the proposed device and show that it outperforms designs in which only plasmons from metallic nanoantennas or plasmons from graphene are utilized. These investigations consider the model system of a representative protein-goat anti-mouse immunoglobulin G (IgG) - in monolayer or sub-monolayer form. Our findings provide guidance for future biosensors for the sensitive quantification and identification of biomolecules.


Assuntos
Grafite , Nanopartículas Metálicas , Espectrofotometria Infravermelho/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Desenho de Equipamento/métodos , Proteínas/análise , Ressonância de Plasmônio de Superfície/métodos
4.
PLoS One ; 15(7): e0235793, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634162

RESUMO

Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi's sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.


Assuntos
Fracionamento Celular/métodos , Células Endoteliais/química , Vesículas Extracelulares/química , Biomarcadores/análise , Centrifugação/métodos , Precipitação Química , DNA/análise , Células Endoteliais/virologia , Vesículas Extracelulares/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas/análise , RNA/análise
5.
PLoS One ; 15(7): e0235838, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678854

RESUMO

We aimed to explore the proteomic profiles of mid-trimester amniotic fluid in pregnant women with systemic lupus erythematosus (SLE) according to the occurrence of adverse pregnancy outcome (APO). The study population included 35 pregnant women with SLE who underwent clinically indicated amniocentesis at 15-24 weeks of gestation. Patients were divided into two groups according to pregnancy outcomes: SLE patients without APO (Group 1) and SLE patients with APO (Group 2). Stored samples of amniotic fluid were analyzed using mass spectrometry (MS)-based proteomics with two-step approach, consisting of discovery and verification phase. In the discovery phase, 44 proteins were differentially expressed between Group 1 and Group 2. In the verification phase, differentially expressed proteins (DEPs) were verified in independent samples using DIA method. Four proteins including filamin A (FLNA), sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1), lecithin-cholesterol acyltransferase (LCAT), and transglutaminase 2 (TGM2) were differentially expressed both in discovery and verification phase. To select the best combination of proteins for discriminating two groups, three-fold cross validation (CV) with repetition of one hundred times was performed. The multi-marker model with three biomarkers (SVEP1, LCAT, TGM2) had a high discriminatory power to distinguish between the two groups (the area under the receiver operating characteristic, AUROC = 0.946, p <0.001). Our results indicate that the expression of FLNA, SVEP1, LCAT, and TGM2 in mid-trimester amniotic fluid was increased in SLE patients with APO (Group 2). A large-scale prospective study is warranted to verify this finding.


Assuntos
Líquido Amniótico/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Proteínas/análise , Adulto , Amniocentese , Biomarcadores/análise , Biomarcadores/metabolismo , Feminino , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Gravidez , Resultado da Gravidez , Trimestres da Gravidez , Prognóstico , Proteínas/metabolismo , Proteômica , Estudos Retrospectivos
6.
PLoS One ; 15(7): e0234993, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645014

RESUMO

The main functions of the choroid plexus (CP) are the production of cerebral spinal fluid (CSF), the formation of the blood-CSF barrier, and regulation of immune response. This barrier allows for the exchange of specific nutrients, waste, and peripheral immune cells between the blood stream and CSF. Borrelia burgdorferi (Bb), the causative bacteria of Lyme disease, is associated with neurological complications including meningitis-indeed, Bb has been isolated from the CSF of patients. While it is accepted that B. burgdorferi can enter the central nervous system (CNS) of patients, it is unknown how the bacteria crosses this barrier and how the pathogenesis of the disease leads to the observed symptoms in patients. We hypothesize that during infection Borrelia burgdorferi will induce an immune response conducive to the chemotaxis of immune cells and subsequently lead to a pro-inflammatory state with the CNS parenchyma. Primary human choroid plexus epithelial cells were grown in culture and infected with B. burgdorferi strain B31 MI-16 for 48 hours. RNA was isolated and used for RNA sequencing and RT-qPCR validation. Secreted proteins in the supernatant were analyzed via ELISA. Transcriptome analysis based on RNA sequencing determined a total of 160 upregulated genes and 98 downregulated genes. Pathway and biological process analysis determined a significant upregulation in immune and inflammatory genes specifically in chemokine and interferon related pathways. Further analysis revealed downregulation in genes related to cell to cell junctions including tight and adherens junctions. These results were validated via RT-qPCR. Protein analysis of secreted factors showed an increase in inflammatory chemokines, corresponding to our transcriptome analysis. These data further demonstrate the role of the CP in the modulation of the immune response in a disease state and give insight into the mechanisms by which Borrelia burgdorferi may disseminate into, and act upon, the CNS. Future experiments aim to detail the impact of B. burgdorferi on the blood-CSF-barrier (BCSFB) integrity and inflammatory response within animal models.


