Genome-wide search for loci controlling serum IGF binding protein levels of mice.

A segregating F(2) pedigree based on two mouse lines (DU6i and DBA/2) with extremely different growth characteristics was generated to search for loci affecting serum levels of insulin-like growth factor (IGF) binding proteins (IGFBPs) and to estimate their effects on growth and body composition. DU6i is characterized by high body mass and obesity associated with hyperinsulinemia, hyperleptinemia, and elevated serum IGF-I concentrations. Furthermore, significantly elevated serum levels of IGFBP-2, IGFBP-3, and IGFBP-4 were found in DU6i vs. DBA/2 mice. Linkage analysis identified loci with major effects on the serum level of IGFBP-3 on Chromosome 5 at 58 cM (Igfbp3q1; F = 9.9) and on Chromosome 10 at 46 cM (Igfbp3q2; F = 33.8). A locus significantly influencing serum IGFBP-2 levels in males was found on Chromosome 7. Additional linkage was detected in males and females for IGFBP-2 on Chromosomes 8, 11, 14, 17, and X, and for IGFBP-4 on Chromosome 4. Additional loci affecting IGFBPs acted in a sex-specific manner. The identified loci coincide in part with chromosomal regions controlling growth and obesity. Thus, multiple genes or pleiotropic gene effects may be assumed for these chromosomal regions. The identification of quantitative trait loci for IGFBPs as subcomponents of growth regulation and differentiation will further improve the understanding of complex trait regulation.     

Genome-wide search for genes that modulate inflammatory arthritis caused by Ali18 mutation in mice

Many of inflammatory diseases, including inflammatory arthritis, are multifactorial bases. The Ali18 semidominant mutation induced by N-ethyl-N-nitrosourea in the C3HeB/FeJ (C3H) genome causes spontaneous inflammation of peripheral limbs and elevated immunoglobulin E (IgE) levels in mice. Although the Ali18 locus was mapped to a single locus on chromosome 4, the arthritic phenotype of Ali18/+ mice was completely suppressed in F1 hybrid genetic backgrounds. To determine the chromosomal locations of the modifier loci affecting the severity of arthritis, an autosomal genome scan of 22 affected Ali18/+ F2 mice was conducted using C57BL/6J as a partner strain. Interestingly, regions on chromosomes 1 and 3 in C3H showed significant genetic interactions. Moreover, 174 N2 (backcross to Ali18/Ali18) and 267 F2 animals were used for measurement of arthritis scores and plasma IgE levels, and also for genotyping with 153 genome-wide single nucleotide polymorphism (SNP) markers. In N2 populations, two significant trait loci for arthritis scores on chromosomes 1 and 15 were detected. Although no significant scores were detected in F2 mice besides chromosome 4, a suggestive score was detected on chromosome 3. In addition, a two-dimensional genome scan using F2 identified five suggestive scores of chromosomal combinations, chromosomes 1 × 10, 2 × 6, 3 × 4, 4 × 9, and 6 × 15. No significant trait loci affecting IgE levels were detected in both N2 and F2 populations. Identification of the Ali18 modifier genes by further detailed analyses such as congenic strains and expression profiling may dissect molecular complexity in inflammatory diseases.     

Modifier genes in mice and humans

An emerging theme of studies with spontaneous, engineered and induced mutant mice is that phenotypes often depend on genetic background, implying that genetic modifiers have a role in guiding the functional consequences of genetic variation. Understanding the molecular and cellular basis by which modifier genes exert their influence will provide insights into developmental and physiological pathways that are critical to fundamental biological processes, as well as into novel targets for therapeutic interventions in human diseases.     

Genome-wide association study identifies candidate genes for male fertility traits in humans

Despite the fact that hundreds of genes are known to affect fertility in animal models, relatively little is known about genes that influence natural fertility in humans. To broadly survey genes contributing to variation in male fertility, we conducted a genome-wide association study (GWAS) of two fertility traits (family size and birth rate) in 269 married men who are members of a founder population of European descent that proscribes contraception and has large family sizes. Associations between ～250,000 autosomal SNPs and the fertility traits were examined. A total of 41 SNPs with p ≤ 1 × 10(-4) for either trait were taken forward to a validation study of 123 ethnically diverse men from Chicago who had previously undergone semen analyses. Nine (22%) of the SNPs associated with reduced fertility in the GWAS were also associated with one or more of the ten measures of reduced sperm quantity and/or function, yielding 27 associations with p values < 0.05 and seven with p values < 0.01 in the validation study. On the basis of 5,000 permutations of our data, the probabilities of observing this many or more small p values were 0.0014 and 5.6 × 10(-4), respectively. Among the nine associated loci, outstanding candidates for male fertility genes include USP8, an essential deubiquitinating enzyme that has a role in acrosome assembly; UBD and EPSTI1, which have potential roles in innate immunity; and LRRC32, which encodes a latent transforming growth factor β (TGF-β) receptor on regulatory T cells. We suggest that mutations in these genes that are more severe may account for some of the unexplained infertility (or subfertility) in the general population.     

