Project description:Intracranial aneurysms tend to form at bifurcation apices, where flow impingement causes high frictional force (or wall shear stress, WSS) and flow acceleration and deceleration that create positive and negative streamwise gradients in WSS (WSSG), respectively. In vivo, intracranial aneurysms initiate under high WSS and positive WSSG. Little is known about the responses of endothelial cells (ECs) to either positive or negative WSSG under high WSS conditions. We used cDNA microarrays to profile EC gene expression exposed to positive WSSG vs. negative WSSG for 24 hours in a flow chamber with converging and diverging channels, respectively. WSS varied between 3.5 and 28.4 Pa in each gradient channel. GO and biological pathway analysis indicated that positive WSSG favored proliferation, apoptosis, and extracellular matrix processing while decreasing expression of pro-inflammatory genes. A subset of characteristic genes was validated using qPCR: Genes for ADAMTS1, CKAP2 and NCEH1 had higher expression under positive WSSG compared to negative WSSG while TAGLN, THBS1, VCAM1, CCL2, and CSF2 had lower expression. To determine if these patterns of expression are also exhibited in vivo, we tested whether the extracellular matrix related protein ADAMTS1 and proliferation were modulated by positive WSSG during intracranial aneurysm initiation. An aneurysm was induced at the basiliar terminus in rabbits by bilateral carotid ligation. WSSG at the bifurcation was determined by computational fluid dynamic simulations from 3D angiography and mapped on immunofluorescence staining for ADAMTS1 and the proliferation marker, Ki-67. Endothelial ADAMTS1 protein and Ki-67 were significantly higher in regions with positive WSSG compared to adjacent sites where WSSG was negative. Our results indicate that WSSG can elicit distinct gene expression profiles in ECs. Increased matrix processing and high levels of proliferation under positive WSSG could contribute to intracranial aneurysm initiation by causing transient gaps in the endothelium or disrupting EC signals to smooth muscle cells. Time-matched bovine aortic endothelial cells were exposed to positive wall shear stress gradient, negative wall shear stress gradient, and two no gradient samples: uniform WSS of 3.5 Pa and high WSS of 28.4 Pa for 24 hrs in an in vitro flow loop system. RNA was extracted and hybridized on Affymetrix microarrays. There were 12 samples in total, four flow conditions with three replicates each.

Project description:Intracranial aneurysms tend to form at bifurcation apices, where flow impingement causes high frictional force (or wall shear stress, WSS) and flow acceleration and deceleration that create positive and negative streamwise gradients in WSS (WSSG), respectively. In vivo, intracranial aneurysms initiate under high WSS and positive WSSG. Little is known about the responses of endothelial cells (ECs) to either positive or negative WSSG under high WSS conditions. We used cDNA microarrays to profile EC gene expression exposed to positive WSSG vs. negative WSSG for 24 hours in a flow chamber with converging and diverging channels, respectively. WSS varied between 3.5 and 28.4 Pa in each gradient channel. GO and biological pathway analysis indicated that positive WSSG favored proliferation, apoptosis, and extracellular matrix processing while decreasing expression of pro-inflammatory genes. A subset of characteristic genes was validated using qPCR: Genes for ADAMTS1, CKAP2 and NCEH1 had higher expression under positive WSSG compared to negative WSSG while TAGLN, THBS1, VCAM1, CCL2, and CSF2 had lower expression. To determine if these patterns of expression are also exhibited in vivo, we tested whether the extracellular matrix related protein ADAMTS1 and proliferation were modulated by positive WSSG during intracranial aneurysm initiation. An aneurysm was induced at the basiliar terminus in rabbits by bilateral carotid ligation. WSSG at the bifurcation was determined by computational fluid dynamic simulations from 3D angiography and mapped on immunofluorescence staining for ADAMTS1 and the proliferation marker, Ki-67. Endothelial ADAMTS1 protein and Ki-67 were significantly higher in regions with positive WSSG compared to adjacent sites where WSSG was negative. Our results indicate that WSSG can elicit distinct gene expression profiles in ECs. Increased matrix processing and high levels of proliferation under positive WSSG could contribute to intracranial aneurysm initiation by causing transient gaps in the endothelium or disrupting EC signals to smooth muscle cells.

