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1.
Prep Biochem Biotechnol ; 50(1): 18-27, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31453751

RESUMO

We report on the development of a functionalized membrane-based technology for selective enrichment of milk fat globules from raw bovine milk. Functionalization was conducted by in situ polymerization of acrylic acid within a polyvinylidene fluoride membrane, followed by the electrostatic attachment of a cationic polymer to impart a net positive charge. The functionalized membrane-based technology enabled a one-step method of selective separation of globules directly from milk-based on size and charge. The presence of globules in the eluate was confirmed by fluorescence microscopy. Quantification of the extracted phospholipids from globules in the eluant revealed a significantly higher amount of polar lipids than the permeate. Our study describes a comprehensive analysis of selective enrichment of fat globules using a functionalized membrane and demonstrates the beneficial effect of extracted phospholipids from enriched fat globules.


Assuntos
Glicolipídeos/isolamento & purificação , Glicoproteínas/isolamento & purificação , Membranas Artificiais , Leite/química , Polivinil/química , Animais , Bovinos , Fracionamento Químico/métodos , Glicolipídeos/análise , Glicoproteínas/análise , Fosfolipídeos/análise
2.
Chem Commun (Camb) ; 55(91): 13673-13676, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31647081

RESUMO

An array sensing platform exploiting phenyl boronic acid-functionalized pyrene amphiphile/surfactant assemblies is constructed for glycoprotein discrimination, achieving a discrimination sensitivity of 50 nM. Gastric cancer cell lines of various differentiation grades are successfully discriminated, demonstrating its great potential in sensitive differentiation of glycoprotein-related samples in biomedical diagnosis.


Assuntos
Glicoproteínas/análise , Pirenos/química , Tensoativos/química , Ácidos Borônicos/química , Diferenciação Celular , Linhagem Celular Tumoral , Análise Discriminante , Difusão Dinâmica da Luz , Glicoproteínas/urina , Humanos , Micelas , Espectrometria de Fluorescência
3.
Anal Chim Acta ; 1089: 1-18, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31627805

RESUMO

Over the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of biosimilar products. As a result, multiple biosimilar mAbs are becoming available for a single originator drug. As opposed to small-molecular drugs, protein biopharmaceuticals do not have fully defined and reproducible structures, making it impossible to create identical copies. Therefore, regulators demand biosimilar sponsors to demonstrate similarity with the reference product to prevent safety and efficacy issues with the proposed product. Protein glycosylation is considered a crucially important quality attribute, because of its major role in immunogenicity and clinical efficacy of therapeutic proteins. However, the intrinsic biological variability of glycan structures creates a significant challenge for the current analytical platforms. In this review, we discuss the importance of glycan characterization on therapeutic proteins, with a particular focus on the analytical techniques applied for glycan profiling of biosimilar mAb products. In addition, we present a case study on infliximab biosimilars to illustrate the potential clinical implications of differences in glycan profile between originator and biosimilar mAb products.


Assuntos
Anticorpos Monoclonais/análise , Medicamentos Biossimilares/análise , Glicoproteínas/análise , Polissacarídeos/análise , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Medicamentos Biossimilares/química , Medicamentos Biossimilares/metabolismo , Cromatografia Líquida , Glicoproteínas/química , Glicosilação , Humanos , Imunoglobulina G/análise , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Infliximab/análise , Infliximab/química , Infliximab/metabolismo , Processamento de Proteína Pós-Traducional , Espectrometria de Massas em Tandem
4.
J Dairy Sci ; 102(12): 11124-11141, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31563305

RESUMO

In genome-wide association studies (GWAS), sample size is the most important factor affecting statistical power that is under control of the investigator, posing a major challenge in understanding the genetics underlying difficult-to-measure traits. Combining data sets available from different populations for joint or meta-analysis is a promising alternative to increasing sample sizes available for GWAS. Simulation studies indicate statistical advantages from combining raw data or GWAS summaries in enhancing quantitative trait loci (QTL) detection power. However, the complexity of genetics underlying most quantitative traits, which itself is not fully understood, is difficult to fully capture in simulated data sets. In this study, population-specific and combined-population GWAS as well as a meta-analysis of the population-specific GWAS summaries were carried out with the objective of assessing the advantages and challenges of different data-combining strategies in enhancing detection power of GWAS using milk fatty acid (FA) traits as examples. Gas chromatography (GC) quantified milk FA samples and high-density (HD) genotypes were available from 1,566 Dutch, 614 Danish, and 700 Chinese Holstein Friesian cows. Using the joint GWAS, 28 additional genomic regions were detected, with significant associations to at least 1 FA, compared with the population-specific analyses. Some of these additional regions were also detected using the implemented meta-analysis. Furthermore, using the frequently reported variants of the diacylglycerol acyltransferase 1 (DGAT1) and stearoyl-CoA desaturase (SCD1) genes, we show that significant associations were established with more FA traits in the joint GWAS than the remaining scenarios. However, there were few regions detected in the population-specific analyses that were not detected using the joint GWAS or the meta-analyses. Our results show that combining multi-population data set can be a powerful tool to enhance detection power in GWAS for seldom-recorded traits. Detection of a higher number of regions using the meta-analysis, compared with any of the population-specific analyses also emphasizes the utility of these methods in the absence of raw multi-population data sets to undertake joint GWAS.