Intraerythrocyte and plasma lactate concentrations during exercise in humans

The purpose of this study was to examine plasma and intraerythrocyte lactate concentrations during graded exercise in humans. Seven adult volunteers performed a maximum O2 uptake (VO2max) test on a cycle ergometer. Plasma and intraerythrocyte lactate concentrations (mmol . L-1 of plasma or cell water) were determined at rest, during exercise, and at 15-min post-exercise. The results show that plasma and intraerythrocyte lactate concentrations were not significantly different from each other at rest or moderate (less than or equal to 50% VO2max) exercise. However, the plasma concentrations were significantly increased over the intraerythrocyte levels at 75% and 100% VO2max. The plasma to red cell lactate gradient reached a mean (+/- SE) 1.7 +/- 0.4 mmol . L-1 of H2O at exhaustion, and was linearly (r = 0.84) related to the plasma lactate concentration during exercise. Interestingly, at 15-min post-exercise the direction of the lactate gradient was reversed, with the mean intraerythrocyte concentration now being significantly increased over that found in the plasma. These results suggest that the erythrocyte membrane provides a barrier to the flux of lactate between plasma and red cells during rapidly changing blood lactate levels. Furthermore, these data add to the growing body of research that indicates that lactate is not evenly distributed in the various water compartments of the body during non-steady state exercise.     

Association between the plasma proteome and plasma α-tocopherol concentrations in humans

Vitamin E is a lipophilic antioxidant that has been inversely associated with certain chronic diseases; however, the biological processes regulated by this vitamin have not been fully elucidated. The objective of the present study was to examine the association between the most biologically active and abundant form of vitamin E in the circulation, α-tocopherol, and the plasma proteome. Subjects were from the Toronto Nutrigenomics and Health Study and included men and women (n=1,022) who completed a general health and lifestyle questionnaire and 196-item food frequency questionnaire, and provided a fasting blood sample. Plasma α-tocopherol concentrations were measured by high-performance liquid chromatography and 54 plasma proteins were assayed by a mass spectrometry-based multiple reaction monitoring method. Analysis of covariance was used to compare mean concentrations of plasma proteins across tertiles of α-tocopherol. Plasma concentrations of apolipoprotein C-III, fibrinogen alpha, beta, and gamma chains, fibronectin and fibrinopeptide A were significantly and positively associated with plasma α-tocopherol, while intermediate levels of α-tocopherol were significantly associated with higher levels of alpha-1B-glycoprotein (all P<.0009). These findings show that circulating levels of α-tocopherol are significantly associated with specific plasma proteins and suggest novel physiological effects of vitamin E.     

Modifier genes and protective alleles in humans and mice

Interest in modifier genes is growing rapidly because of their ability to modulate the phenotype of individuals with monogenic and multigenic traits and diseases. A neglected class of modifiers is protective alleles that can suppress disease in otherwise susceptible individuals. Together these modifier genes and protective alleles provide important glimpses into the molecular and cellular basis for the functional networks that provide robustness and homeostasis in complex biological systems.     

Angptl3-null mice show low plasma lipid concentrations by enhanced lipoprotein lipase activity.

Angiopoietin-like 3 (ANGPTL3) is a secreted protein with both angiogenesis and lipid metabolism functions. We generated knockout mice that failed to express the Angptl3 gene, and analyzed the lipid metabolism. Angptl3-null mice, fed a normal diet or a high-fat, high-calorie (HFC) diet, revealed markedly low plasma lipid concentrations, especially plasma triglyceride concentration, although the body weight and liver weight were not different between Angptl3-null mice and wild-type mice. Angptl3-null mice fed an HFC diet also revealed a significantly reduced epididymal adipose tissue weight despite there being no difference in adipocyte size between them and wild-type mice. A triglyceride clearance study indicated that the lower plasma triglyceride concentration in Angptl3-null mice was caused by an accelerated clearance of triglyceride. In fact, lipoprotein lipase and hepatic lipase activities in the post-heparin plasma of Angptl3-null mice were 1.57 times and 1.42 times higher than those of wild-type mice, respectively. These results suggest that ANGPTL3 may have an effect not only on lipid metabolism but also on adipose formation.     

