Elucidating the Molecular Composition of Cartilage by Proteomics.

Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers.

QTL associated with barley (Hordeum vulgare) feed quality traits measured through in situ digestion

Barley (Hordeum vulgare) is a major feed source for the intensive livestock industry. Competitiveness against other cereal grains depends largely on the price per unit of expressed feed quality. The traits which contribute to feed quality in barley are largely quantitative in nature but little is known about their genetic control. A study to identify the quantitative trait loci (QTLs) associated with feed quality was performed using a F6-derived recombinant inbred barley population. Samples from each line were incubated in the rumen of fistulated cattle, recovered, washed and dried for determination of in situ dry matter digestibility. Additionally, both pre- and post-digestion samples were analysed to quantify the content of key quality components relating to acid detergent fibre, total starch and protein. The data was used to identify trait-associated QTLs. Genetic analysis identified significant QTLs on chromosomes 2H, 5H and 7H. Genetic markers linked to these QTL should provide an effective tool for the selection and improvement of feed barley in the future.

Modifications in Trypsin Digestion Protocol for Increasing the Efficiency and Coverage

Standard trypsin digestion protocol of proteins followed by MALDI-MS analysis has been realized as an important tool for the identification and characterization of proteins. In this article, we proposed the elimination of the step of `staining/de-staining of gel pieces' in in-gel digestion protocol in order to improve the efficiency of trypsin digestion. Coomassie dye is known to interfere with digestion of proteins by trypsin and the procedure of staining-de-staining could result in loss of photoaffinity probe, post translational modifications and catalytic activities of enzymes. Further, we studied parameters like hydrophobicity and isoelectric point, and attempted to quantitatively relate it to the efficiency of trypsin digestion. We suggest that properties of proteins should be considered and trypsin digestion protocol should be appropriately modified as per sequence and other information.

The effect of condensed tannins in fresh sainfoin (Onobrychis viciifolia) on in vivo and in situ digestion in sheep

This study focused on effects of structure, content and biological activity of condensed tannins (CT) in leaves, stems and whole plant of sainfoin (Onobrychis viciifolia) on its in vivo and in situ digestive characteristics in sheep. Sainfoin was studied as fresh forage during the first vegetation cycle at two phenological stages (i.e., end of flowering and green seeds) and during the second vegetation cycle (i.e., start of flowering). The feeding experiment used 12 sheep; with six dosed, through the rumen cannula, with polyethylene glycol (PEG) to neutralise CT effects. Organic matter digestibility (OMD), total tract N disappearance and N balance were measured in sheep fed the whole plant. The residues of dry matter (DM) and N from nylon bags suspended in the rumen were determined on leaves and stems. Intestinal digestibility was measured using other, intestinally fistulated sheep. PEG addition and vegetation cycle increased total tract N digestibility (P<0.001) but PEG affected OMD only at the end of flowering. PEG inactivated the CT and increased urinary N excretion (P<0.05) but this was offset by lower faecal N excretion (P<0.001). Feeding sainfoin can be used to alter the form of excreted N (i.e., urine vs faeces) and thus potentially reduce environmental N pollution without affecting body N retention. Kinetic studies of total N, ammonia N (NH3-N) and volatile fatty acids (VFA) in rumen fluid were made before and 1.5, 3 and 6 h after feeding. Sainfoin CT decreased rumen fluid soluble N (P<0.05) and NH3-N (P<0.01). Ruminal N disappearance (DisN) of leaves or stems was lower in the presence of active CT compared to PEG-inactivated CT (P<0.001) for both vegetation cycles. PEG also increased intestinal digestibility (P<0.05) of leaves and stems. Leaves had lower ruminal DisN, but higher N disappearing from intestine than stems. The biological activity and content of CT in the whole plant decreased as phenological stage increased. Prodelphinidin:procyanidin (PD:PC) ratios of leaves varied with vegetation cycle and phenological stage. The molecular size of CT in the whole plant, as indicated by their mean degree of polymerisation (mDP), was lowest at the start of flowering and coincided with the higher biological activity and content of CT.

In situ hybridization of bat chromosomes with human (TTAGGG)n probe, after previous digestion with Alu I

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Validation and in situ application of an automated dissolved nickel monitor for estuarine studies

The validation of a fully automated dissolved Ni monitor for in situ estuarine studies is presented, based on adsorptive cathodic stripping voltammetry (AdCSV). Dissolved Ni concentrations were determined following on-line filtration and UV digestion, and addition of an AdCSV ligand (dimethyl glyoxime) and pH buffer (N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid). The technique is capable of up to six fully quantified Ni measurements per hour. The automated in situ methodology was applied successfully during two surveys on the Tamar estuary (south west Britain). The strongly varying sample matrix encountered in the estuarine system did not present analytical interferences, and each sample was quantified using internal standard additions. Up to 37 Ni measurements were performed during each survey, which involved 13 h of continuous sampling and analysis. The high resolution data from the winter and summer tidal cycle studies allowed a thorough interpretation of the biogeochemical processes in the studied estuarine system.

