Effect of sex and RYR1 gene mutation on the muscle proteomic profile and main physiological biomarkers in pigs at slaughter.

Gender and RYR1 gene mutation might have an effect on the muscle metabolic characteristics and on the animal's stress at slaughter, which could influence the process of muscle-to-meat conversion. Forty-eight pigs were distributed in a design including two factors: sex (male/female) and RYR1 genotype (NN/Nn). At slaughter, physiological blood biomarkers and muscle proteome were analyzed and carcass and meat quality traits were registered. Females had higher serum levels of glucose, urea, C-reactive protein "CRP", Pig-MAP and glutation-peroxidase "GPx" and lower levels of lactate, showed faster muscle pH decline and higher meat exudation. RYR1 mutation increased serum creatinine, creatine kinase and CRP and decreased GPx. The proteomic study highlighted significant effects of gender and RYR1 genotype on proteins related to fibre composition, antioxidant defense and post mortem glycolytic pathway, which correlate to differences of meat quality. This study provides interesting information on muscle biomarkers of the ultimate meat quality that are modulated by the animal's individual susceptibility to stress at slaughter.

Primary bovine skeletal muscle cells enters apoptosis rapidly via the intrinsic pathway when available oxygen is removed.

Muscle cells undergo changes post-mortem during the process of converting muscle into meat, and this complex process is far from revealed. Recent reports have suggested programmed cell death (apoptosis) to be important in the very early period of converting muscle into meat. The dynamic balance that occurs between anti-apoptotic members, such as Bcl-2, and pro-apoptotic members (Bid, Bim) helps determine whether the cell initiates apoptosis. In this study, we used primary bovine skeletal muscle cells, cultured in monolayers in vitro, to investigate if apoptosis is induced when oxygen is removed from the growth medium. Primary bovine muscle cells were differentiated to form myotubes, and anoxia was induced for 6h. The anoxic conditions significantly increased (P<0.05) the relative gene expression of anti- and pro-apoptotic markers (Aif, Bcl-2, Bid and Bim), and the PARK7 (P<0.05) and Grp75 (Hsp70) protein expressions were transiently increased. The anoxic conditions also led to a loss of mitochondrial membrane potential, which is an early apoptotic event, as well as cytochrome c release from the mitochondria. Finally, reorganization and degradation of cytoskeletal filaments occurred. These results suggest that muscle cells enters apoptosis via the intrinsic pathway rapidly when available oxygen in the muscle diminishes post-mortem.

Sequence Analysis of Bitter Taste Receptor Gene Repertoires in Different Ruminant Species.

Bitter taste has been extensively studied in mammalian species and is associated with sensitivity to toxins and with food choices that avoid dangerous substances in the diet. At the molecular level, bitter compounds are sensed by bitter taste receptor proteins (T2R) present at the surface of taste receptor cells in the gustatory papillae. Our work aims at exploring the phylogenetic relationships of T2R gene sequences within different ruminant species. To accomplish this goal, we gathered a collection of ruminant species with different feeding behaviors and for which no genome data is available: American bison, chamois, elk, European bison, fallow deer, goat, moose, mouflon, muskox, red deer, reindeer and white tailed deer. The herbivores chosen for this study belong to different taxonomic families and habitats, and hence, exhibit distinct foraging behaviors and diet preferences. We describe the first partial repertoires of T2R gene sequences for these species obtained by direct sequencing. We then consider the homology and evolutionary history of these receptors within this ruminant group, and whether it relates to feeding type classification, using MEGA software. Our results suggest that phylogenetic proximity of T2R genes corresponds more to the traditional taxonomic groups of the species rather than reflecting a categorization by feeding strategy.

Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, ß1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy.