Assuntos
Borrelia burgdorferi/patogenicidade , Plexo Corióideo/patologia , Células Epiteliais/patologia , Doença de Lyme/microbiologia , Barreira Hematoencefálica , Borrelia burgdorferi/imunologia , Células Cultivadas , Plexo Corióideo/imunologia , Plexo Corióideo/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Inflamação/metabolismo , Doença de Lyme/imunologia , Doença de Lyme/patologia , Proteínas/análise , RNA/análise
7.
An Acad Bras Cienc ; 92 Suppl 1: e20180757, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32491136

RESUMO

Pereskia grandifolia Haworth (PGH) and Pereskia aculeata Miller (PAM) are recognized sources of proteins; dietary fiber; vitamins and minerals make this plant leaves, raw, cooked, and braised, an important ally against protein and micronutrient deficiencies. One of the main problems is the presence of antinutritional factors that may interfere in the digestibility and bioavailability of some nutrients. The objective was to evaluate the amino acid profile and the chemical score of the raw leaves and the effects of heating media and time on the total dietary fiber, minerals, trypsin inhibition, oxalic acid and tannins of leaves of PGH and PAM. The samples had similar amino acid profiles and total dietary fiber. With regard to antinutritional compounds, heating the leaves of PGH led to a decrease in trypsin inhibition, primarily after the first minutes of wet cooking. Oxalic acid and tannins predominated in both species. Considering the interaction with time, the variables related to iron and zinc minimized the tannin responses in PGH and PAM, respectively. Heating media and times interfered with the chemical components present in the leaves of Pereskia species and led to high antinutrient retention after heat treatment.


Assuntos
Cactaceae/química , Valor Nutritivo , Verduras/química , Aminoácidos/análise , Cactaceae/classificação , Fibras na Dieta/análise , Manipulação de Alimentos , Minerais/análise , Proteínas/análise , Taninos/análise , Verduras/classificação
8.
PLoS One ; 15(5): e0233513, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437403

RESUMO

Due to climate change, numerous ice bodies have been lost in the West Antarctic Peninsula (WAP). As a consequence, deglaciation is expected to impact the marine environment and its biota at physiological and ecosystem levels. Nuculana inaequisculpta is a marine bivalve widely distributed around Antarctica that plays an important role for ecosystem functioning. Considering that N. inaequisculpta inhabits coastal areas under effect of glacial melt and retreat, impacts on its nutritional condition are expected due to alterations on its physiology and food availability. To test this hypothesis, biochemical composition (lipids, proteins, and fatty acids) and energy content were measured in individuals of N. inaequisculpta collected in a fjord at different distances to the retreating glacier in the WAP. Oceanographic parameters of the top and bottom-water layers (temperature, salinity, dissolved oxygen, and chlorophyll-a) were measured to investigate how the environment changes along the fjord. Results showed that surface oceanographic parameters displayed a lower temperature and dissolved oxygen, but a higher salinity and chlorophyll-a content at nearest compared to farthest sites to the glacier. In contrast, a lower temperature and chlorophyll-a, and a higher salinity and dissolved oxygen was measured in the bottom-water layer toward the glacier. N. inaequisculpta had a higher amount of lipids (17.42 ± 3.24 vs. 12.16 ± 3.46%), protein (24.34 ± 6.12 vs. 21.05 ± 2.46%) and energy content (50.57 ± 6.97 J vs. 39.14 ± 5.80 J) in the farthest compared to the nearest site to the glacier. No differences were found in total fatty acids among all sites. It seems likely that lower individual fitness related to proximity to the glacier would not be related to nutritional quality of sediment food, but rather to food quantity.


