Menstrual cycle phase and oral contraceptive effects on triglyceride mobilization during exercise.

We examined the effects of menstrual cycle phase and oral contraceptive (OC) use on triglyceride mobilization during 90 min of rest and 60 min of leg ergometry exercise at 45 and 65% peak O(2) uptake (Vo(2 peak)) in eight moderately physically active, eumenorrheic women (24.8 +/- 1.2 yr). Subjects were tested during the follicular phase (FP) and the luteal phase (LP) before OC use and during the inactive phase (IP) and high-dose phase (HP) after 4 complete mo of OC use. Glycerol rate of appearance (R(a)), a measure of triglyceride mobilization, was determined in a 3-h postabsorptive state using a primed constant infusion of [1,1,2,3,3-(2)H]glycerol. Before OC use (BOC), there were no significant differences between FP and LP in any of the variables studied. Dietary composition, exercise patterns, plasma glycerol concentrations, growth hormone concentrations, and exercise respiratory exchange ratio did not change with OC use. However, 4 mo of OC use significantly (P < 0.05) increased glycerol R(a) in HP during exercise at 45% Vo(2 peak) (6.2 +/- 0.2, 6.5 +/- 0.4, and 7.7 +/- 1.1 micromol.kg(-1).min(-1) for BOC, IP, and HP, respectively) and in IP and HP at 65% Vo(2 peak) (6.6 +/- 0.1, 8.2 +/- 0.6, and 8.1 +/- 0.7 micromol.kg(-1).min(-1) for BOC, IP, and HP, respectively). Plasma cortisol concentrations were significantly higher with OC use at rest and during exercise at 45 and 65% Vo(2 peak). In summary, although fluctuations of endogenous ovarian steroids have little effect on triglyceride mobilization, the synthetic ovarian steroids found in OCs increase triglyceride mobilization and plasma cortisol concentrations in exercising women. We conclude that the hierarchy of effects of ovarian steroids and their analogs on triglyceride mobilization in exercising women is as follows: energy flux > OC use > recent carbohydrate nutrition, menstrual cycle effects.     

Enhancement of Extra Chromosomal Recombination in Somatic Cells by Affecting the Ratio of Homologous Recombination (HR) to Non-Homologous End Joining (NHEJ)

Advancements in somatic cell gene targeting have been slow due to the finite lifespan of somatic cells and the overall inefficiency of homologous recombination. The rate of homologous recombination is determined by mechanisms of DNA repair, and by the balance between homologous recombination (HR) and non-homologous end joining (NHEJ). A plasmid-to-plasmid, extra chromosomal recombination system was used to study the effects of the manipulation of molecules involved in NHEJ (Mre11, Ku70/80, and p53) on HR/NHEJ ratios. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. While a mutant Mre11 and an anti-Ku aptamer did not significantly affect the rate of NHEJ or HR, transient expression of a p53 mutant increased overall HR/NHEJ by 2.5 fold. However, expression of the mutant p53 resulted in increased aneuploidy of the cultured cells. Additionally, we found no relationship between telomerase expression and changes in HR/NHEJ. In contrast, cell synchrony by thymidine incorporation did not induce chromosomal abnormalities, and increased the ratio of HR/NHEJ 5-fold by reducing the overall rate of NHEJ. Overall our results show that attempts at reducing NHEJ by use of Mre11 or anti-Ku aptamers were unsuccessful. Cell synchrony via thymidine incorporation, however, does increase the ratio of HR/NHEJ and this indicates that this approach may be of use to facilitate targeting in somatic cells by reducing the numbers of colonies that need to be analyzed before a HR is identified.     