Assuntos
Conjuntos de Dados como Assunto , Estudo de Associação Genômica Ampla/veterinária , Glicolipídeos/análise , Glicoproteínas/análise , Metanálise como Assunto , Leite/química , Animais , Bovinos , Cromatografia Gasosa , Diacilglicerol O-Aciltransferase/genética , Ácidos Graxos/análise , Feminino , Estudo de Associação Genômica Ampla/métodos , Genótipo , Locos de Características Quantitativas , Estearoil-CoA Dessaturase/genética
5.
J Dairy Sci ; 102(11): 10460-10470, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31495611

RESUMO

The objective of this study was to investigate the potential of milk mid-infrared (MIR) spectroscopy, MIR-derived traits including milk composition, milk fatty acids, and blood metabolic profiles (fatty acids, ß-hydroxybutyrate, and urea), and other on-farm data for discriminating cows of good versus poor likelihood of conception to first insemination (i.e., pregnant vs. open). A total of 6,488 spectral and milk production records of 2,987 cows from 19 commercial dairy herds across 3 Australian states were used. Seven models, comprising different explanatory variables, were examined. Model 1 included milk production; concentrations of fat, protein, and lactose; somatic cell count; age at calving; days in milk at herd test; and days from calving to insemination. Model 2 included, in addition to the variables in model 1, milk fatty acids and blood metabolic profiles. The MIR spectrum collected before first insemination was added to model 2 to form model 3. Fat, protein, and lactose percentages, milk fatty acids, and blood metabolic profiles were removed from model 3 to create model 4. Model 5 and model 6 comprised model 4 and either fertility genomic estimated breeding value or principal components obtained from a genomic relationship matrix derived using animal genotypes, respectively. In model 7, all previously described sources of information, but not MIR-derived traits, were used. The models were developed using partial least squares discriminant analysis. The performance of each model was evaluated in 2 ways: 10-fold random cross-validation and herd-by-herd external validation. The accuracy measures were sensitivity (i.e., the proportion of pregnant cows that were correctly classified), specificity (i.e., the proportion of open cows that were correctly classified), and area under the curve (AUC) for the receiver operating curve. The results showed that in all models, prediction accuracy obtained through 10-fold random cross-validation was higher than that of herd-by-herd external validation, with the difference in AUC ranging between 0.01 and 0.09. In the herd-by-herd external validation, using basic on-farm information (model 1) was not sufficient to classify good- and poor-fertility cows; the sensitivity, specificity, and AUC were around 0.66. Compared with model 1, adding milk fatty acids and blood metabolic profiles (model 2) increased the sensitivity, specificity, and AUC by 0.01, 0.02, and 0.02 unit, respectively (i.e., 0.65, 0.63, and 0.678). Incorporating MIR spectra into model 2 resulted in sensitivity, specificity, and AUC values of 0.73, 0.63, and 0.72, respectively (model 3). The comparable prediction accuracies observed for models 3 and 4 mean that useful information from MIR-derived traits is already included in the spectra. Adding the fertility genomic estimated breeding value and animal genotypes (model 7) produced the highest prediction accuracy, with sensitivity, specificity, and AUC values of 0.75, 0.66, and 0.75, respectively. However, removing either the fertility estimated breeding value or animal genotype from model 7 resulted in a reduction of the prediction accuracy of only 0.01 and 0.02, respectively. In conclusion, this study indicates that MIR and other on-farm data could be used to classify cows of good and poor likelihood of conception with promising accuracy.