Very limited information on the post-implantatory effects of vitrification has been published till now. We observed in a previous study that the vitrification procedure for the cryopreservation of embryos introduced transcriptomic and proteomic modifications in the rabbit foetal placenta at the middle of gestation. Now, we have conducted a proteomic study to determine whether protein alterations in the foetal placenta induced by the vitrification procedure remain during pregnancy. In this study, we used 2D-DIGE and mass spectrometry (MALDI-TOF-TOF and LC-MS/MS analysis) to identify the protein changes during middle and late stages of gestation (Day 14 and Day 24, respectively) in rabbit foetal placenta. We identified 11 differentially expressed proteins at Day 14 and 13 proteins at Day 24. Data are available via ProteomeXchange with identifiers PXD001840 and PXD001836. In addition, we demonstrate the presence of three proteins, serum albumin, isocitrate dehydrogenase 1 [NADP+], and phosphoglycerate mutase 1, which were altered during pregnancy. We demonstrate the existence of changes in foetal placental protein during pregnancy induced by the vitrification procedure, which brings into question whether vitrification effects observed during foetal development could lead to physiological and metabolic disorders in adulthood. This effect, taken together with other effects reported in the literature, suggests that embryo cryopreservation is not neutral.

Systems biology: a new tool for farm animal science.

It is rapidly emerging that the tender meat phenotype is affected by an enormous amount of variables, not only tied to genetics (livestock breeding selection), but also to extrinsic factors, such as feeding conditions, physical activity, rearing environment, administration of hormonal growth promotants, pre-slaughter handling and stress. Proteomics has been widely accepted by meat scientists over the last years and is now commonly used to shed light on the postmortem processes involved in meat tenderization. This review discusses the latest findings with the use of proteomics and systems biology to study the different biochemical pathways postmortem aiming at understanding the concerted action of different molecular mechanisms responsible for meat quality. The conversion of muscle to meat postmortem can be described as a sequence of events involving molecular pathways controlled by a complex interplay of many factors. Among the different pathways emerging are the influence of apoptosis and lately also the role of autophagy in muscle postmortem development. This review thus, focus on how systems-wide integrated investigations (metabolomics, transcriptomics, interactomics, phosphoproteomics, mathematical modeling), which have emerged as complementary tools to proteomics, have helped establishing a few milestones in our understanding of the events leading from muscle to meat conversion.

The combination of glycosaminoglycans and fibrous proteins improves cell proliferation and early differentiation of bovine primary skeletal muscle cells.

Primary muscle cell model systems from farm animals are widely used to acquire knowledge about muscle development, muscle pathologies, overweight issues and tissue regeneration. The morphological properties of a bovine primary muscle cell model system, in addition to cell proliferation and differentiation features, were characterized using immunocytochemistry, western blotting and real-time PCR. We observed a reorganization of the Golgi complex in differentiated cells. The Golgi complex transformed to a highly fragmented network of small stacks of cisternae positioned throughout the myotubes as well as around the nucleus. Different extracellular matrix (ECM) components were used as surface coatings in order to improve cell culture conditions. Our experiments demonstrated improved proliferation and early differentiation for cells grown on surface coatings containing a mixture of both glycosaminoglycans (GAGs) and fibrous proteins. We suggest that GAGs and fibrous proteins mixed together into a composite biomaterial can mimic a natural ECM, and this could improve myogenesis for in vitro cell cultures.

Muscle and meat: new horizons and applications for proteomics on a farm to fork perspective.

Meat consumption is an important part of human diet with strong implications in health, economy and culture worldwide. Meat is a proteinaceous product and therefore proteomics holds a considerable value to the study of the protein events underlying meat production and processing. In this article we will review this subject in an integrated "farm to fork" perspective, i.e. focusing on all the major levels of the meat producing chain: farm, abattoir and transformation industry. We will focus on the use, importance and applications of proteomics, providing clear examples of the most relevant studies in the field. A special attention will be given to meat production, as well as quality control. In the latter, a particular emphasis will be given to microbial safety and the detection of frauds.

Differential proteomic analysis of drought stress response in leaves of common bean (Phaseolus vulgaris L.).

