Poor performance of bootstrap confidence intervals for the location of a quantitative trait locus

The aim of many genetic studies is to locate the genomic regions (called quantitative trait loci, QTL) that contribute to variation in a quantitative trait (such as body weight). Confidence intervals for the locations of QTL are particularly important for the design of further experiments to identify the gene or genes responsible for the effect. Likelihood support intervals are the most widely used method to obtain confidence intervals for QTL location, but the nonparametric bootstrap has also been recommended. Through extensive computer simulation, we show that bootstrap confidence intervals behave poorly and so should not be used in this context. The profile likelihood (or LOD curve) for QTL location has a tendency to peak at genetic markers, and so the distribution of the maximum-likelihood estimate (MLE) of QTL location has the unusual feature of point masses at genetic markers; this contributes to the poor behavior of the bootstrap. Likelihood support intervals and approximate Bayes credible intervals, on the other hand, are shown to behave appropriately.     

Long-Term Effects of Sulfadiazine-Trimethoprim Medicated Diet on Cardiac Function,Hematology, and Weight Gain in Hsd:ICR (CD1) and Tac:SW Mice

With the alarming increase in heart disease and heart failure, the need for appropriate and ethical animal models of cardiac dysfunction continues to grow. Currently, many animal models of cardiomyopathy require either invasive procedures or genetic manipulation, both of which require extensive expertise, time, and cost. Serendipitous findings at our institution revealed a possible correlation between sulfadiazine-trimethoprim (SDZ-TMP) medicated diet and the development of cardiomyopathy in IcrTac:ICR mice. We hypothesized that mice fed SDZ-TMP medicated diet continuously for 3 to 6 mo would develop cardiomyocyte degeneration and fibrosis, eventually leading to dilated cardiomyopathy. A total of 44 mice (22 Hsd:ICR (CD1) and 22 Tac:SW) were enrolled in the study. Half of these 44 mice were fed standard rodent diet and the other half were fed SDZ-TMP medicated diet. Baseline samples, including weights, CBCs, select biochemistry parameters, and echocardiography were performed prior to the start of either diet. Weights were obtained monthly and all other parameters were measured at least once during the study, and again at its conclusion. After 42 wk, mice were euthanized, and heart, lung and bone marrow tissue were submitted for histopathologic evaluation. Histologically, hearts were scored for the degree of degeneration, fibrosis, inflammation, and vacuolation. The data showed that SDZ-TMP did not have a significant effect on cardiac function, RBC parameters, biochemistry parameters (ALT, AST, calcium, magnesium, creatine kinase, and creatinine), hematopoiesis, or histologic heart scores. In addition, mice fed the SDZ-TMP medicated diet gained less weight over time. In summary, we were unable to reproduce the previous findings and thus could not use this approach to develop a novel model of cardiomyopathy. However, these results indicate that SDZ-TMP medicated diet containing 1,365 ppm of SDZ and 275 ppm of TMP does not appear to have long-term detrimental effects in mice.

Heart disease is currently the leading cause of death in the United States. The American Heart Association estimates that the prevalence of heart failure will increase to 46% by 2030,⁴ and the cost associated with heart disease is expected to rise from $55 billion in 2016 to $1.2 trillion (US dollars) by 2030.⁴ The existing animal models of heart disease have enhanced our knowledge of its pathophysiology and has led to the development of numerous therapies.^9,30,47,54 However, many animal models of cardiomyopathy require invasive procedures such as aortic constriction,^16,58 left anterior descending artery ligation,^15,31,52 cryogenic injury,^27,57 or renal ischemia⁶⁸ to induce the cardiac changes associated with heart failure. The chemotherapeutic agent doxorubicin has also been used to induce heart failure in mice,^66,69 but a major disadvantage is the risk it poses to workers as a hazardous agent. Genetically engineered mice can replace invasive surgical models of cardiac disease,¹⁴ but they also have significant limitations including cost, time, and failure to express the anticipated phenotype.^3,38,39 Despite advancements in cardiac research, reproducible and economical animal models of heart disease are still needed to further our understanding and explore novel therapeutic solutions.Researchers at the University of Chicago reported that IcrTac:ICR sentinel mice that were accidently fed sulfadiazine trimethoprim (SDZ-TMP) medicated diet for 3 to 6 mo developed cardiomyopathy and subsequently cardiac dysfunction.⁴⁸ Prior to the onset of cardiomyopathy, these animals showed no signs of clinical disease.⁴⁸ SDZ-TMP is a potentiated sulfonamide, and the synergistic activity of sulfadiazine and trimethoprim allows it to not only inhibit folic acid metabolism in bacteria but also in some protozoa and fungi.²⁵ Potentiated sulfonamides are commonly administered to immunocompromised mice to prevent or treat opportunistic infections such as Pneumocystis murina.²⁰ However, SDZ-TMP may have contributed to the development of cardiomyopathy in these mice,⁴⁸ as potentiated sulfonamides are known to have adverse side effects in other animals, such as keratitis sicca, hypothyroidism, anemia, leukopenia, and hypersensitivity reactions.⁵⁰Based on the previous findings,⁴⁸ the goal of this study was to develop a cost-effective, reproducible, and noninvasive animal model of dilated cardiomyopathy. The prior data suggested that this model would have a slow and progressive onset, mimicking many aspects of heart disease and heart failure in humans.⁴⁸ We hypothesized that mice fed SDZ-TMP medicated diet continuously for 3 to 6 mo would develop cardiomyocyte degeneration and fibrosis, eventually progressing to dilated cardiomyopathy. Initial findings were discovered in the outbred stock IcrTac:ICR, but at the time of the present study, live production of this stock had ceased. Therefore, we elected to investigate our hypothesis in 2 readily available outbred stocks. We first attempted to monitor the progression of cardiomyopathy over time via echocardiography in Hsd:ICR (CD1) and Tac:SW mice fed SDZ-TMP medicated diet. In addition to capturing this progression, other objectives of this study were to investigate the long-term effects of SDZ-TMP medicated diet on other health parameters, including CBCs, a subset of biochemistry parameters to assess kidney and liver function, and weight gain. Thus, our goals were to both create an improved animal model of cardiomyopathy and enhance our understanding of prolonged prophylactic antibiotic use and its potential effects on biomedical research models and results.     