Assuntos
Bovinos/fisiologia , Fertilidade , Leite/diagnóstico por imagem , Ácido 3-Hidroxibutírico/sangue , Animais , Área Sob a Curva , Austrália , Ácidos Graxos/análise , Feminino , Glicolipídeos/análise , Glicoproteínas/análise , Inseminação , Lactação , Lactose/análise , Análise dos Mínimos Quadrados , Leite/química , Proteínas do Leite/análise , Valor Preditivo dos Testes , Gravidez , Sensibilidade e Especificidade , Espectrofotometria Infravermelho/veterinária , Ureia/sangue
6.
J Anim Sci ; 97(11): 4647-4656, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31560748

RESUMO

This experiment was conducted to determine the effects of stearic acid (SA; C18:0) or rumen-protected oleic acid (OA; C18:1 cis-9) on milk performance and energy partitioning of early lactation cows when supplemented in diets with low and high level of rumen unsaturated fatty acids (RUFA). In low RUFA experiment (LRUFA), FA supplement rich in either SA or calcium salts OA was added to a basal diet with a low concentration of RUFA (0.75% vs. 1.4%, LRUFA-SA vs. LRUFA-OA). In high RUFA experiment (HRUFA), 2% soybean oil was added to the diet fed in the LRUFA experiment. In each experiment, 30 multiparous cows were blocked by parity and predicted transmitting ability for milk yield and were randomly fed 1 of 2 treatment diets from 2 to 13 wk postpartum. In the LRUFA experiment, LRUFA-SA had 2.4 kg/d more dry matter intake (DMI) (P < 0.01), 3.8 kg/d more energy-corrected milk (P < 0.01), and 0.3% units more milk fat percentage (P < 0.01) and 0.2 kg/d more milk fat yield (P < 0.01). Dietary treatments did not affect body weight, energy balance, and energy intake partitioning into milk, maintenance, and body tissues (P > 0.1). In the HRUFA experiment, HRUFA-SA had 1.4 kg/d more DMI (P = 0.03) but similar milk and milk components yields (P > 0.1). HRUFA-SA had a tendency to gain more body weight (P = 0.07) and had more positive energy balance (P = 0.01) and decreased gross feed efficiency (milk yield/DMI) (P = 0.01). Consistently, HRUFA-SA increased intake energy partitioning into body tissues (P = 0.02) and decreased energy partitioning into milk (P = 0.01). In summary, SA supplementation had more DMI relative to OA, but the effects on milk and milk fat production were different and affected by the level of RUFA in the basal diet. In application, SA supplementation was more effective to improve milk production when included in the basal diet with the low RUFA.


Assuntos
Bovinos/fisiologia , Suplementos Nutricionais/análise , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos Insaturados/administração & dosagem , Leite/metabolismo , Ácido Oleico/administração & dosagem , Ácidos Esteáricos/administração & dosagem , Animais , Peso Corporal , Dieta/veterinária , Feminino , Glicolipídeos/análise , Glicoproteínas/análise , Lactação , Leite/química , Período Pós-Parto , Gravidez , Rúmen/metabolismo , Óleo de Soja/administração & dosagem
7.
Nat Methods ; 16(9): 902-910, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31384044

RESUMO

We report a liquid chromatography coupled to tandem mass spectrometry O-glycoproteomics strategy using data-independent acquisition (DIA) mode for direct analysis of O-glycoproteins. This approach enables characterization of glycopeptides and structures of O-glycans on a proteome-wide scale with quantification of stoichiometries (though it does not allow for direct unambiguous glycosite identification). The method relies on a spectral library of O-glycopeptides; the Glyco-DIA library contains sublibraries obtained from human cell lines and human serum, and it currently covers 2,076 O-glycoproteins (11,452 unique glycopeptide sequences) and the 5 most common core1 O-glycan structures. Applying the Glyco-DIA library to human serum without enrichment for glycopeptides enabled us to identify and quantify 269 distinct glycopeptide sequences bearing up to 5 different core1 O-glycans from 159 glycoproteins in a SingleShot analysis.


Assuntos
Biologia Computacional/métodos , Simulação por Computador , Glicopeptídeos/análise , Glicoproteínas/análise , Polissacarídeos/análise , Proteoma/análise , Proteômica/métodos , Glicosilação , Humanos
8.
Glycoconj J ; 36(4): 259-266, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31270739