Characterization of glutaraldehyde-immobilized chymotrypsin and an in-situ immobilized enzyme reactor using capillary electrophoresis-based peptide mapping

La digestion enzymatique des protéines est une méthode de base pour les études protéomiques ainsi que pour le séquençage en mode « bottom-up ». Les enzymes sont ajoutées soit en solution (phase homogène), soit directement sur le gel polyacrylamide selon la méthode déjà utilisée pour l’isolation de la protéine. Les enzymes protéolytiques immobilisées, c’est-à-dire insolubles, offrent plusieurs avantages tels que la réutilisation de l’enzyme, un rapport élevé d’enzyme-sur-substrat, et une intégration facile avec les systèmes fluidiques. Dans cette étude, la chymotrypsine (CT) a été immobilisée par réticulation avec le glutaraldehyde (GA), ce qui crée des particules insolubles. L’efficacité d’immobilisation, déterminée par spectrophotométrie d’absorbance, était de 96% de la masse totale de la CT ajouté. Plusieurs différentes conditions d’immobilisation (i.e., réticulation) tels que la composition/pH du tampon et la masse de CT durant la réticulation ainsi que les différentes conditions d’entreposage tels que la température, durée et humidité pour les particules GA-CT ont été évaluées par comparaison des cartes peptidiques en électrophorèse capillaire (CE) des protéines standards digérées par les particules. Les particules de GA-CT ont été utilisés pour digérer la BSA comme exemple d’une protéine repliée large qui requit une dénaturation préalable à la digestion, et pour digérer la caséine marquée avec de l’isothiocyanate de fluorescéine (FITC) comme exemple d’un substrat dérivé afin de vérifier l’activité enzymatique du GA-CT dans la présence des groupements fluorescents liés au substrat. La cartographie peptidique des digestions par les particules GA-CT a été réalisée par CE avec la détection par absorbance ultraviolet (UV) ou fluorescence induite par laser. La caséine-FITC a été, en effet, digérée par GA-CT au même degré que par la CT libre (i.e., soluble). Un microréacteur enzymatique (IMER) a été fabriqué par immobilisation de la CT dans un capillaire de silice fondu du diamètre interne de 250 µm prétraité avec du 3-aminopropyltriéthoxysilane afin de fonctionnaliser la paroi interne avec les groupements amines. Le GA a été réagit avec les groupements amine puis la CT a été immobilisée par réticulation avec le GA. Les IMERs à base de GA-CT étaient préparé à l’aide d’un système CE automatisé puis utilisé pour digérer la BSA, la myoglobine, un peptide ayant 9 résidus et un dipeptide comme exemples des substrats ayant taille large, moyenne et petite, respectivement. La comparaison des cartes peptidiques des digestats obtenues par CE-UV ou CE-spectrométrie de masse nous permettent d’étudier les conditions d’immobilisation en fonction de la composition et le pH du tampon et le temps de réaction de la réticulation. Une étude par microscopie de fluorescence, un outil utilisé pour examiner l’étendue et les endroits d’immobilisation GA-CT dans l’IMER, ont montré que l’immobilisation a eu lieu majoritairement sur la paroi et que la réticulation ne s’est étendue pas si loin au centre du capillaire qu’anticipée.

Efeito do emurchecimento e da adição de fubá na degradabilidade in situ da silagem de alfafa (Medicago sativa L.)

O objetivo deste ensaio foi avaliar o efeito do emurchecimento e da adição de fubá durante o processo de ensilagem da alfafa sobre a degradabilidade in situ da matéria seca (MS), da fibra em detergente neutro (FDN) e da proteína bruta (PB). Foram utilizados três bovinos adultos mestiços fistulados no rúmen, nos quais foram incubados seis tipos de silagens (1-alfafa fresca; 2-alfafa fresca +5% de fubá; 3-alfafa fresca +10% de fubá; 4-alfafa emurchecida; 5-alfafa emurchecida + 5% de fubá ; 6-alfafa emurchecida + 10% de fubá) durante três tempos de incubação (6, 24, 96 horas). O delineamento experimental foi em parcelas subdivididas, com tratamentos em parcelas, tempos em subparcelas e animais como blocos. A adição de fubá à silagem aumentou (P<0,05) a degradação da MS e FDN com o tempo de incubação. O emurchecimento proporcionou diminuição (P<0,05) na degradabilidade dos nutrientes, entretanto esse efeito foi ligeiramente contornado pela adição de 10% de fubá. Os tratamentos com a adição de fubá e o emurchecimento proporcionaram maior taxa de degradabilidade da MS de fração b, entretanto o emurchecimento diminuiu o potencial de degradação a, enquanto que a adição de níveis de fubá não alterou a degradabilidade da MS dessa fração. A degradação da fração b da FDN aumentou com a adição de fubá e diminuiu com o emurchecimento, entretanto houve aumento dessa fração quando se adicionou fubá à forragem emurchecida. A degradação da fração a da PB aumentou com a adição de fubá e diminuiu com o emurchecimento; para a fração b ocorreu o inverso. Concluiu-se que a prática do emurchecimento, por reduzir a fração solúvel da forragem, diminui a degradabilidade dos nutrientes da silagem de alfafa, que no entanto pode ser melhorada com a adição de fubá.