The majority of common bean plants are cultivated under drought conditions. Maintaining crop yields under drought stress is thus one of the biggest challenges facing bean production. In order to improve our understanding of the complex mechanisms involved in the response of common bean (Phaseolus vulgaris) to drought stress, a proteomic approach was used to identify drought-responsive proteins in leaves of two cultivars differing in their response to drought, Tiber and more sensitive Starozagorski cern. 2D-DIGE was used to compare differences in protein abundance between control and stressed plants. Fifty-eight proteins whose abundance changed significantly were identified by LC-MS/MS in Tiber and 64 in Starozagorski cern. The majority of identified proteins were classified into functional categories that include energy metabolism, photosynthesis, ATP interconversion, protein synthesis and proteolysis, stress and defence related proteins. Details of the function of the identified proteins and their abundance profiles in Tiber and Starozagorski are discussed. Interactions between identified proteins were demonstrated by bioinformatics analysis, enabling a more complete insight into biological pathways and molecular functions affected by drought stress. The results form the basis for a further understanding of the biochemical mechanisms of drought response in common bean.

Proteome changes in the insoluble protein fraction of bovine Longissimus dorsi muscle as a result of low-voltage electrical stimulation.

Changes induced by low-voltage electrical stimulation (ES; 0-95 V for 8 s; 95 V for 32 s) in the insoluble protein fraction of bovine longissimus dorsi (LD) muscle at 1 and 24h post-ES were investigated by proteomics. Protein abundance patterns from ten Norwegian Red (NRF) young bulls were compared, and significant changes due to ES were found by rotation test and partial least square (PLS) regression analyses. Five protein spots showed lower abundance in ES samples at both sampling times, and in addition, 10 proteins at 1 h post-ES and 13 proteins at 24 h post-ES changed significantly in abundance due to ES. Reduced abundance of full-length structural proteins in ES samples indicates an accelerated proteolysis due to ES. Moreover, increased abundance of small heat shock proteins indicates earlier initiation of stress responses due to ES. These findings provide a better understanding of the biochemical processes taking place as a result of ES during post mortem storage of meat.

Farm animal proteomics--a review.

In agricultural sciences as in all other areas of life science, the implementation of proteomics and other post-genomic tools is an important step towards more detailed understanding of the complex biological systems that control physiology and pathology of living beings. Farm animals are raised in large-scale operations, with the aim to obtain animal products for human consumption. Hence, understanding the biological traits that impact yield and quality of these products is the specific aim of much biological experimentation. However, most of the data gathered from experiments on e.g. swine and cattle are relevant not only for farm animal sciences, but also for adding to our understanding of complex biological mechanisms of health and disease in humans. The aim of this review is to present an overview of the specific topics of interest within farm animal proteomics, and to highlight some of the areas where synergy between classic model organism proteomics and farm animal proteomics is rapidly emerging. Focus will be on introducing the special biological traits that play an important role in food production, and on how proteomics may help optimize farm animal production.

Proteome changes in bovine longissimus thoracis muscle during the first 48 h postmortem: shifts in energy status and myofibrillar stability.

Changes in the insoluble protein fraction of bovine longissimus thoracis muscle from eight Norwegian Red (NRF) dual-purpose young bulls during the first 48 h postmortem were investigated by two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF MS/MS). Significant changes were observed in a total of 35 proteins, and of those, 26 were identified and divided into three different groups: metabolic enzymes, cellular defense/stress proteins, and structural proteins, according to their predicted function. The majority of the metabolic enzymes identified are involved in the energy metabolism of the cell, while the cellular defense/stress proteins can be related to regulation and stabilization of the myofibrillar proteins. Both easily soluble proteins as well as structural proteins were identified in the insoluble protein fraction. We have studied the changes in solubility during postmortem storage by comparing the postmortem changes in protein composition between the soluble and insoluble protein fractions. We have identified two metabolic enzymes (2,3-bisphosphoglycerat mutase and NADH dehydrogenase) and one protein involved in the stress responses/apoptosis of the cell (Hsp70) that have not been identified previously in the insoluble protein fraction. The occurrence of these easily soluble proteins in the insoluble protein fraction could be due to precipitation or aggregation, thereby going from a soluble to an insoluble state.