Arsenic and high affinity phosphate uptake gene distribution in shallow submarine hydrothermal sediments

The toxicity of arsenic (As) towards life on Earth is apparent in the dense distribution of genes associated with As detoxification across the tree of life. The ability to defend against As is particularly vital for survival in As-rich shallow submarine hydrothermal ecosystems along the Hellenic Volcanic Arc (HVA), where life is exposed to hydrothermal fluids containing up to 3000 times more As than present in seawater. We propose that the removal of dissolved As and phosphorus (P) by sulfide and Fe(III)(oxyhydr)oxide minerals during sediment–seawater interaction, produces nutrient-deficient porewaters containing?<?2.0 ppb P. The porewater arsenite-As(III) to arsenate-As(V) ratios, combined with sulfide concentration in the sediment and/or porewater, suggest a hydrothermally-induced seafloor redox gradient. This gradient overlaps with changing high affinity phosphate uptake gene abundance. High affinity phosphate uptake and As cycling genes are depleted in the sulfide-rich settings, relative to the more oxidizing habitats where mainly Fe(III)(oxyhydr)oxides are precipitated. In addition, a habitat-wide low As-respiring and As-oxidizing gene content relative to As resistance gene richness, suggests that As detoxification is prioritized over metabolic As cycling in the sediments. Collectively, the data point to redox control on Fe and S mineralization as a decisive factor in the regulation of high affinity phosphate uptake and As cycling gene content in shallow submarine hydrothermal ecosystems along the HVA.     

Deep‐biosphere consortium of fungi and prokaryotes in Eocene subseafloor basalts

The deep biosphere of the subseafloor crust is believed to contain a significant part of Earth's biomass, but because of the difficulties of directly observing the living organisms, its composition and ecology are poorly known. We report here a consortium of fossilized prokaryotic and eukaryotic micro‐organisms, occupying cavities in deep‐drilled vesicular basalt from the Emperor Seamounts, Pacific Ocean, 67.5 m below seafloor (mbsf). Fungal hyphae provide the framework on which prokaryote‐like organisms are suspended like cobwebs and iron‐oxidizing bacteria form microstromatolites (Frutexites). The spatial inter‐relationships show that the organisms were living at the same time in an integrated fashion, suggesting symbiotic interdependence. The community is contemporaneous with secondary mineralizations of calcite partly filling the cavities. The fungal hyphae frequently extend into the calcite, indicating that they were able to bore into the substrate through mineral dissolution. A symbiotic relationship with chemoautotrophs, as inferred for the observed consortium, may be a pre‐requisite for the eukaryotic colonization of crustal rocks. Fossils thus open a window to the extant as well as the ancient deep biosphere.     

The response of the seated human to sinusoidal vibration and impact

Low back pain has been shown to occur more frequently among vehicle drivers than in representative control groups. Thus the response of the human to vibration and impact is of interest. This study investigated the response of the spine to both impact and sinusoidal excitation in either a relaxed or erect seated posture. The sinusoidal testing apparatus used was a resonating system consisting of two parallel wooden beams, simply supported, and the impact testing apparatus a bearing-guided, spring-suspended platform, struck from below. Ten subjects (5 males, 5 females) were evaluated using both methods. Transfer functions were compared at 2-4 Hz, 4-8 Hz and 8-16 Hz intervals using a sign test. Although in 24 comparisons of either test method (vibration or impact) or posture (erect or relaxed) where eleven showed differences significant at the p less than .05 level, only 2 out of 24 comparisons were the differences distinct enough to be significant (at the p less than .01 level). Both of these latter differences were due to test method while the subjects were sitting erect. In those instances where there were no significant differences due to test method, the impact method may be a viable replacement for the vibration test method. Where the levels of significance are higher (p less than .01 or p less than .05), further study of the magnitude of the differences is indicated and may reveal further insight into the seated individual as a system.     

