Project description:Background: Demographic studies have shown that the mean age of patients requiring coronary artery bypass surgery (CABG) is 68 years with approximately 44% of these patients being female . In recent studies, women have been shown to have a significantly greater operative risk as compared to men and that women have worse outcomes after cardiac surgery. Recently we have shown that cardioprotection afforded by cardioplegia is modulated by RNA and protein dependent mechanisms and is significantly decreased with age and in particular is significantly decreased in the aged female. Methods: The mechanisms through which cardioplegia affords cardioprotection are complex and can not be investigated as a single entity but rather must be investigated using a systems approach. To meet these needs we have used microarray and proteomic analysis. The combined use of these two techniques allows for the identification of functionally related gene groups and protein biomarkers. Mature 15 to 20 weeks; 3 to 4 kg) and aged (not senescent, >32 months; 5 to 6 kg) male and female New Zealand White rabbits were used for in situ blood perfused cardiopulmonary bypass. Control hearts received 30 minutes sham ischemia and 120 minutes sham reperfusion. Global ischemia (GI) hearts received 30 minutes of global ischemia. GI was achieved by crossclamping of the aorta. Cardioplegia hearts received cold blood cardioplegia (Deaconess Surgical Associates (DSA) solution (containing 50 uM diazoxide) prior to GI. Following 30 minutes of GI the crossclamp was released, the cardiopulmonary bypass was ceased and the hearts were reperfused for 120 minutes. The hearts were removed flushed of blood and then quick frozen in liquid nitrogen prior to isolation of RNA and protein. Microarray and high throughput proteomic analysis was performed as previously described. Results: Our studies demonstrate that the cardioprotection afforded by cardioplegia is modulated by RNA and protein dependent mechanisms. Our data also show that there are significant differences in the proteins expressed in the mature and aged. Principal component analysis indicated that in the mature male, mature female, and aged male the proteins expressed in control, global ischemia, and cardioplegia were diverse and clustered for individual treatments suggesting that the proteins had different expression levels in each treatment. In contrast, in the aged female the proteins expressed in global ischemia and in cardioplegia were clustered together, suggesting there was no difference in the proteins expressed during global ischemia and with cardioplegia. Glycolysis/gluconeogensis was a significant functional pathway for the aged male but not the mature male or female. Glycolytic and gluconeogenic proteins were significantly up-regulated in the aged male compared to the female and significantly up-regulated in the aged male with cardioplegia as compared to global ischemia. In the aged female 9 unique mitochondrial proteins were up-regulated with cardioplegia. These proteins were associated with oxidative phosphorylation, the electron transport chain, the formation of pyruvate, and the tricarboxylic acid (TCA) cycle, but not glycolysis nor gluconeogenesis. Conclusions: Our study demonstrates that the mitochondrion plays a key role in age and gender specific cardioprotection. The identification of these specific pathways and biomarkers should allow for the development of age and gender specific cardioprotection and significantly decrease morbidity and mortality in the aged female. Rabbit heart (atria and ventricles):control vs. global ischemia; control vs. cardioplegia; cardioplegia vs. global ischemia