Assuntos
Bivalves/fisiologia , Mudança Climática , Camada de Gelo , Fenômenos Fisiológicos da Nutrição Animal , Animais , Regiões Antárticas , Bivalves/química , Ecossistema , Estuários , Ácidos Graxos/análise , Camada de Gelo/química , Lipídeos/análise , Proteínas/análise
9.
Nihon Yakurigaku Zasshi ; 155(3): 155-158, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32378634

RESUMO

Histidine-rich glycoprotein (HRG) is a 75 kDa plasma glycoprotein synthesized in liver mainly, which exists at approximately 60-100 µg/ml in human plasma. HRG is known to bind to several ligands and cells, leading to exert coagulation, fibrinolysis, immune and inflammation regulatory activity in septic condition. Thus, decreased plasma HRG level induces the dysregulations of coagulation, fibrinolysis and immune system, resulting in disseminated intravascular coagulation and multiple organ failure. This article focuses on the physiological activity of HRG and the potential of HRG as the biomarker and therapeutic drug for sepsis.


Assuntos
Proteínas Sanguíneas/análise , Proteínas Sanguíneas/fisiologia , Proteínas/análise , Proteínas/fisiologia , Sepse/diagnóstico , Biomarcadores/sangue , Coagulação Sanguínea , Humanos
10.
Food Chem ; 324: 126664, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32380410

RESUMO

Authentication of meat products is critical in the food industry. Meat adulteration may lead to religious apprehensions, financial gain and food-toxicities such as meat allergies. Thus, empirical validation of the quality and constituents of meat is paramount. Various analytical methods often based on protein or DNA measurements are utilized to identify meat species. Protein-based methods, including electrophoretic and immunological techniques, are at times unsuitable for discriminating closely related species. Most of these methods have been replaced by more accurate and sensitive detection methods, such as DNA-based techniques. Emerging technologies like DNA barcoding and mass spectrometry are still in their infancy when it comes to their utilization in meat detection. Gold nanobiosensors have shown some promise in this regard. However, its applicability in small scale industries is distant. This article comprehensively reviews the recent developments in the field of analytical methods used for porcine identification.


Assuntos
Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Produtos da Carne/análise , Suínos , Animais , Técnicas Biossensoriais , Cromatografia/métodos , DNA/análise , Análise de Alimentos/instrumentação , Espectrometria de Massas , Carne/análise , Reação em Cadeia da Polimerase , Proteínas/análise , Análise Espectral/métodos , Suínos/genética
11.
Nat Commun ; 11(1): 2197, 2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32366845

RESUMO

Emerging urinary biomarkers continue to show promise in evaluating lupus nephritis (LN). Here, we screen urine from active LN patients for 1129 proteins using an aptamer-based platform, followed by ELISA validation in two independent cohorts comprised of 127 inactive lupus, 107 active LN, 67 active non-renal lupus patients and 74 healthy controls, of three different ethnicities. Urine proteins that best distinguish active LN from inactive disease are ALCAM, PF-4, properdin, and VCAM-1 among African-Americans, sE-selectin, VCAM-1, BFL-1 and Hemopexin among Caucasians, and ALCAM, VCAM-1, TFPI and PF-4 among Asians. Most of these correlate significantly with disease activity indices in the respective ethnic groups, and surpass conventional metrics in identifying active LN, with better sensitivity, and negative/positive predictive values. Several elevated urinary molecules are also expressed within the kidneys in LN, based on single-cell RNAseq analysis. Longitudinal studies are warranted to assess the utility of these biomarkers in tracking lupus nephritis.


Assuntos
Aptâmeros de Peptídeos/metabolismo , Biomarcadores/urina , Nefrite Lúpica/diagnóstico , Proteínas/análise , Molécula de Adesão de Leucócito Ativado/urina , Adulto , Afro-Americanos/estatística & dados numéricos , Grupo com Ancestrais do Continente Asiático/estatística & dados numéricos , Selectina E/análise , Grupo com Ancestrais do Continente Europeu/estatística & dados numéricos , Feminino , Humanos , Nefrite Lúpica/etnologia , Nefrite Lúpica/urina , Properdina/urina , Sensibilidade e Especificidade , Molécula 1 de Adesão de Célula Vascular/urina , Adulto Jovem
12.
Nucleic Acids Res ; 48(11): e63, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32383757