The Use of Positive Reinforcement in Training Zebra Sharks (Stegostoma fasciatum)

Few studies have examined how personality traits may be related to the amounts and types of attachments humans have toward companion animals (pets). In this study, 1,098 companion animal guardians (owners) completed a survey that included the Big Five Inventory, the Lexington Attachment to Pets Scale, and the Pet Attachment Questionnaire. Each participant chose whether he or she identified as a Cat Person, Dog Person, Both, or Neither. Results indicated that neuroticism, conscientiousness, choosing a dog as a favorite pet, and identifying as a Cat Person, Dog Person, or Both predicted affection for a pet. Conscientiousness, extraversion, and openness decreased avoidant attachment to pets, and neuroticism increased anxious attachment to pets. Both dogs and cats could benefit from pet owners who are conscientious, and there may be some benefits of neuroticism in pet owners. The findings of this study will advance understanding of the human–animal bond. As this understanding increases, measurements of human attachment and personality may be useful for the development of tools that could assist shelter employees and veterinarians in counseling people about pet ownership.     

A Label-free Selected Reaction Monitoring Workflow Identifies a Subset of Pregnancy Specific Glycoproteins as Potential Predictive Markers of Early-onset Pre-eclampsia

Pre-eclampsia (PE) is a serious complication of pregnancy with potentially life threatening consequences for both mother and baby. Presently there is no test with the required performance to predict which healthy first-time mothers will go on to develop PE. The high specificity, sensitivity, and multiplexed nature of selected reaction monitoring holds great potential as a tool for the verification and validation of putative candidate biomarkersfor disease states. Realization of this potential involves establishing a high throughput, cost effective, reproducible sample preparation workflow. We have developed a semi-automated HPLC-based sample preparation workflow before a label-free selected reaction monitoring approach. This workflow has been applied to the search for novel predictive biomarkers for PE.To discover novel candidate biomarkers for PE, we used isobaric tagging to identify several potential biomarker proteins in plasma obtained at 15 weeks gestation from nulliparous women who later developed PE compared with pregnant women who remained healthy. Such a study generates a number of “candidate” biomarkers that require further testing in larger patient cohorts. As proof-of-principle, two of these proteins were taken forward for verification in a 100 women (58 PE, 42 controls) using label-free SRM. We obtained reproducible protein quantitation across the 100 samples and demonstrated significant changes in protein levels, even with as little as 20% change in protein concentration. The SRM data correlated with a commercial ELISA, suggesting that this is a robust workflow suitable for rapid, affordable, label-free verification of which candidate biomarkers should be taken forward for thorough investigation. A subset of pregnancy-specific glycoproteins (PSGs) had value as novel predictive markers for PE.The identification of clinically relevant plasma biomarkers with diagnostic and/or predictive value continues to challenge the proteomics field. Whereas once the biomarker pipeline was described as a two part discovery and validation process, there is increasing consensus that an intermediate step is required in which the proteins identified in the discovery phase are technically verified in 50 to 200 samples. This verification step identifies false positives from the discovery phase and allows prioritization of proteins to be taken into large-scale clinical validation studies (1). Although commercial ELISA kits may be used in this phase, these are unavailable for many proteins, are expensive, and may lack specificity. In addition, sample requirements may be too high to perform ELISA on all candidates, especially if many proteins are identified as potential markers by low powered, high penetration discovery workflows.Selected reaction monitoring (SRM)¹ mass spectrometry has great potential as an alternative verification method (2–6) as it can be multiplexed, customized, and is highly specific. This potential has not been exploited to date, largely because of technical issues developing a low-cost, reproducible workflow encompassing plasma and serum preparation and LC/MS analysis with the capability to measure protein levels reproducible in hundreds of samples. With traditional stable isotope dilution SRM (SID-SRM), the high cost of accurately quantified, purified stable isotope encoded peptides or proteins may be prohibitive for the verification of multiple peptides from many proteins. Label-free relatively quantitative methods are increasingly popular in discovery proteomics but to a much lesser extent in targeted SRM studies (7, 8).For any SRM method, sample preparation workflows must balance the extent of enrichment and fractionation to enable quantification of lower abundance proteins, against increased technical variability (which is influenced by the number of sample handling steps) and reduced multiplexed potential as a consequence of fractionating peptides from the protein of interest into several distinct fractions. It is also essential that the true technical variation in the workflow is quantitatively evaluated from freezer to MS analysis, rather than just the variation within the LC-SRM part of the experiment. As a paradigm for a label-free SRM assay, we developed our workflow and applied it to the verification of candidate biomarkers that indicate the risk of pre-eclampsia (PE).PE affects 2–8% of pregnancies, and is characterized by hypertension and proteinuria, which may progress to severe maternal complications or death (9). Because delivery of the infant is the only effective intervention, a third of babies are born premature and fetal or newborn mortality is increased three- to 10-fold (10). Its complex etiology involves abnormal placentation, an altered immune response and a sensitized maternal vascular endothelium (11). Prediction of the condition in early pregnancy would allow prevention strategies, such as low dose aspirin, to be targeted to high risk women. In first-time pregnant women, a group particularly at risk, biomarkers continue to fall short of a test that would be useful or cost effective in clinical practice (12–14). Better-performing novel biomarkers are required.The aim of this study was to identify candidate predictive biomarkers for PE and then develop a verification assay using mass spectrometry to determine whether these should be taken forward into more extensive and expensive validation studies. Initial discovery experiments were employed using a pooled sample iTRAQ approach using two different MS platforms to increase plasma proteome coverage. Among the set of proteins discovered, we then developed a label-free SRM assay for relative quantification of CXCL7 (Platelet basic protein; PBP) and members of the Pregnancy specific glycoprotein (PSG) family in a 100-sample set from the international SCreeningfOr Pregnancy Endpoints (SCOPE) study (www.scopestudy.net). Our workflow allowed the specificity and linearity of response for each peptide to be determined, along with true technical variability. Although absolute concentration and LOD/LOQ cannot be calculated using this approach, we aimed to test the hypothesis that a label-free SRM approach could provide a rapid, robust, and efficient screen of candidate plasma biomarkers.     