RESUMO

Protein glycosylation is increasingly recognised as an essential requirement for effective microbial infections. Within microbial pathogen's protein glycosylation is used for both defensive and offensive purposes; enabling pathogens to fortify themselves against the host immune response or to disarm the host's ability to resist infection. Although microbial protein glycosylation systems have been recognised for nearly two decades only recently has the true extend of protein glycosylation within microbes begun to be appreciated. A key enabler for this conceptual shift has been the development and application of modern approaches for the characterisation of glycosylation. Over the last decade my research has focused on the development of proteomic tools to probe microbial glycosylation. By developing workflows for glycopeptide enrichment and identification we have demostrated that it is now possible to characterise the glycoproteomes of microbial species in a truely high-throughput manner. Using these high-throughput approaches we have shown a number of bacterial species modify multiple proteins including members of the Campylobacter genus and the pathogens A. baumannii, R. solanacearum and B. cenocepacia. These studies have established that bacterial glycosylation is widespread, that glycan microheterogeneity is common place and that an extensive array of glycans are used to decorate protein compared to Eukaryotic glycosylation systems. Excitingly these approaches developed to characterise O- and N-linked bacterial glycosylation systems are equally amenable to studying newly discovered forms of microbial glycosylation such as Arginine glycosylation as well as glycosylation within the parasitic eukaryotic organisms T. gondii and P. falciparum. This work demonstrates that MS approaches can now be considered an indispensable tool for the elucidation and tracking of microbial glycosylation events.


Assuntos
Glicoproteínas/análise , Espectrometria de Massas/métodos , Proteômica/métodos , Campylobacter/metabolismo , Glicopeptídeos/análise , Glicosilação
9.
Adv Exp Med Biol ; 1140: 155-167, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31347047

RESUMO

Endoplasmic reticulum (ER) resident and secretory proteins that fail to reach their native conformation are selected for degradation through the ER-Associated Degradation (ERAD) pathway. The ER degradation-enhancing alpha-mannosidase-like proteins (EDEMs) were shown to be involved in this pathway but their precise role is still under investigation. Mass spectrometry analysis has contributed significantly to the characterization of protein complexes in the last years. The recent advancements in instrumentation, especially within resolution and speed can provide unique insights concerning the molecular architecture of protein-protein interactions in systems biology. Previous reports have suggested that several protein complexes in ERAD are sensitive to the extraction conditions. Indeed, whilst EDEM proteins can be recovered in most detergents, some of their partners are not solubilized, which further emphasizes the importance of the experimental setup. Here, we define such dynamic interactions of EDEM proteins by employing offline protein fractionation, nanoLC-MS/MS and describe how mass spectrometry can contribute to the characterization of such complexes, particularly within a disease context like melanoma.


Assuntos
Retículo Endoplasmático/fisiologia , Melanoma , Espectrometria de Massas em Tandem , Glicoproteínas/análise , Humanos , Proteínas de Membrana/análise , alfa-Manosidase/análise
10.
J Dairy Sci ; 102(10): 8670-8690, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31351726

RESUMO

Our goal was to determine the effect of systematically controlled variation in milk fat, true protein, casein, and serum protein concentrations on the sensory color, flavor and texture properties, instrumental color and viscosity, and milk fat globule size distribution of milk-based beverages. Beverage formulations were based on a complete balanced 3-factor (fat, true protein, and casein as a percentage of true protein) design with 3 fat levels (0.2, 1.0, and 2.0%), 4 true protein (TP) levels (3.00, 3.67, 4.34, and 5.00%) within each fat level, and 5 casein as a percentage of true protein (CN%TP) levels (5, 25, 50, 75, and 80%) within each protein level (for a total of 60 formulations within each of 2 replicates). Instrumental measures of Hunter L and a values and Commission Internationale de l'Éclairage (CIE) b* values, instrumental viscosity, particle size, flavor, sensory texture and sensory appearance evaluations were done on each pasteurized/homogenized beverage formulation. Within each of the 3 fat levels, higher serum protein concentration drove higher aroma intensity, sweet aromatic, cooked/sulfur, cardboard/doughy flavors, and sensory yellowness scores, whereas higher casein concentration drove higher instrumental viscosity in milk protein beverages. Increasing serum protein concentration increased yellowness, sweet aromatic, aroma intensity, cooked/sulfur, and cardboard/doughy flavors across all fat levels and also had the largest effect on L, a, and b* values, sensory whiteness, and opacity within each fat level. Increases in true protein increased throat cling and astringency intensities. Increases in fat concentration were correlated with higher L, a, and b* values, larger particle size, and increased sensory whiteness, mouth coating, cooked/milky, and milkfat flavors. Multiple linear regression of L, a, and b* values produced better predictions of sensory whiteness and yellowness of pasteurized milk protein beverages than simple linear regression of L or b* values, respectively. Formulating milk protein beverages to a higher true protein level increased astringency regardless of fat level. When formulating milk protein beverages, a product developer has a wide range of milk-based protein ingredient choices that differ in price and change price relationship across time. Understanding the expected relative effect of different milk protein ingredients on the textural and flavor characteristics of milk-based beverages could be used to help guide product reformulation decisions and ingredient choices to achieve a specific sensory profile while controlling total beverage ingredient cost.


