Effects of the number of markers per haplotype and clustering of haplotypes on the accuracy of QTL mapping and prediction of genomic breeding values

The aim of this paper was to compare the effect of haplotype definition on the precision of QTL-mapping and on the accuracy of predicted genomic breeding values. In a multiple QTL model using identity-by-descent (IBD) probabilities between haplotypes, various haplotype definitions were tested i.e. including 2, 6, 12 or 20 marker alleles and clustering base haplotypes related with an IBD probability of > 0.55, 0.75 or 0.95. Simulated data contained 1100 animals with known genotypes and phenotypes and 1000 animals with known genotypes and unknown phenotypes. Genomes comprising 3 Morgan were simulated and contained 74 polymorphic QTL and 383 polymorphic SNP markers with an average r² value of 0.14 between adjacent markers. The total number of haplotypes decreased up to 50% when the window size was increased from two to 20 markers and decreased by at least 50% when haplotypes related with an IBD probability of > 0.55 instead of > 0.95 were clustered. An intermediate window size led to more precise QTL mapping. Window size and clustering had a limited effect on the accuracy of predicted total breeding values, ranging from 0.79 to 0.81. Our conclusion is that different optimal window sizes should be used in QTL-mapping versus genome-wide breeding value prediction.     

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.     

Automated affinity chromatography measurements of compound mixtures using a lab-on-valve apparatus coupled to electrospray ionization mass spectrometry

We report a fully automated affinity chromatography system using a lab-on-valve (LOV) apparatus coupled to an electrospray ionization ion-trap mass spectrometer (ESI-MS). The system allows simultaneous measurements of multiple ligand affinities to proteins immobilized on beads. Bead regeneration, column repacking, and repetitive measurements are achieved on the time scale of several minutes. In this study, the system was used to screen the binding of a peptide mixture to human and Trypanosoma brucei (T. brucei) truncated Pex5 (tPex5) proteins. Equilibrium dissociation constants (K(d)) were measured for T. brucei tPex5 and compared to the values obtained by a fluorescence-based competition assay. The three peptides that showed affinity toward tPex5 had K(d) values that were comparable in magnitude (within a factor of 5) and showed the same ranking order as those from manual fluorescence measurements. With 12 min of sample infusion, the entire sample-to-sample cycle takes about 15 min and can be repeated without any preparation between runs. For T. brucei tPex5 affinity measurements, 1 mg of protein was sufficient for 35 repetitive analyses in the automated LOV-ESI-MS apparatus. The system allows rapid determination of K(d) in the range of 10(-5)-10(-7) M for sample mixtures and is suitable for screening a large number of compounds against multiple proteins.     

Epidemiology of training injuries in amateur taekwondo athletes: a retrospective cohort study

The objectives of this study were to estimate the incidence and describe the pattern and severity of training injuries in taekwondo, and to compare pattern and severity of training injuries with competition injuries. One hundred and fifty-two active Australian amateur taekwondo athletes, aged 12 years or over, completed an online survey comprising questions on training exposure and injury history over the preceding 12 months. The main outcome measures were: overall injury incidence rate per athlete-year; training injury incidence rate per athlete-year, per 1000 athlete-training-sessions, and per 1000 athlete-hours of training; injury severity; and injury proportions by anatomical region and by type of injury. Injury incidence rates were calculated with 95% confidence intervals using standard methods, while injury proportions were compared using Fisher''s exact test. The vast majority (81.5%) of taekwondo injuries in an average athlete-year occurred during training. The training injury incidence rate was estimated to be 1.6 (95% CI: 1.4, 1.9) per athlete-year, 11.8 (95% CI: 10.4, 13.4) per 1000 athlete-training-sessions, and 7.0 (95% CI: 6.1, 7.9) per 1000 athlete-hours of training. Among athletes with five or fewer injuries, the severity and injury pattern of training injuries were, by and large, the same as for competition injuries. Approximately sixty percent (60.3%) of training injuries required treatment by a health professional. Considering the burden of training injuries exceeds that of competition injuries, taekwondo governing bodies and stakeholders are encouraged to devote more efforts towards the identification of risk factors for, and prevention of, training injuries in the sport of taekwondo.     

Urinary protein profiling in hyperactive delirium and non-delirium cardiac surgery ICU patients

Background  

Suitable biomarkers associated with the development of delirium are still not known. Urinary proteomics has successfully been applied to identify novel biomarkers associated with various disease states, but its value has not been investigated in delirium patients.     