RESUMO

Site-saturation libraries reduce protein screening effort in directed evolution campaigns by focusing on a limited number of rationally chosen residues. However, uneven library synthesis efficiency leads to amino acid bias, remedied at high cost by expensive custom synthesis of oligonucleotides, or through use of proprietary library synthesis platforms. To address these shortcomings, we have devised a method where DNA libraries are constructed on the surface of microbeads by ligating dsDNA fragments onto growing, surface-immobilised DNA, in iterative split-and-mix cycles. This method-termed SpliMLiB for Split-and-Mix Library on Beads-was applied towards the directed evolution of an anti-IgE Affibody (ZIgE), generating a 160,000-membered, 4-site, saturation library on the surface of 8 million monoclonal beads. Deep sequencing confirmed excellent library balance (5.1% ± 0.77 per amino acid) and coverage (99.3%). As SpliMLiB beads are monoclonal, they were amenable to direct functional screening in water-in-oil emulsion droplets with cell-free expression. A FACS-based sorting of the library beads allowed recovery of hits improved in Kd over wild-type ZIgE by up to 3.5-fold, while a consensus mutant of the best hits provided a 10-fold improvement. With SpliMLiB, directed evolution workflows are accelerated by integrating high-quality DNA library generation with an ultra-high throughput protein screening platform.


Assuntos
DNA/química , DNA/metabolismo , Biblioteca Gênica , Ensaios de Triagem em Larga Escala/métodos , Microesferas , Proteínas/análise , Proteínas/metabolismo , Clonagem Molecular , Sequência Consenso , DNA/genética , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Mutação , Fosforilação , Proteínas/química
13.
Biomed Khim ; 66(2): 167-173, 2020 Feb.
Artigo em Russo | MEDLINE | ID: mdl-32420899

RESUMO

In this work, we have compared malignant and non-malignant diseases of the mammary gland using 8 proteins: HRG, MUC1, PAI-1, HSP90αA1, CDH1, ERα, PGR and IL-12. Their concentrations in the supernatants of blood cells and breast biopsies were compared in terms of spontaneous production, induced by a polyclonal activator and after exposure to biopsy samples of the HLDF differentiation factor, as well as the indices of the effect of the polyclonal activator and HLDF on the protein production. In addition, the correlation relationships of the above indicators with the expression of markers of the epithelial-mesenchymal transition: collagen type II (CII), ß-1 integrin (CD29) and cadherin-E (CDH1) were studied. The study revealed statistically significant differences in the concentration of HRG in the supernatant of blood cells, IL-12 during spontaneous production by biopsy specimens, PGR production of biopsy specimens induced by the polyclonal activator, CDH1 and IL-12 production biopsy specimens exposed to HLDF. According to the influence index of the polyclonal activator and HLDF, statistically significant differences were found for CDH1production. Comparison of non-specific invasive carcinoma biopsy specimens and non-malignant breast diseases by means of the markers of the epithelial-mesenchymal transition revealed statistically significant differences in CD29 expression and the lack of differences in the expression of CDH1 and CII. This indicates the presence of cell atypia in samples of non-malignant breast diseases; it is confirmed by the recognized correlation between the production of certain proteins and the expression of the epithelial-mesenchymal transition markers.


Assuntos
Biomarcadores , Doenças Mamárias/diagnóstico , Neoplasias da Mama/diagnóstico , Proteínas/análise , Transição Epitelial-Mesenquimal , Feminino , Humanos
14.
J Chromatogr A ; 1621: 461064, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32336499

RESUMO

The performance of columns packed with 1.7 µm particles for aqueous size-exclusion chromatography was assessed at high-pressure conditions and linked to particle- and column-bed integrity. Decreasing the particle size from 3.5 µm to 1.7 µm increases the resolution due to the improved mass-transfer characteristics, allowing to significantly speed-up analysis without compromising the selectivity. A sub-minute separation of intact proteins was realized on a 4.6 mm i.d × 75 mm long column packed with 1.7 µm SEC particles applying a flow rate of 1.8 mL/min, corresponding to a column pressure of 530 bar. Ultra-high pressure operation (exceeding manufacturer's recommendations) resulted in peak deformation, a shift towards earlier retention times, and an alteration in selectivity. To gain insights in the mechanisms of column deterioration, short 30 mm long columns were operated at UHPLC conditions, maximizing the pressure drop over individual particles. This resulted in the presence of fractured particles situated at the column outlet, as verified by scanning electron micrographs. Mercury-intrusion porosimetry and argon-adsorption measurements did not reveal significant differences in intraparticle volume between particle batches sampled before and after pressure stress testing. As particles at the column outlet fracture (but not collapse) at high pressure operation, a void was formed at the column inlet. The degradation of the separation performance appeared to be the result of a decrease in interparticle pore volume.