Assuntos
Bebidas , Proteínas Sanguíneas/análise , Proteínas do Leite/análise , Leite , Paladar , Adulto , Animais , Bebidas/análise , Caseínas , Bovinos , Cor , Feminino , Glicolipídeos/análise , Glicoproteínas/análise , Humanos , Masculino , Pessoa de Meia-Idade , Leite/química , Tamanho da Partícula , Pasteurização , Viscosidade
11.
Ann Lab Med ; 39(6): 561-565, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31240884

RESUMO

POEMS syndrome is a rare paraneoplastic syndrome, which includes polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes due to plasma cell (PC) neoplasm. Diagnosis of this disease is challenging because of its rarity and complex clinical manifestations. We attempted to identify the key clinical features and characteristic bone marrow (BM) findings of POEMS syndrome, by reviewing the medical records and BM analyses of 24 Korean patients. Frequent clinical manifestations included polyneuropathy (100%), monoclonal gammopathy (100%), organomegaly (92%), extravascular volume overload (79%), and endocrinopathy (63%). The BM analyses revealed mild PC hyperplasia (median PCs: 5.5%) and frequent megakaryocytic hyperplasia (88%), megakaryocyte clusters (88%), and hyperlobation (100%). Flow cytometry of BM aspirates using CD138/CD38/CD45/CD19/CD56 showed normal (67%, 4/6) or neoplastic PC immunophenotypes (33%, 2/6). A diagnosis of POEMS syndrome must be considered when a patient suspected of having PC dyscrasia shows the above clinical presentation and BM findings.


Assuntos
Medula Óssea/metabolismo , Síndrome POEMS/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD19/metabolismo , Medula Óssea/patologia , Feminino , Glicoproteínas/análise , Humanos , Imunofenotipagem , Linfócitos/citologia , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Paraproteinemias/diagnóstico , República da Coreia , Estudos Retrospectivos , Adulto Jovem
12.
Chem Commun (Camb) ; 55(55): 7934-7937, 2019 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-31219116

RESUMO

High-throughput proteome-level characterization of stemness markers of MCF-7 cancer stem cells was carried out using our recently developed site- and structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. 144 differentially expressed intact N-glycopeptides were quantified all at once yet with good accurate consistency with the individual biochemical measurements of proteins, RNA or DNA.


Assuntos
Glicoproteínas/análise , Proteoma/análise , Sequência de Aminoácidos , Biomarcadores/análise , Regulação para Baixo , Ontologia Genética , Humanos , Marcação por Isótopo , Células MCF-7 , Células-Tronco Neoplásicas/química , Proteômica/métodos , Regulação para Cima
13.
J Dairy Sci ; 102(8): 7189-7203, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31178181

RESUMO

The aim of this study was to investigate the feasibility of using mid-infrared (MIR) spectroscopy analysis of milk samples to increase the power and precision of genome-wide association studies (GWAS) for milk composition and to better distinguish linked quantitative trait loci (QTL). To achieve this goal, we analyzed phenotypic data of milk composition traits, related MIR spectra, and genotypic data comprising 626,777 SNP on 5,202 Holstein, Jersey, and crossbred cows. We performed a conventional GWAS on protein, lactose, fat, and fatty acid concentrations in milk, a GWAS on individual MIR wavenumbers, and a partial least squares regression (PLS), which is equivalent to a multi-trait GWAS, exploiting MIR data simultaneously to predict SNP genotypes. The PLS detected most of the QTL identified using single-trait GWAS, usually with a higher significance value, as well as previously undetected QTL for milk composition. Each QTL tends to have a different pattern of effects across the MIR spectrum and this explains the increased power. Because SNP tracking different QTL tend to have different patterns of effect, it was possible to distinguish closely linked QTL. Overall, the results of this study suggest that using MIR data through either GWAS or PLS analysis applied to genomic data can provide a powerful tool to distinguish milk composition QTL.


Assuntos
Bovinos/fisiologia , Estudo de Associação Genômica Ampla/veterinária , Leite/química , Locos de Características Quantitativas/genética , Animais , Bovinos/genética , Ácidos Graxos/análise , Feminino , Genótipo , Glicolipídeos/análise , Glicoproteínas/análise , Raios Infravermelhos , Lactose/análise , Leite/efeitos da radiação , Proteínas do Leite/análise , Fenótipo
14.
J Dairy Sci ; 102(8): 7118-7133, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31155249