Enhanced antiviral activity against foot-and-mouth disease virus by a combination of type I and II porcine interferons

Previously, we showed that type I interferon (alpha/beta interferon [IFN-alpha/beta]) can inhibit foot-and-mouth disease virus (FMDV) replication in cell culture, and swine inoculated with 10(9) PFU of human adenovirus type 5 expressing porcine IFN-alpha (Ad5-pIFN-alpha) were protected when challenged 1 day later. In this study, we found that type II pIFN (pIFN-gamma) also has antiviral activity against FMDV in cell culture and that, in combination with pIFN-alpha, it has a synergistic antiviral effect. We also observed that while each IFN alone induced a number of IFN-stimulated genes (ISGs), the combination resulted in a synergistic induction of some ISGs. To extend these studies to susceptible animals, we inoculated groups of swine with a control Ad5, 10(8) PFU of Ad5-pIFN-alpha, low- or high-dose Ad5-pIFN-gamma, or a combination of Ad5-pIFN-alpha and low- or high-dose Ad5-pIFN-gamma and challenged all groups with FMDV 1 day later. The control group and the groups inoculated with either Ad5-pIFN-alpha or a low dose of Ad5-pIFN-gamma developed clinical disease and viremia. However, the group that received the combination of both Ad5-IFNs with the low dose of Ad5-pIFN-gamma was completely protected from challenge and had no viremia. Similarly the groups inoculated with the combination of Ad5s with the higher dose of Ad5-pIFN-gamma or with only high-dose Ad5-pIFN-gamma were protected. The protected animals did not develop antibodies against viral nonstructural (NS) proteins, while all infected animals were NS protein seropositive. No antiviral activity or significant levels of IFNs were detected in the protected groups, but there was an induction of some ISGs. The results indicate that the combination of type I and II IFNs act synergistically to inhibit FMDV replication in vitro and in vivo.     

Peripheral natural killer cell maturation depends on the transcription factor Aiolos

Natural killer (NK) cells are an innate lymphoid cell lineage characterized by their capacity to provide rapid effector functions, including cytokine production and cytotoxicity. Here, we identify the Ikaros family member, Aiolos, as a regulator of NK-cell maturation. Aiolos expression is initiated at the point of lineage commitment and maintained throughout NK-cell ontogeny. Analysis of cell surface markers representative of distinct stages of peripheral NK-cell maturation revealed that Aiolos was required for the maturation in the spleen of CD11b^highCD27⁻ NK cells. The differentiation block was intrinsic to the NK-cell lineage and resembled that found in mice lacking either T-bet or Blimp1; however, genetic analysis revealed that Aiolos acted independently of all other known regulators of NK-cell differentiation. NK cells lacking Aiolos were strongly hyper-reactive to a variety of NK-cell-mediated tumor models, yet impaired in controlling viral infection, suggesting a regulatory function for CD27⁻ NK cells in balancing these two arms of the immune response. These data place Aiolos in the emerging gene regulatory network controlling NK-cell maturation and function.     

Microchip capillary gel electrophoresis of multiply PEGylated high-molecular-mass glycoproteins

PEGylation is the most successful approach, to date, to prolong the in vivo survival of recombinant proteins. The conjugation of the polymer to glycoproteins results in challenging analysis, and furthermore, requires a wide variety of analytical tools for the determination of the extent of PEGylation. Herein, we present microchip capillary gel electrophoresis (MCGE) with a non-commercial high-molecular-weight protein assay for the analysis of the PEGylation degree with a focus on multiple PEGylation. To show the potential of the modified MCGE system, high-mass PEGylated glycoproteins (e.g. coagulation factor VIII) were analyzed. For the von Willebrand factor, the influence of glycans and the hydrodynamic radius on migration time and molecular weight determination is shown. The modified MCGE assay system is a powerful tool for the rapid assessment of the degree of PEGylation, demonstrating conjugate quality or reaction control of PEGylated proteins. This is the main advantage over time-consuming conventional SDS-PAGE. Furthermore, electrophoretic separation, staining, destaining, and fluorescence detection in one step combined with automated data analysis show that the MCGE system is a promising technique for high-throughput monitoring. The MCGE system can be used for rapid structure confirmation ("MCGE fingerprinting") of multiply PEGylated glycoproteins beyond the 230 kDa molecular mass range.     

The artiodactyl <Emphasis Type="Italic">APOBEC3</Emphasis> innate immune repertoire shows evidence for a multi-functional domain organization that existed in the ancestor of placental mammals

Background  

APOBEC3 (A3) proteins deaminate DNA cytosines and block the replication of retroviruses and retrotransposons. Each A3 gene encodes a protein with one or two conserved zinc-coordinating motifs (Z1, Z2 or Z3). The presence of one A3 gene in mice (Z2–Z3) and seven in humans, A3A-H (Z1a, Z2a-Z1b, Z2b, Z2c-Z2d, Z2e-Z2f, Z2g-Z1c, Z3), suggests extraordinary evolutionary flexibility. To gain insights into the mechanism and timing of A3 gene expansion and into the functional modularity of these genes, we analyzed the genomic sequences, expressed cDNAs and activities of the full A3 repertoire of three artiodactyl lineages: sheep, cattle and pigs.     

Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.

We describe an approach for the accurate quantification and concurrent sequence identification of the individual proteins within complex mixtures. The method is based on a class of new chemical reagents termed isotope-coded affinity tags (ICATs) and tandem mass spectrometry. Using this strategy, we compared protein expression in the yeast Saccharomyces cerevisiae, using either ethanol or galactose as a carbon source. The measured differences in protein expression correlated with known yeast metabolic function under glucose-repressed conditions. The method is redundant if multiple cysteinyl residues are present, and the relative quantification is highly accurate because it is based on stable isotope dilution techniques. The ICAT approach should provide a widely applicable means to compare quantitatively global protein expression in cells and tissues.