Assuntos
Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Tamanho da Partícula , Proteínas/análise , Proteínas/isolamento & purificação , Água/química
15.
Ecotoxicol Environ Saf ; 197: 110597, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32311613

RESUMO

Dissolved organic matter (DOM) plays an important role in the migration and transformation of nutrients and pollutants. Recently, DOM derived from biochar has the potential to determine the application of biochar and has attracted much researcher's attention. However, the effects of pyrolysis temperature on the composition evolution of DOM in manure-derived biochar are still unclear. In this study, DOM solutions extracted from a series of biochars derived from three kinds of manure (chicken, swine and dairy) at six pyrolysis temperature (200-700 °C) were analyzed using UV-Visible, Fourier transform infrared and fluorescence spectroscopy, aiming to investigate the effects of pyrolysis temperature on the composition evolution of DOM. The results showed that, with the increased of pyrolysis temperature, the dissolved organic matter (DOC) content sharply declined to reach stable. High DOC content was obtained at low pyrolysis temperature. Moreover, the DOM mainly contained humic acid-like and protein-like substances. With the pyrolysis temperature increased, the protein-like substances firstly decreased and then increased, while there was an opposite trend for the humic acid-like substances. Moreover, functional groups evolution of DOM depended on the pyrolysis temperature and manure type, evidenced by the Fourier transform infrared spectroscopy with two-dimensional correlation analysis. This study highlights the importance of optical analysis and may provide valuable information regarding the characteristics evolution of biochar-derived DOM.


Assuntos
Carvão Vegetal/química , Esterco/análise , Pirólise , Animais , Bovinos , Galinhas , Substâncias Húmicas/análise , Proteínas/análise , Suínos , Temperatura
16.
PLoS Negl Trop Dis ; 14(4): e0007802, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32236099

RESUMO

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors.


Assuntos
Anticoagulantes/análise , Fatores Biológicos/análise , Coagulantes/análise , Peptídeos/análise , Proteínas/análise , Venenos de Serpentes/química , Animais , Coagulação Sanguínea/efeitos dos fármacos , Fracionamento Químico , Cromatografia Líquida , Humanos , Plasma/efeitos dos fármacos , Proteômica , Espectrometria de Massas em Tandem
17.
Food Chem ; 324: 126786, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32344353

RESUMO

The effects of Na2CO3 on the quality, change of protein subunits and volatile compounds of sourdough leavened Chinese steamed bread (sourdough-CSB) and yeast leavened CSB (yeast-CSB) were investigated. Results suggested that, low Na2CO3 level endowed both CSB with softer crumb and little change of surface color. Besides, Na2CO3 addition improved the overall aroma profile by inhibiting the production of aroma-negative compounds (butanoic acid, 1-octen-3-ol, hexanal and heptanal). Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed an obvious increase in intensity of protein bands with low molecular weight, consistent with the result of size-exclusion high-performance liquid chromatography analysis and free sulfhydryl group (SH) content, indicating the hydrolysis of glutenin macropolymer (GMP) under alkaline condition in yeast-CSB. While in sourdough-CSB, GMP and SH content firstly decreased at low Na2CO3 level (0-0.2%) and then increased at high Na2CO3 level (0.3%-0.5%).


Assuntos
Pão , Carbonatos , Odorantes/análise , Compostos Orgânicos Voláteis/análise , Aldeídos/análise , Pão/análise , Culinária , Eletroforese em Gel de Poliacrilamida , Fermentação , Alimentos e Bebidas Fermentados , Qualidade dos Alimentos , Glutens/análise , Hidrólise , Peso Molecular , Proteínas/análise , Proteínas/metabolismo , Saccharomyces cerevisiae , Vapor
18.
PLoS Comput Biol ; 16(4): e1007870, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32339173