Project description:Proteins are the major constituents of muscle and are key molecules regulating the metabolic changes during conversion of muscle to meat. Brazil is one of the largest exporters of beef and most Brazilian cattle are composed by zebu (Nellore) genotype. Bos indicus beef is generally leaner and tougher than Bos taurus such as Angus. The aim of this study was to compare the muscle proteomic and phosphoproteomic profile of Angus and Nellore. Seven animals of each breed previously subjected the same growth management were confined for 84 days. Proteins were extracted from Longissimus lumborum samples collected immediately after slaughter and separated by two-dimensional electrophoresis. Pro-Q Diamond stain was used in phosphoproteomics. Proteins identification was performed using matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Tropomyosin alpha-1 chain, troponin-T, myosin light chain-1 fragment, cytoplasmic malate dehydrogenase, alpha-enolase and 78 kDa glucose-regulated protein were more abundant in Nellore, while myosin light chain 3, prohibitin, mitochondrial stress-70 protein and heat shock 70 kDa protein 6 were more abundant in Angus (P<0.05). Nellore had higher phosphorylation of myosin regulatory light chain-2, alpha actin-1, triosephosphate isomerase and 14-3-3 protein epsilon. However, Angus had greater phosphorylation of phosphoglucomutase-1 and troponin-T (P<0.05). Therefore, proteins involved in contraction and muscle organization, myofilaments expressed in fast or slow-twitch fibers and heat shock proteins localized in mitochondria or sarcoplasmic reticulum and involved in cell flux of calcium and apoptosis might be associated with differences in beef quality between Angus and Nellore. Furthermore, prohibitin appears to be a potential biomarker of intramuscular fat in cattle. Additionally, differences in phosphorylation of myofilaments and glycolytic enzymes could be involved with differences in muscle contraction force, susceptibility to calpain, apoptosis and postmortem glycolysis, which might also be related to differences in beef quality among Angus and Nellore.

Project description:BackgroundReference genomes are essential in the analysis of genomic data. As the cost of sequencing decreases, multiple reference genomes are being produced within species to alleviate problems such as low mapping accuracy and reference allele bias in variant calling that can be associated with the alignment of divergent samples to a single reference individual. The latest reference sequence adopted by the scientific community for the analysis of cattle data is ARS_UCD1.2, built from the DNA of a Hereford cow (Bos taurus taurus-B. taurus). A complementary genome assembly, UOA_Brahman_1, was recently built to represent the other cattle subspecies (Bos taurus indicus-B. indicus) from a Brahman cow haplotype to further support analysis of B. indicus data. In this study, we aligned the sequence data of 15 B. taurus and B. indicus breeds to each of these references.ResultsThe alignment of B. taurus individuals against UOA_Brahman_1 detected up to five million more single-nucleotide variants (SNVs) compared to that against ARS_UCD1.2. Similarly, the alignment of B. indicus individuals against ARS_UCD1.2 resulted in one and a half million more SNVs than that against UOA_Brahman_1. The number of SNVs with nearly fixed alternative alleles also increased in the alignments with cross-subspecies. Interestingly, the alignment of B. taurus cattle against UOA_Brahman_1 revealed regions with a smaller than expected number of counts of SNVs with nearly fixed alternative alleles. Since B. taurus introgression represents on average 10% of the genome of Brahman cattle, we suggest that these regions comprise taurine DNA as opposed to indicine DNA in the UOA_Brahman_1 reference genome. Principal component and admixture analyses using genotypes inferred from this region support these taurine-introgressed loci. Overall, the flagged taurine segments represent 13.7% of the UOA_Brahman_1 assembly. The genes located within these segments were previously reported to be under positive selection in Brahman cattle, and include functional candidate genes implicated in feed efficiency, development and immunity.ConclusionsWe report a list of taurine segments that are in the UOA_Brahman_1 assembly, which will be useful for the interpretation of interesting genomic features (e.g., signatures of selection, runs of homozygosity, increased mutation rate, etc.) that could appear in future re-sequencing analysis of indicine cattle.

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Bos taurus

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