Improved dynamic range of protein quantification in silver-stained gels by modelling gel images over time.

Silver staining is a commonly used protein stain to visualise proteins separated by 2-DE. Despite this, the technique suffers from a limited dynamic range, making the simultaneous quantification of high- and low-abundant proteins difficult. In this paper we take advantage of the fact that silver staining is not an end-point stain by photographing the gels during development. This procedure provides information about the change in measured absorbance for each pixel in the protein spots on the gel. The maximum rate of change was found to be correlated with the amount of applied protein, providing a new way of estimating protein amount in 2-DE gels. We observed an improvement in the dynamic range of silver staining by up to two orders of magnitude.

Proteome changes in wheat subjected to different nitrogen and sulfur fertilizations.

Controlling the quality of wheat for breadmaking is a major concern for the milling and baking industry. Wheat flour quality depends on both the genetic background and environmental factors during growth and storage. Amount and timing of application of fertilizer are factors that affect wheat quality. This study investigated the effect of different levels of nitrogen and sulfur on the tris-soluble and glutenin protein fractions by 2D-electrophoresis. Multivariate analysis was performed to study changes in the proteome pattern. In the tris-soluble fraction 20 proteins were changed in abundance due to S fertilization, whereas 16 proteins were changed in the glutenin protein fraction. It was found that induced sulfur deficiency during growth resulted in the most pronounced effect on protein composition. Understanding which proteins are affected by varying levels of fertilizers may help tailor specific traits in various wheat varieties.

Retinoic acid dampens LPS-induced NF-kappaB activity: results from human monoblasts and in vivo imaging of NF-kappaB reporter mice.

Bacterial lipopolysaccharide (LPS) is a major inducer of systemic inflammatory reactions and oxidative stress in response to microbial infections and may cause sepsis. In the present study, we demonstrate that retinoic acid inhibits LPS-induced activation in transgenic reporter mice and human monoblasts through inhibition of nuclear factor kappaB (NF-kappaB). By using noninvasive molecular imaging of NF-kappaB luciferase reporter mice, we showed that administration of retinoic acid repressed LPS-induced whole-body luminescence, demonstrating in vivo the dynamics of retinoic acid's ability to repress physiologic response to LPS. Retinoic acid also inhibited LPS-induced NF-kappaB activity in the human myeloblastic cell line U937. Retinoic-acid-receptor-selective agonists mimicked - while specific antagonists inhibited - the effects of retinoic acid, suggesting the involvement of nuclear retinoic acid receptors. Retinoic acid also repressed LPS-induced transcription of NF-kappaB target genes such as IL-6, MCP-1 and COX-2. The effect of retinoic acid was dependent on new protein synthesis, was obstructed by a deacetylase inhibitor and was partly eliminated by a signal transducer and activator of transcription-1 (STAT1)/methyltransferase inhibitor, indicating that retinoic acid induces a new protein, possibly STAT1, that is involved in inhibiting NF-kappaB. This provides more evidence for retinoic acid's anti-inflammatory potential, which may have clinical implications in terms of fighting microbial infections.

Comparison of muscle proteome profiles in pure breeds of Norwegian Landrace and Duroc at three different ages.

We have used proteomics as a tool to unravel the changes in protein composition between two pure pig breeds and three age groups. Forty two female pigs of Norwegian Landrace and Duroc breed slaughtered at 6, 9 and 12 months age were included in the study. Each of the breeds was raised in separate farms and was slaughtered at the same day in a commercial abattoir. A sample from the adductor muscle was collected approximately 45min postmortem. Proteome analyses of the water soluble proteins using 2D electrophoresis showed that of the 1125 analyzed protein spots, 94 and 41 proteins are changed in abundance according to breed and age, respectively. A total of 63 changed proteins were identified by mass spectrometry. The identified proteins were classified as structural proteins, metabolic proteins, stress/defense proteins and other proteins. This demonstrates a difference in metabolism and muscle composition between breeds and age groups and shows that proteomics is a useful tool to uncover the molecular basis for physiological differences in muscles between pig breeds and age groups.