RESUMO

Many proteins contain multiple folded domains separated by flexible linkers, and the ability to describe the structure and conformational heterogeneity of such flexible systems pushes the limits of structural biology. Using the three-domain protein TIA-1 as an example, we here combine coarse-grained molecular dynamics simulations with previously measured small-angle scattering data to study the conformation of TIA-1 in solution. We show that while the coarse-grained potential (Martini) in itself leads to too compact conformations, increasing the strength of protein-water interactions results in ensembles that are in very good agreement with experiments. We show how these ensembles can be refined further using a Bayesian/Maximum Entropy approach, and examine the robustness to errors in the energy function. In particular we find that as long as the initial simulation is relatively good, reweighting against experiments is very robust. We also study the relative information in X-ray and neutron scattering experiments and find that refining against the SAXS experiments leads to improvement in the SANS data. Our results suggest a general strategy for studying the conformation of multi-domain proteins in solution that combines coarse-grained simulations with small-angle X-ray scattering data that are generally most easy to obtain. These results may in turn be used to design further small-angle neutron scattering experiments that exploit contrast variation through 1H/2H isotope substitutions.


Assuntos
Simulação de Dinâmica Molecular , Proteínas , Espalhamento a Baixo Ângulo , Difração de Raios X , Algoritmos , Biologia Computacional , Nêutrons , Conformação Proteica , Domínios Proteicos , Proteínas/análise , Proteínas/química
19.
Arch Gynecol Obstet ; 301(5): 1219-1225, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32266526

RESUMO

OBJECTIVE: To determine the diagnostic value and clinical significance of serum HE4 levels in differentiating between benign and malignant ovarian disease in patients with elevated CA125 levels. METHODS: The levels and positive expression rate of HE4 were compared between 371 patients with elevated CA125 levels and benign ovarian disease, and 132 patients with epithelial ovarian cancer to determine the diagnostic value of HE4. RESULTS: The level and positive expression rate of HE4 differed significantly between the benign and malignant groups, in that, there was no significant difference in HE4 expression between CA125 low- and high-level groups within the benign ovarian disease group, with levels of HE4 being in the normal range in both groups. However, the positive expression rates and levels of HE4 in the malignant group were significantly different between the serum CA125 low- and high-level groups. ROC curve analysis showed that optimal HE4 cutoff values for increased accuracy in diagnosis were 78.03 pmol/L and 119.70 pmol/L before and after menopause, respectively. CONCLUSIONS: Serum HE4 levels can potentially be used as a marker to differentiate between benign and malignant ovarian disease with elevated serum CA125 levels. The high specificity of HE4 was superior in identifying benign ovarian disease. We recommend increasing the cutoff values of HE4 in premenopausal patients and decreasing the cutoff values in postmenopausal patients for increased accuracy in the differential diagnosis of patients with elevated CA125 levels.


Assuntos
Biomarcadores Tumorais/sangue , Antígeno Ca-125/sangue , Carcinoma Epitelial do Ovário/diagnóstico , Neoplasias Ovarianas/diagnóstico , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/metabolismo , Adulto , Biomarcadores Tumorais/análise , Carcinoma Epitelial do Ovário/sangue , Diagnóstico Diferencial , Feminino , Humanos , Menopausa , Pessoa de Meia-Idade , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Pré-Menopausa , Proteínas/análise , Proteínas/metabolismo , Curva ROC , Sensibilidade e Especificidade , Proteína 2 do Domínio Central WAP de Quatro Dissulfetos/análise
20.
BMC Bioinformatics ; 21(1): 117, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32192430

RESUMO

BACKGROUND: Two-dimensional electrophoresis (2DE) is one of the most widely applied techniques in comparative proteomics. The basic task of 2DE is to identify differential protein expression by quantitative analysis of 2DE images. To reduce the errors of spot quantification in 2DE images, a novel brightness correction method based on gradient interval histogram (GIH) is proposed in this paper. RESULTS: First, GIH equalization is proposed to enhance the protein spot edges, especially the weak protein spots in the 2DE image. Second, to eliminate the overall brightness shift, GIH matching is applied to the 2DE images that need to be compared. Finally, the proposed method is verified by subjective quality evaluation and quantitative analysis of protein spots in real 2DE images. CONCLUSIONS: The experimental results show that the average error of the quantification of corresponding protein spots in the resulting image pairs is less than 3%, which is significantly superior to that of the existing methods.


Assuntos
Eletroforese em Gel Bidimensional , Proteínas/análise , Proteômica/métodos , Algoritmos , Processamento de Imagem Assistida por Computador
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