Comparação do sistema de monitoramento computadorizado de digestão in vitro com os métodos in vivo e in situ: 2. Uso do resíduo da matéria seca de forragens

RESUMO - O objetivo deste trabalho foi comparar o sistema de monitoramento computadorizado da produção de gás in vitro com os métodos in vivo e in situ. Nas comparações, foram utilizadas silagens de milho com alto/baixo teores de matéria seca, com/sem inoculante. A digestibilidade das silagens com alto teor de matéria seca (MS), com/sem inoculação, não apresentou diferenças entre os métodos analisados. Quando avaliadas separadamente do efeito do inóculo, essas silagens diferiram nos métodos in vitro/gás e in situ, mas não no in vivo. Entretanto, quando analisadas somente sob efeito do inoculante, apenas no método in situ houve diferença. Não foram encontradas diferenças na digestibilidade da fibra em detergente neutro (FDN) das silagens com alta ou baixa MS, inoculadas ou não, bem como em relação ao pH ao final da digestão. em conclusão, o desaparecimento da MS e FDN quantificada pelo resíduo no sistema in vitro/gás foi semelhante aos demais métodos avaliados.

Degradabilidade ruminal e digestibilidade intestinal de alimentos por intermédio da técnica in situ associada à do saco de náilon móvel

Objetivou-se no presente trabalho verificar a degradação ruminal e a digestibilidade intestinal e total da matéria seca (MS) e da proteína bruta (PB) do farelo de soja, do grão de milho, do melaço em pó, da farinha de peixe, da farinha de penas e do feno de alfafa, por intermédio da técnica de degradabilidade ruminal in situ associada à técnica do saco de náilon móvel. As amostras dos alimentos foram moídas e colocadas em duplicata em sacos de náilon de 10x5 cm (48 micras) nas quantidades de 15 e 5 mg de MS/cm² para os alimentos concentrados e feno de alfafa, respectivamente. Os sacos de náilon permaneceram incubados no rúmen de bois holandeses por 0; 2; 6; 8; 24 e 48 h; e 0; 8; 12; 24; 48; 72 e 96 horas, respectivamente, sendo depois retirados e sua duplicata inserida no duodeno através de uma cânula. Posteriormente, os sacos foram coletados junto com as fezes. Os valores de degradabilidade efetiva da PB para uma velocidade de passagem de 5%/hora, para o melaço em pó, grão de milho, farelo de soja, farinha de peixe, farinha de penas e feno de alfafa, foram de 100,00; 62,50; 57,90; 39,30; 34,20 e 60,90%, respectivamente; a digestibilidade intestinal de 100,00; 96,05; 99,79; 98,19; 96,07 e 94,64%, respectivamente; e a digestibilidade total de 100,00; 97,86; 99,87; 98,88; 97,35 e 98,09%, respectivamente. Verificou-se que as proteínas do melaço foram totalmente solúveis no rúmen, sendo as do milho, feno e farelo de soja bastante degradadas, além de possuírem um aproveitamento quase total no intestino. As proteínas das farinhas de peixe e de penas apresentaram baixa solubilidade ruminal e alta digestibilidade intestinal, sendo a farinha de peixe levemente mais digerida no intestino do que a farinha de penas.

Aplicação da técnica de RT-PCR in situ na detecção da co-infecção pelo Coronavírus grupo 3 (TCoV) e Astrovírus (TAstV-2) em perus com quadro agudo de enterite

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Buffalo cheese whey proteins, identification of a 24 kda protein and characterization of their hydrolysates: in vitro gastrointestinal digestion

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In Situ Observation of the Thermal Decomposition of Weddelite by Heating Stage Environmental Scanning Electron Microscopy

The morphological and chemical changes occurring during the thermal decomposition of weddelite, CaC2O4·2H2O, have been followed in real time in a heating stage attached to an Environmental Scanning Electron Microscope operating at a pressure of 2 Torr, with a heating rate of 10 °C/min and an equilibration time of approximately 10 min. The dehydration step around 120 °C and the loss of CO around 425 °C do not involve changes in morphology, but changes in the composition were observed. The final reaction of CaCO3 to CaO while evolving CO2 around 600 °C involved the formation of chains of very small oxide particles pseudomorphic to the original oxalate crystals. The change in chemical composition could only be observed after cooling the sample to 350 °C because of the effects of thermal radiation.