Something Old and Something New: Wedding Recombinant Inbred Lines with Traditional Line Cross Analysis Increases Power to Describe Gene Interactions

In this paper we present a novel approach to quantifying genetic architecture that combines recombinant inbred lines (RIL) with line cross analysis (LCA). LCA is a method of quantifying directional genetic effects (i.e. summed effects of all loci) that differentiate two parental lines. Directional genetic effects are thought to be critical components of genetic architecture for the long term response to selection and as a cause of inbreeding depression. LCA typically begins with two inbred parental lines that are crossed to produce several generations such as F1, F2, and backcrosses to each parent. When a RIL population (founded from the same P1 and P2 as was used to found the line cross population) is added to the LCA, the sampling variance of several nonadditive genetic effect estimates is greatly reduced. Specifically, estimates of directional dominance, additive x additive, and dominance x dominance epistatic effects are reduced by 92%, 94%, and 56% respectively. The RIL population can be simultaneously used for QTL identification, thus uncovering the effects of specific loci or genomic regions as elements of genetic architecture. LCA and QTL mapping with RIL provide two qualitatively different measures of genetic architecture with the potential to overcome weaknesses of each approach alone. This approach provides cross-validation of the estimates of additive and additive x additive effects, much smaller confidence intervals on dominance, additive x additive and dominance x dominance estimates, qualitatively different measures of genetic architecture, and the potential when used together to balance the weaknesses of LCA or RIL QTL analyses when used alone

Effect of Animal and Industrial Trans Fatty Acids on HDL and LDL Cholesterol Levels in Humans – A Quantitative Review

Background: Trans fatty acids are produced either by industrial hydrogenation or by biohydrogenation in the rumens of cows and sheep. Industrial trans fatty acids lower HDL cholesterol, raise LDL cholesterol, and increase the risk of coronary heart disease. The effects of conjugated linoleic acid and trans fatty acids from ruminant animals are less clear. We reviewed the literature, estimated the effects trans fatty acids from ruminant sources and of conjugated trans linoleic acid (CLA) on blood lipoproteins, and compared these with industrial trans fatty acids. Methodology/Principal Findings: We searched Medline and scanned reference lists for intervention trials that reported effects of industrial trans fatty acids, ruminant trans fatty acids or conjugated linoleic acid on LDL and HDL cholesterol in humans. The 39 studies that met our criteria provided results of 29 treatments with industrial trans fatty acids, 6 with ruminant trans fatty acids and 17 with CLA. Control treatments differed between studies; to enable comparison between studies we recalculated for each study what the effect of trans fatty acids on lipoprotein would be if they isocalorically replaced cis mono unsaturated fatty acids. In linear regression analysis the plasma LDL to HDL cholesterol ratio increased by 0.055 (95% CI 0.044-0.066) for each % of dietary energy from industrial trans fatty acids replacing cis monounsaturated fatty acids The increase in the LDL to HDL ratio for each % of energy was 0.038 (95% CI 0.012-0.065) for ruminant trans fatty acids, and 0.043 (95% CI 0.012-0.074) for conjugated linoleic acid (p = 0.99 for difference between CLA and industrial trans fatty acids; p = 0.37 for ruminant versus industrial trans fatty acids). Conclusions/Significance: Published data suggest that all fatty acids with a double bond in the trans configuration raise the ratio of plasma LDL to HDL cholesterol

Platelets Retain High Levels of Active Plasminogen Activator Inhibitor 1

The vascular fibrinolytic system is crucial for spontaneous lysis of blood clots. Plasminogen activator inhibitor 1 (PAI-1), the principal inhibitor of the key fibrinolytic enzyme tissue-type plasminogen activator (tPA), is present in platelets at high concentrations. However, the majority of PAI-1 stored in platelets has been considered to be inactive. Our recent finding (Brogren H, et al. Blood 2004) that PAI-1 de novo synthesized in platelets remained active for over 24 h, suggested that PAI-1 stored in the α-granules might be active to a larger extent than previously reported. To re-evaluate this issue, we performed experiments where the fraction of active PAI-1 was estimated by analyzing the tPA-PAI-1 complex formation. In these experiments platelets were lysed with Triton X-100 in the presence of serial dilutions of tPA and subsequently the tPA-PAI-1 complex was evaluated by Western blot. Also, using a non-immunologic assay, tPA was labeled with 125I, and 125I-tPA and 125I-tPA-PAI-1 was quantified by scintigraphy. Interestingly, both methods demonstrated that the majority (>50%) of platelet PAI-1 is active. Further analyses suggested that pre-analytical procedures used in previous studies (sonication or freezing/thawing) may have substantially reduced the activity of platelet PAI-1, which has lead to an underestimation of the proportion of active PAI-1. Our in vitro results are more compatible with the role of PAI-1 in clot stabilization as demonstrated in physiological and pathophysiological studies