RESUMO

Grass-based production systems use concentrate supplementation primarily when pasture quality and availability have declined. Barley is a common concentrate ingredient; however, oat grain grows well in Ireland, is a source of lipids and fiber, and may provide an alternative to barley. The antioxidant α-tocopherol (α-TOC) plays a role in cell membrane structure, and it has the potential to improve tight junction structures of the mammary gland that deteriorate in late lactation. The objective of this research was to investigate the effect of cereal type and α-TOC level on milk yield, milk composition, rumen fermentation, and N excretion in late-lactation dairy cows at pasture and when housed indoors on grass silage. Forty-eight Holstein Friesian dairy cows were blocked on days in milk (+185 d in milk) and balanced for parity, pre-experimental milk yield, milk composition, and body condition score and assigned to 1 of 4 dietary treatments in a randomized complete block design (n = 12). The dietary treatments were control (C) base diet; base diet + barley-based concentrate + low α-TOC (350 IU/kg) (B); base diet + oat-based concentrate + low α-TOC (350 IU/kg) (O); and base diet + oat-based concentrate + high α-TOC (1,050 IU/kg) (O+T). Following a 14-d acclimation period, diets were offered for a 49-d experimental period at pasture (P1) and a 21-d experimental period indoors (P2). The base diet was grazed grass in P1 and grass silage in P2. In P2, cows on C also received 2.65 kg (dry matter) of a standard concentrate. In P1, supplementation increased milk and milk solids yield (B: 20.7 kg/d, 1.74 kg/d; O: 20.6 kg/d, 1.81 kg/d; O+T: 20.5 kg/d, 1.77 kg/d, respectively) compared with C (17.8 kg/d, 1.60 kg/d). Cows offered B had a lower milk fat (4.60%) concentration than C (5.00%) and O (4.90%). In P2, cereal type and α-TOC level did not alter milk production. In conclusion, concentrate supplementation increased milk and milk solids yield and cows offered O had a higher milk fat concentration than cows offered B. Increasing the level of α-TOC had no major effect on production parameters measured in P1 or in P2.


Assuntos
Antioxidantes/administração & dosagem , Bovinos/fisiologia , Leite/metabolismo , Nitrogênio/metabolismo , alfa-Tocoferol/administração & dosagem , Animais , Dieta/veterinária , Fibras na Dieta/administração & dosagem , Grão Comestível/química , Feminino , Fermentação/efeitos dos fármacos , Glicolipídeos/análise , Glicoproteínas/análise , Irlanda , Lactação , Metabolismo dos Lipídeos , Leite/química , Poaceae , Gravidez , Rúmen/metabolismo , Silagem/análise
15.
APMIS ; 127(8): 588-593, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31233243

RESUMO

Microfibrillar-associated protein 4 (MFAP4) is a non-structural matrix protein with cell regulatory activities and a potential as seromarker for fibrosis. We aimed to study the occurrence of MFAP4 in the synovial membrane from patients with rheumatoid arthritis (RA) vs osteoarthritis (OA). Formaldehyde-fixed synovial tissue sections, from patients with RA (N = 6) and OA (N = 6) undergoing total hip arthroplasty, were deparaffinized and immunostained with monoclonal antibodies against MFAP4. Elastin was detected using ElastiKit. MFAP4 in serum (sMFAP4) and synovial fluid was measured by an immunoassay. MFAP4 was present in synovial biopsies from both RA and OA patients, particularly prominent in deep arterioles where it colocalized with elastin. Notably however, MFAP4 was absent from the internal elastic lamina in RA arterioles irrespective of disease duration and synovitis activity, while present although with irregular staining patterns in OA specimens. sMFAP4 was increased in RA and OA serum vs healthy controls: median (interquartile range) 29.8 (25.3-39.1) and 25.5 U/L (18.1-43.3) vs 17.7 U/L (13.7-21.2), p = 0.006 and p = 0.02, respectively The concentration of synovial fluid was lower than in serum in both RA and OA. These findings may suggest that MFAP4 is involved in adaptive vessel wall remodeling associated with chronic joint disease.


Assuntos
Artrite Reumatoide/imunologia , Proteínas de Transporte/análise , Proteínas da Matriz Extracelular/análise , Glicoproteínas/análise , Osteoartrite/imunologia , Membrana Sinovial/imunologia , Idoso , Anticorpos Monoclonais/imunologia , Biomarcadores/análise , Proteínas de Transporte/imunologia , Estudos de Casos e Controles , Proteínas da Matriz Extracelular/imunologia , Feminino , Glicoproteínas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Líquido Sinovial/química , Membrana Sinovial/patologia
16.
Animal ; 13(12): 2802-2810, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31113503