Genetic resistance to diet-induced obesity in chromosome substitution strains of mice

Discovery of genes that confer resistance to diseases such as diet-induced obesity could have tremendous therapeutic impact. We previously demonstrated that the C57BL/6J-Chr^A/J/NaJ panel of chromosome substitution strains (CSSs) is a unique model for studying resistance to diet-induced obesity. In the present study, three replicate CSS surveys showed remarkable consistency, with 13 A/J-derived chromosomes reproducibly conferring resistance to high-fat-diet-induced obesity. Twenty CSS intercrosses, one derived from each of the 19 autosomes and chromosome X, were used to determine the number and location of quantitative trait loci (QTLs) on individual chromosomes and localized six QTLs. However, analyses of mean body weight in intercross progeny versus C57BL/6J provided strong evidence that many QTLs discovered in the CSS surveys eluded detection in these CSS intercrosses. Studies of the temporal effects of these QTLs suggest that obesity resistance was dynamic, with QTLs acting at different ages or after different durations of diet exposure. Thus, these studies provide insight into the genetic architecture of complex traits such as resistance to diet-induced obesity in the C57BL/6J-Chr^A/J/NaJ CSSs. Because some of the QTLs detected in the CSS intercrosses were not detected using a traditional C57BL/6J × A/J intercross, our results demonstrate that surveys of CSSs and congenic strains derived from them are useful complementary tools for analyzing complex traits.     

Variation in Genome-Wide Levels of Meiotic Recombination Is Established at the Onset of Prophase in Mammalian Males

Segregation of chromosomes during the first meiotic division relies on crossovers established during prophase. Although crossovers are strictly regulated so that at least one occurs per chromosome, individual variation in crossover levels is not uncommon. In an analysis of different inbred strains of male mice, we identified among-strain variation in the number of foci for the crossover-associated protein MLH1. We report studies of strains with “low” (CAST/EiJ), “medium” (C3H/HeJ), and “high” (C57BL/6J) genome-wide MLH1 values to define factors responsible for this variation. We utilized immunofluorescence to analyze the number and distribution of proteins that function at different stages in the recombination pathway: RAD51 and DMC1, strand invasion proteins acting shortly after double-strand break (DSB) formation, MSH4, part of the complex stabilizing double Holliday junctions, and the Bloom helicase BLM, thought to have anti-crossover activity. For each protein, we identified strain-specific differences that mirrored the results for MLH1; i.e., CAST/EiJ mice had the lowest values, C3H/HeJ mice intermediate values, and C57BL/6J mice the highest values. This indicates that differences in the numbers of DSBs (as identified by RAD51 and DMC1) are translated into differences in the number of crossovers, suggesting that variation in crossover levels is established by the time of DSB formation. However, DSBs per se are unlikely to be the primary determinant, since allelic variation for the DSB-inducing locus Spo11 resulted in differences in the numbers of DSBs but not the number of MLH1 foci. Instead, chromatin conformation appears to be a more important contributor, since analysis of synaptonemal complex length and DNA loop size also identified consistent strain-specific differences; i.e., crossover frequency increased with synaptonemal complex length and was inversely related to chromatin loop size. This indicates a relationship between recombination and chromatin compaction that may develop as DSBs form or earlier during establishment of the meiotic axis.     

Mounting of the transducers in measurement of segmental motion of the spine

A seated female subject was subjected to sinusoidal whole body vibration at 2, 4, 5 and 6 Hz. Accelerations were measured by accelerometers on pins screwed into the spinous process. The pins were also fitted with light emitting diodes (LED). The displacement of these LEDs were compared to LEDs attached directly to the skin. Substantial differences in measured displacements were noted between surface mounted LEDs and those mounted on pins rigidly attached to the skeleton. These differences were more marked further out from the center line.     

Correlations between Synaptic Initiation and Meiotic Recombination: A Study of Humans and Mice

Meiotic recombination is initiated by programmed double strand breaks (DSBs), only a small subset of which are resolved into crossovers (COs). The mechanism determining the location of these COs is not well understood. Studies in plants, fungi, and insects indicate that the same genomic regions are involved in synaptic initiation and COs, suggesting that early homolog alignment is correlated with the eventual resolution of DSBs as COs. It is generally assumed that this relationship extends to mammals, but little effort has been made to test this idea. Accordingly, we conducted an analysis of synaptic initiation sites (SISs) and COs in human and mouse spermatocytes and oocytes. In contrast to our expectation, we observed remarkable sex- and species-specific differences, including pronounced differences between human males and females in both the number and chromosomal location of SISs. Further, the combined data from our studies in mice and humans suggest that the relationship between SISs and COs in mammals is a complex one that is not dictated by the sites of synaptic initiation as reported in other organisms, although it is clearly influenced by them.