Suspected survivor bias in case–control studies: stratify on survival time and use a negative control

AbstractObjectivesSelection bias in case–control studies occurs when control selection is inappropriate. However, selection bias due to improper case sampling is less well recognized. We describe how to recognize survivor bias (i.e., selection on exposed cases) and illustrate this with an example study.Study Design and SettingA case–control study was used to analyze the effect of statins on major bleedings during treatment with vitamin K antagonists. A total of 110 patients who experienced such bleedings were included 18–1,018 days after the bleeding complication and matched to 220 controls.ResultsA protective association of major bleeding for exposure to statins (odds ratio [OR]: 0.56; 95% confidence interval: 0.29–1.08) was found, which did not become stronger after adjustment for confounding factors. These observations lead us to suspect survivor bias. To identify this bias, results were stratified on time between bleeding event and inclusion, and repeated for a negative control (an exposure not related to survival): blood group non-O. The ORs for exposure to statins increased gradually to 1.37 with shorter time between outcome and inclusion, whereas ORs for the negative control remained constant, confirming our hypothesis.ConclusionWe recommend the presented method to check for overoptimistic results, that is, survivor bias in case–control studies

Quality of DNA Extracted from Mouthwashes

Background A cost effective, safe and efficient method of obtaining DNA samples is essential in large scale genetic analyses. Buccal cells are an attractive source of DNA, as their collection is non-invasive and can be carried out by mail. However, little attention has been given to the quality of DNA extracted from mouthwashes. Methodology Mouthwash-derived DNA was extracted from 500 subjects participating in a genetic study of high myopia. DNA quality was investigated using two standard techniques: agarose gel electrophoresis and quantitative polymerase chain reaction (qPCR). Principal Findings Whereas the majority of mouthwash-derived DNA samples showed a single band of high molecular weight DNA by gel electrophoresis, 8.9% (95% CI: 7.1–10.7%) of samples contained only a smear of low-to-medium molecular weight, degraded DNA. The odds of DNA degradation in a subject's second mouthwash sample, given degradation of the first, was significantly greater than one (OR = 3.13; 95% CI: 1.22–7.39; Fisher's test P = 0.009), suggesting that DNA degradation was at least partially a subject-specific phenomenon. Approximately 12.4% (95% CI: 10.4–14.4%) of mouthwash-derived DNA failed to PCR amplify efficiently (using an ~200 bp microsatellite marker). However, we found there was no significant difference in amplification success rate between DNA samples judged to be degraded or non-degraded by gel electrophoresis (Fisher's test P = 0.5). Conclusions This study demonstrated that DNA degradation affects a significant minority of saline mouthwashes, and that the phenomenon is partially subject-specific. Whilst the level of degradation did not significantly prevent successful amplification of short PCR fragments, previous studies suggest that such DNA degradation would compromise more demanding applications

Temperature-Dependence of Weibel-Palade Body Exocytosis and Cell Surface Dispersal of von Willebrand Factor and Its Propolypeptide

Background: Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca2+-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca2+-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs. Principal Findings: Ca2+-driven WPB exocytosis occurred at all temperatures studied (7–37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s−1 (37°C) to 0.80±0.14 s−1 (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca2+-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75–80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60–120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself. Conclusions : We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased

Analysis of blood coagulation in mice: pre-analytical conditions and evaluation of a home-made assay for thrombin-antithrombin complexes

BACKGROUND: The use of mouse models for the study of thrombotic disorders has gained increasing importance. Methods for measurement of coagulation activation in mice are, however, scarce. The primary aim of this study was to develop a specific mouse thrombin-antithrombin (TAT) ELISA for measurement of coagulation activation and to compare it with two commercially available assays for human TAT complexes. In addition, we aimed to improve methods for mouse plasma anticoagulation and preparation. METHODS AND RESULTS: First, for the measurement of TAT-complexes in plasma a mouse specific TAT-ELISA was developed using rabbit polyclonal antibodies raised against mouse thrombin and rat antithrombin, respectively. This ELISA detected an increase in TAT levels in a mouse model of endotoxemia. Two commercial human TAT ELISAs appeared to be less specific for mouse thrombin-rat antithrombin complexes. Second, to prevent clotting of mouse blood sodium citrate was either mixed with blood during collection in a syringe or was injected intravenously immediately prior to blood collection. Intravenous sodium citrate completely inhibited blood coagulation resulting in plasma with consistently low TAT levels. Sodium citrate mixed with blood during collection resulted in increased TAT levels in 4 out of 16 plasma samples. Third, heparinase was added to plasma samples after in vivo injection of different heparin doses to test its neutralizing effect. Heparinase neutralized up to a 20 U of heparin/mouse and resulted in accurate APTT and factor VIII determinations. CONCLUSION: These procedures and reagents for plasma preparation and coagulation testing will improve studies on thrombotic disorders in mice