RESUMO

The majority of New Zealand dairy goat farmers utilise cultivated green-fed fodder dominated by perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.), but evidence from other ruminant species suggests that milk production may be improved when using a more diverse array of species within the green fodder. The aim of this experiment was to determine whether feeding lactating dairy goats a mixed-species green fodder (MF, consisting of perennial ryegrass, timothy (Phleum pratense L.), prairie grass (Bromus willdenowii Kunth), white clover, red clover (Trifolium pratense L.), lucerne (Medicago sativa L.), chicory (Cichorium intybus L.) and plantain (Plantago lanceolata L.) improves dietary intake, milk yield and composition compared with a standard ryegrass and white clover green fodder (SF). Thirty-six mid-lactation goats were housed indoors in pairs and split into two groups (A and B). The trial was split into three periods - firstly a uniformity period of 6 days, in which all goats were fed a combination of both green fodder types, followed by two treatment periods (P1 and P2) of 12 days, respectively. For P1, group A was fed MF and group B was fed SF, and then the group diets were switched for P2. Goats fed MF had 13% greater dry matter intake and 7% greater milk yield than goats fed SF. In addition, the milk protein and fat concentration of goats fed MF were 4% greater than for those fed SF, whereas there was no effect on milk lactose concentration. There was no treatment effect on the levels of protein, glucose, urea or non-esterified fatty acids in the blood of the goats. An effect of green fodder type on milk fat profile was demonstrated, with proportions of pentadecylic acid (C15:0), cis-vaccenic acid (C18:1 c11), linoleic acid (C18:2 n6) and α-linolenic acid (C18:3 n3) being increased in response to MF consumption. In contrast, iso-C15 and iso-C17 proportions were lesser. In summary, this study demonstrated that goats fed MF increased green fodder intake and milk production compared with goats fed SF. The green fodder type affected the fatty acid profile of goat's milk, with MF increasing the levels of beneficial polyunsaturated omega fatty acids (linoleic and α-linolenic acids).


Assuntos
Ração Animal/análise , Ácidos Graxos Insaturados/análise , Cabras/fisiologia , Leite/química , Animais , Chicória , Dieta/veterinária , Fabaceae , Feminino , Glicolipídeos/análise , Glicoproteínas/análise , Lactação/efeitos dos fármacos , Lactose/análise , Leite/metabolismo , Proteínas do Leite/análise , Nova Zelândia , Poaceae
17.
Anal Bioanal Chem ; 411(17): 3941-3949, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31119348

RESUMO

Glycoprotein detection holds great potential for early diagnosis of diverse diseases. For this purpose, the combination of quartz crystal microbalance (QCM) sensor and molecular imprinting has attracted increasing attention. Nonetheless, the recently common imprinted films fabricated on QCM electrode are thick and rigid, lacking flexibility in aqueous phase. Alternatively, small molecules immobilized on the electrode to construct molecular scale film could address this problem, while stabilization of the imprinted sites remains challenging. Herein, a co-assembly complex was obtained by the mixture of template and multifunctional oligomer, which was then immobilized on the amino-modified transducer surface through epoxy-amino reaction to form a protein-imprinted film. Afterward, the remaining epoxy groups in oligomer chains were cross-linked to conserve and stabilize the orientation of imprinted sites after template elution. Template rebinding tests show that cross-linked film has much higher imprinting factors than that of the non-cross-linked counterpart. Furthermore, control proteins that are distinct in properties and structures were employed to demonstrate the selectivity of this approach, and the imprinted assay reveals high affinity and specificity towards template protein. Graphical Abstract.


Assuntos
Glicoproteínas/análise , Impressão Molecular , Polímeros/química , Técnicas de Microbalança de Cristal de Quartzo , Eletrodos
18.
J Chromatogr A ; 1600: 46-54, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31036360

RESUMO

Glycosylation, as a biologically important protein post-translational modification, often alters on both glycosites and glycans, simultaneously. However, most of current approaches focused on biased profiling of either glycosites or glycans, and limited by time-consuming process and milligrams of starting protein material. We describe here a simple and integrated spintip-based glycoproteomics technology (termed Glyco-SISPROT) for achieving a comprehensive view of glycoproteome with shorter sample processing time and low microgram starting material. By carefully integrating and optimizing SCX, C18 and Concanavalin A (Con A) packing material and their combination in spintip format, both predigested peptides and protein lysates could be processed by Glyco-SISPROT with high efficiency. More importantly, deglycopeptide, intact glycopeptide and glycans released by multiple glycosidases could be readily collected from the same Glyco-SISPROT workflow for LC-MS analysis. In total, above 1850 glycosites in ˜1770 unique deglycopeptides were characterized from mouse liver by using either 100 µg of predigested peptides or directly using 100 µg of protein lysates, in which about 30% of glycosites were released by both PNGase F and Endos. To the best of our knowledge, this approach should be one of the most comprehensive glycoproteomic approaches by using limited protein starting material. One significant benefit of Glyco-SISPROT is that whole processing time is dramatically reduced from a few days to less than 6 h with good reproducibility when protein lysates were directly processed by Glyco-SISPROT. We expect that this method will be suitable for multi-level glycoproteome analysis of rare biological samples with high sensitivity.