Project description:Background: Demographic studies have shown that the mean age of patients requiring coronary artery bypass surgery (CABG) is 68 years with approximately 44% of these patients being female . In recent studies, women have been shown to have a significantly greater operative risk as compared to men and that women have worse outcomes after cardiac surgery. Recently we have shown that cardioprotection afforded by cardioplegia is modulated by RNA and protein dependent mechanisms and is significantly decreased with age and in particular is significantly decreased in the aged female. Methods: The mechanisms through which cardioplegia affords cardioprotection are complex and can not be investigated as a single entity but rather must be investigated using a systems approach. To meet these needs we have used microarray and proteomic analysis. The combined use of these two techniques allows for the identification of functionally related gene groups and protein biomarkers. Mature 15 to 20 weeks; 3 to 4 kg) and aged (not senescent, >32 months; 5 to 6 kg) male and female New Zealand White rabbits were used for in situ blood perfused cardiopulmonary bypass. Control hearts received 30 minutes sham ischemia and 120 minutes sham reperfusion. Global ischemia (GI) hearts received 30 minutes of global ischemia. GI was achieved by crossclamping of the aorta. Cardioplegia hearts received cold blood cardioplegia (Deaconess Surgical Associates (DSA) solution (containing 50 uM diazoxide) prior to GI. Following 30 minutes of GI the crossclamp was released, the cardiopulmonary bypass was ceased and the hearts were reperfused for 120 minutes. The hearts were removed flushed of blood and then quick frozen in liquid nitrogen prior to isolation of RNA and protein. Microarray and high throughput proteomic analysis was performed as previously described. Results: Our studies demonstrate that the cardioprotection afforded by cardioplegia is modulated by RNA and protein dependent mechanisms. Our data also show that there are significant differences in the proteins expressed in the mature and aged. Principal component analysis indicated that in the mature male, mature female, and aged male the proteins expressed in control, global ischemia, and cardioplegia were diverse and clustered for individual treatments suggesting that the proteins had different expression levels in each treatment. In contrast, in the aged female the proteins expressed in global ischemia and in cardioplegia were clustered together, suggesting there was no difference in the proteins expressed during global ischemia and with cardioplegia. Glycolysis/gluconeogensis was a significant functional pathway for the aged male but not the mature male or female. Glycolytic and gluconeogenic proteins were significantly up-regulated in the aged male compared to the female and significantly up-regulated in the aged male with cardioplegia as compared to global ischemia. In the aged female 9 unique mitochondrial proteins were up-regulated with cardioplegia. These proteins were associated with oxidative phosphorylation, the electron transport chain, the formation of pyruvate, and the tricarboxylic acid (TCA) cycle, but not glycolysis nor gluconeogenesis. Conclusions: Our study demonstrates that the mitochondrion plays a key role in age and gender specific cardioprotection. The identification of these specific pathways and biomarkers should allow for the development of age and gender specific cardioprotection and significantly decrease morbidity and mortality in the aged female.

Project description:A greater understanding of the proteins involved in reproduction can benefit animal production. New advances in proteomics are having a major impact on our understanding of how spermatozoa acquire their capacity for fertilization [1]. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function [2]. The sperm cell is one of the most highly differentiated cells and is composed of a head with a highly compacted chromatin structure and a large flagellum with midpiece that contains the required machinery for movement and therefore to deliver the paternal genetic and epigenetic content to the oocyte [3]. By being so highly differentiated, spermatozoa are advantageous cells to study proteomics of specific compartments such as the membrane, which basically is the area of major importance for its role in interacting with the surroundings and the oocyte [4]. The fusion of a sperm and an oocyte is a sophisticated process that must be preceded by suitable changes in the sperm's membrane composition [5]. Recent studies of spermatozoa from the proteomic point of view have allowed the identification of different proteins in spermatozoa that are responsible for the regulation of normal/defective sperm functions [6]. While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics [7]. Using this method, detailed proteomic data are now available for human [8], macaque [9,10], mouse [11], rat [12], bull [13-15], stallion [16], fruit fly [17], Caenorhabditis elegans [18], carp [19], rainbow trout [20], mussel [21], ram [22], honeybee [23] and rooster [24] sperm membrane proteins. Rabbit (Oryctolagus cuniculus) is an important mammalian species worldwide, being at the same time of commercial interest and a research model animal. European rabbit meat production is approximately 500 thousand tons, corresponding to a 30% share of world production [25]. Besides, rabbits account for the seventh highest number of animals slaughtered per year in the European Union-27, with 347,603 × 1000 head in 2014 [26]. In a previous work, we identified and quantified rabbit seminal plasma proteins between two different genotypes [27], concluding the clear effect of genotype in the abundance of certain seminal plasma proteins. However, it is unknown at present whether these differences also exist at sperm proteome level. Therefore, the aim of the present study was to characterise rabbit sperm membrane proteins through NanoLC-MS/MS analysis focusing on the influence of the genetic origin.

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Oryctolagus cuniculus

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