A Copy Number Variant at the KITLG Locus Likely Confers Risk for Canine Squamous Cell Carcinoma of the Digit

The domestic dog is a robust model for studying the genetics of complex disease susceptibility. The strategies used to develop and propagate modern breeds have resulted in an elevated risk for specific diseases in particular breeds. One example is that of Standard Poodles (STPOs), who have increased risk for squamous cell carcinoma of the digit (SCCD), a locally aggressive cancer that causes lytic bone lesions, sometimes with multiple toe recurrence. However, only STPOs of dark coat color are at high risk; light colored STPOs are almost entirely unaffected, suggesting that interactions between multiple pathways are necessary for oncogenesis. We performed a genome-wide association study (GWAS) on STPOs, comparing 31 SCCD cases to 34 unrelated black STPO controls. The peak SNP on canine chromosome 15 was statistically significant at the genome-wide level (P_raw = 1.60×10⁻⁷; P_genome = 0.0066). Additional mapping resolved the region to the KIT Ligand (KITLG) locus. Comparison of STPO cases to other at-risk breeds narrowed the locus to a 144.9-Kb region. Haplotype mapping among 84 STPO cases identified a minimal region of 28.3 Kb. A copy number variant (CNV) containing predicted enhancer elements was found to be strongly associated with SCCD in STPOs (P = 1.72×10⁻⁸). Light colored STPOs carry the CNV risk alleles at the same frequency as black STPOs, but are not susceptible to SCCD. A GWAS comparing 24 black and 24 light colored STPOs highlighted only the MC1R locus as significantly different between the two datasets, suggesting that a compensatory mutation within the MC1R locus likely protects light colored STPOs from disease. Our findings highlight a role for KITLG in SCCD susceptibility, as well as demonstrate that interactions between the KITLG and MC1R loci are potentially required for SCCD oncogenesis. These findings highlight how studies of breed-limited diseases are useful for disentangling multigene disorders.