Assuntos
Glicoproteínas/análise , Proteômica/métodos , Animais , Cromatografia Líquida , Glicopeptídeos/análise , Glicoproteínas/química , Fígado/química , Camundongos , Polissacarídeos/análise , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem
19.
Clin Chim Acta ; 495: 309-317, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31014754

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) due to Hepatitis B viral (HBV) infection is a major cause in Asia-Pacific countries. Its early detection is of paramount importance using a marker having both sensitivity and specificity. The present study promises diagnostic and prognostic markers by the identification of site-specific glycoforms on Haptoglobin (Hp) using LC-MS/MS and lectin ELISA in liver diseased conditions in HBV infection. METHODS: Three groups of patients: chronic, liver cirrhosis and HCC with HBV infection along with controls were enrolled. Hp was purified using affinity column chromatography and, peptide sequence, N-glycosylation site, glycan composition and glycoforms were identified using mass spectrometry. Quantitative lectin ELISA was used to measure levels of fucosylation on Hp in liver diseases due to HBV. RESULTS: Hp levels were significantly lower in HCC when compared with Non-HCC cases (p < .05). Fucosylated glycoforms were significantly increased at site Asn184, Asn207 and Asn211 in liver diseased stages versus controls. A significant association was observed between the Fuc-Hp/Hp Elisa index and, advanced liver disease stages and controls using lectin Elisa (p < .001). CONCLUSION: Quantitation of fucosylation levels on Hp protein using Lectin ELISA may be useful glycobiomarker either alone or in combination (AFP + DCP + FucHp; AUC = 0.94) in HBV HCC diagnosis in clinical practice.


Assuntos
Carcinoma Hepatocelular/diagnóstico , Haptoglobinas/análise , Hepatite B/complicações , Imunoensaio/métodos , Hepatopatias/diagnóstico , Neoplasias Hepáticas/diagnóstico , Proteômica/métodos , Adulto , Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/análise , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/virologia , Cromatografia Líquida , Feminino , Fucose/metabolismo , Glicoproteínas/análise , Glicosilação , Humanos , Lectinas , Hepatopatias/virologia , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem
20.
mBio ; 10(2)2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015322

RESUMO

In eukaryotes, glycosylation plays a role in proteome stability, protein quality control, and modulating protein function; however, similar studies in bacteria are lacking. Here, we investigate the roles of general protein glycosylation systems in bacteria using the enteropathogen Campylobacter jejuni as a well-defined example. By using a quantitative proteomic strategy, we were able to monitor changes in the C. jejuni proteome when glycosylation is disrupted. We demonstrate that in C. jejuni , N-glycosylation is essential to maintain proteome stability and protein quality control. These findings guided us to investigate the role of N-glycosylation in modulating bacterial cellular activities. In glycosylation-deficient C. jejuni, the multidrug efflux pump and electron transport pathways were significantly impaired. We demonstrate that in vivo, fully glycosylation-deficient C. jejuni bacteria were unable to colonize its natural avian host. These results provide the first evidence of a link between proteome stability and complex functions via a bacterial general glycosylation system.IMPORTANCE Advances in genomics and mass spectrometry have revealed several types of glycosylation systems in bacteria. However, why bacterial proteins are modified remains poorly defined. Here, we investigated the role of general N-linked glycosylation in a major food poisoning bacterium, Campylobacter jejuni The aim of this study is to delineate the direct and indirect effects caused by disrupting this posttranslational modification. To achieve this, we employed a quantitative proteomic strategy to monitor alterations in the C. jejuni proteome. Our quantitative proteomic results linked general protein N-glycosylation to maintaining proteome stability. Functional analyses revealed novel roles for bacterial N-glycosylation in modulating multidrug efflux pump, enhancing nitrate reduction activity, and promoting host-microbe interaction. This work provides insights on the importance of general glycosylation in proteins in maintaining bacterial physiology, thus expanding our knowledge of the emergence of posttranslational modification in bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/fisiologia , Processamento de Proteína Pós-Traducional , Proteostase , Animais , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/veterinária , Campylobacter jejuni/patogenicidade , Galinhas , Cromatografia Líquida , Glicoproteínas/análise , Glicosilação , Proteoma/análise , Espectrometria de Massas em Tandem , Virulência
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