Combination of statistical approaches for analysis of 2-DE data gives complementary results.

Five methods for finding significant changes in proteome data have been used to analyze a two-dimensional gel electrophoresis data set. We used both univariate (ANOVA) and multivariate (Partial Least Squares with jackknife, Cross Model Validation, Power-PLS and CovProc) methods. The gels were taken from a time-series experiment exploring the changes in metabolic enzymes in bovine muscle at five time-points after slaughter. The data set consisted of 1377 protein spots, and for each analysis, the data set were preprocessed to fit the requirements of the chosen method. The generated results were one list from each analysis method of proteins found to be significantly changed according to the experimental design. Although the number of selected variables varied between the methods, we found that this was dependent on the specific aim of each method. CovProc and P-PLS focused more on getting the minimum necessary subset of proteins to explain properties of the samples. These methods ended up with less selected proteins. There was also a correlation between level of significance and frequency of selection for the selected proteins.

Pixel-based analysis of multiple images for the identification of changes: a novel approach applied to unravel proteome patterns [corrected] of 2-D electrophoresis gel images.

A novel approach for revealing patterns of proteome variation among series of 2-DE gel images is presented. The approach utilises image alignment to ensure that each pixel represents the same information across all gels. Gel images are normalised, and background corrected, followed by unfolding of the images to 1-D pixel vectors and analysing pixel vectors by multivariate data modelling. Information resulting from the data analysis is refolded back to the image domain for visualisation and interpretation. The method is rapid and suitable for automatic routines applied after the gel alignment. The approach is compared with spot volume analysis to illustrate how this approach can solve persistent problems like mismatch of protein spots, erroneous missing values and failure to detect variation in overlapping proteins. The method may also detect variation in the border area of saturated proteins. The approach is given the name pixel-based analysis of multiple images for the identification of changes (PMC). The method can be used for multiple images in general. Effects of pretreatment of the images are discussed.

Proteome changes in bovine longissimus thoracis muscle during the early postmortem storage period.

Postmortem changes in protein composition up to 24 h in bovine longissimus thoracis muscle were investigated by two-dimensional gel electrophoresis and MALDI-TOF MS/MS. A total of 47 spots were significantly changed the first 24 h postmortem. The 39 identified proteins can be divided into five groups: metabolic enzymes, defense and stress proteins, structural proteins, proteolytic enzymes, and unclassified proteins. The identified metabolic enzymes are all associated with ATP-generating pathways, either the glycolytic pathway or energy metabolism. In addition, several defense and stress proteins were changed in abundance in this study. These findings contribute to a better understanding of the biochemical processes during postmortem storage of meat.

Multivariate analysis of 2-DE protein patterns--practical approaches.

Practical approaches to the use of multivariate data analysis of 2-DE protein patterns are demonstrated by three independent strategies for the image analysis and the multivariate analysis on the same set of 2-DE data. Four wheat varieties were selected on the basis of their baking quality. Two of the varieties were of strong baking quality and hard wheat kernel and two were of weak baking quality and soft kernel. Gliadins at different stages of grain development were analyzed by the application of multivariate data analysis on images of 2-DEs. Patterns related to the wheat varieties, harvest times and quality were detected on images of 2-DE protein patterns for all the three strategies. The use of the multivariate methods was evaluated in the alignment and matching procedures of 2-DE gels. All the three strategies were able to discriminate the samples according to quality, harvest time and variety, although different subsets of protein spots were selected. The explorative approach of using multivariate data analysis and variable selection in the analyses of 2-DEs seems to be promising as a fast, reliable and convenient way of screening and transforming many gel images into spot quantities.