The Bovine HapMap Consortium* The imprints of domestication and breed development on the genomes of livestock likely differ from those of companion animals. A deep draft sequence assembly of shotgun reads from a single Hereford female and comparative sequences sampled from six additional breeds were used to develop probes to interrogate 37,470 single-nucleotide polymorphisms (SNPs) in 497 cattle from 19 geographically and biologically diverse breeds. These data show that cattle have undergone a rapid recent decrease in effective population size from a very large ancestral population, possibly due to bottlenecks associated with domestication, selection, and breed formation. Domestication and artificial selection appear to have left detectable signatures of selection within the cattle genome, yet the current levels of diversity within breeds are at least as great as exists within humans.T he emergence of modern civilization was accompanied by adaptation, assimilation, and interbreeding of captive animals. In cattle (Bos taurus), this resulted in the development of individual breeds differing in, for example, milk yield, meat quality, draft ability, and tolerance or resistance to disease and pests. However, despite mapping and diversity studies (1-5) and the identification of mutations affecting some quantitative phenotypes (6-8), the detailed genetic structure and history of cattle are not known.Cattle occur as two major geographic types, the taurine (humpless-European, African, and Asian) and indicine (humped-South Asian, and East African), which diverged >250 thousand years ago (Kya) (3). We sampled individuals representing 14 taurine (n = 376), three indicine (n = 73) (table S1), and two hybrid breeds (n = 48), as well as two individuals each of Bubalus quarlesi and Bubalus bubalis, which diverged from Bos taurus~1.25 to 2.0 Mya (9, 10). All breeds except Red Angus (n = 12) were represented by at least 24 individuals. We preferred individuals that were unrelated for ≥4 generations; however, each breed had one or two sire, dam, and progeny trios to allow assessment of genotype quality.Single-nucleotide polymorphisms (SNPs) that were polymorphic in many populations were primarily derived by comparing whole-genome sequence reads representing five taurine and one indicine breed to the reference genome assembly obtained from a Hereford cow (10) (table S2). This led to the ascertainment of SNPs with high minor allele frequencies (MAFs) within the discovery breeds (table S5). Thus, as expected, with trio progeny removed, SNPs discovered within the taurine breeds had higher average MAFs

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3β (GSK-3β). Alternatively, receptor tyrosine kinase or certain G protein-coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3β, via protein kinase B/Akt and mTOR/p70 s6k pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3β on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection.

Background-Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor-␤ (TGF-␤). Recent evidence suggests that losartan, an angiotensin II type 1 blocker that blunts TGF-␤ activation, may be an effective treatment for MFS. We hypothesized that dysregulation of TGF-␤ might be mirrored in circulating TGF-␤ concentrations. Methods and Results-Serum obtained from MFS mutant mice (Fbn1C1039G/ϩ ) treated with losartan was analyzed for circulating TGF-␤1 concentrations and compared with those from placebo-treated and wild-type mice. Aortic root size was measured by echocardiography. Data were validated in patients with MFS and healthy individuals. In mice, circulating total TGF-␤1 concentrations increased with age and were elevated in older untreated Fbn1 C1039G/ϩ mice compared with wild-type mice (Pϭ0.01; nϭ16; meanϮSEM, 115Ϯ8 ng/mL versus nϭ17; meanϮSEM, 92Ϯ4 ng/mL). Losartan-treated Fbn1 C1039G/ϩ mice had lower total TGF-␤1 concentrations compared with age-matched Fbn1

This unit outlines the steps required to prepare a sample for MS analysis following protein separation or enrichment by gel electrophoresis, liquid chromatography, and affinity capture within the context of a bottom-up proteomics workflow in which the protein is first broken up into peptides, either by chemical or enzymatic digestion, prior to MS analysis. Also included are protocols for enrichment at the peptide level, including phosphopeptide enrichment and reversed-phase chromatography for sample purification immediately prior to MS analysis. Finally, there is a discussion regarding the types of MS technologies commonly used to analyze proteomics samples, as well as important parameters that should be considered when analyzing the MS data to ensure stringent and robust protein identifications and characterization. Keywordsin-solution digestion; in-gel digestion; peptide desalting proteomics; mass spectrometry Thingholm et al., 2008. See above. This is a landmark paper in which both IMAC and TiO 2 are combined in a sequential manner to allow expanded phosphopeptide enrichment. Shevchenko et al., 1996. See above. This is the most commonly used silver staining and peptide extraction method for MS compatible samples. Key References Internet Resources

Background: Candidate biomarkers discovered with high-throughput proteomic techniques (along with many biomarkers reported in the literature) must be rigorously validated. The simultaneous quantitative assessment of multiple potential biomarkers across large cohorts presents a major challenge to the field. Multiplex immunoassays represent a promising solution, with the potential to provide quantitative data via parallel analyses. These assays also require substantially less sample and reagents than the traditional ELISA (which is further limited by its ability to measure only a single antigen). We have measured the reproducibility, reliability, robustness, accuracy, and throughput of commercially available multiplex immunoassays to ascertain their suitability for serum biomarker analysis and validation. Methods: Assay platforms MULTI-ARRAY (Meso Scale Discovery), Bio-Plex (Bio-Rad Laboratories), A2 (Beckman Coulter), FAST Quant (Whatman Schleicher & Schuell BioScience), and FlowCytomix (Bender MedSystems) were selected as representative examples of technologies currently used for high-throughput immunoanalysis. All assays were performed according to protocols specified by the manufacturers and with the reagents (diluents, calibrators, blocking reagents, and detecting-antibody mixtures) included with their kits. Results: The quantifiable interval determined for each assay and antigen was based on precision (CV < 25%) and percentage recovery (measured concentration within 20% of the actual concentration). The MULTI-ARRAY and Bio-Plex assays had the best performance with the lowest limits of detection, and the MULTI-ARRAY system had the most linear signal output over the widest concentration range (105 to 106). Cytokine concentrations in unspiked and cytokine-spiked serum samples from healthy individuals were further investigated with the MULTI-ARRAY and Bio-Plex assays. Conclusions: The MULTI-ARRAY and Bio-Plex multiplex immunoassay systems are the most suitable for biomarker analysis or quantification.