Adrenergic mechanisms in control of plasma lipid concentrations.

The mechanisms of the changes in plasma lipids concentrations observed after beta-blockade were examined in 53 patients with hypertension receiving treatment with atenolol, metoprolol, propranolol, and oxprenolol in a randomised cross-over trial. Significant increases in mean plasma total and very-low-density lipoprotein (VLDL) triglyceride and reductions in high-density lipoprotein (HDL) cholesterol and free fatty acids concentrations wer observed with all four drugs, the increase in plasma triglyceride concentration being greatest after propranolol and oxprenolol. No significant changes were observed in total of LDL cholesterol concentrations, but HDL:LDL ratios and HDL cholesterol as a proportion of total cholesterol fell significantly. Thus plasma lipid concentrations should be monitored after three to six months of long-term treatment. Changes in triglyceride, HDL cholesterol and free fatty acid concentrations were associated with a highly significant reduction in clearance of soya oil (Intralipid) in 25 patients studied but were unrelated to changes in blood pressure. The fall in HDL cholesterol and rise in free fatty acid concentrations were significantly less in those with initially reduced HDL cholesterol or raised free fatty acid concentrations respectively. It is proposed that unopposed alpha stimulation inhibits lipoprotein lipase with a subsequent rise in plasma triglyceride and fall in HDL cholesterol concentration. Analysis of the relation between pretreatment concentrations and subsequent changes suggests that excessive alpha stimulation may impair production of HDL cholesterol in those with low HDL cholesterol concentrations before treatment. Subtle catecholamine-mediated changes in plasma lipid concentrations might provide a mechanism for the relation between stress and the development of cardiovascular events.     

Association between the plasma proteome and serum ascorbic acid concentrations in humans

Vitamin C has been associated with a reduced risk of chronic diseases, but the biological pathways regulated by vitamin C are not all known. The objective was to use a proteomics approach to identify plasma proteins associated with circulating levels of ascorbic acid. Men and women (n= 1022) 20–29 years of age from the Toronto Nutrigenomics and Health Study completed a general health and lifestyle questionnaire and a 196-item food frequency questionnaire and provided a fasting blood sample. Circulating ascorbic acid was analyzed by high-performance liquid chromatography, and a mass-spectrometry-based multiple reaction monitoring method was used to measure 54 proteins abundant in plasma that are involved in numerous physiologic pathways. Mean protein concentrations were compared across tertiles of serum ascorbic acid using analysis of covariance adjusted for sex, ethnocultural group, season of blood draw, hormonal contraceptive use among women, waist circumference and tertiles of plasma α-tocopherol. A Bonferroni significance level of P<.0009 was applied, and analyses were adjusted for multiple comparisons using the Tukey–Kramer procedure. Levels of complement C9, ceruloplasmin, alpha-1-anti-trypsin, angiotensinogen, complement C3, vitamin D binding protein and plasminogen were inversely associated with levels of ascorbic acid. The inverse association between ascorbic acid and vitamin D binding protein was highest in those with higher levels of serum 25-hydroxyvitamin D. In conclusion, several plasma proteins from various physiologic pathways are significantly associated with circulating levels of ascorbic acid. These findings suggest that vitamin C may have novel physiological effects.     

Genome-wide analysis of Brucella melitensis genes required throughout intranasal infection in mice

Brucellae are facultative intracellular Gram-negative coccobacilli that chronically infect various mammals and cause brucellosis. Human brucellosis is among the most common bacterial zoonoses and the vast majority of cases are attributed to B. melitensis. Using transposon sequencing (Tn-seq) analysis, we showed that among 3369 predicted genes of the B. melitensis genome, 861 are required for optimal growth in rich medium and 186 additional genes appeared necessary for survival of B. melitensis in RAW 264.7 macrophages in vitro. As the mucosal immune system represents the first defense against Brucella infection, we investigated the early phase of pulmonary infection in mice. In situ analysis at the single cell level indicates a succession of killing and growth phases, followed by heterogenous proliferation of B. melitensis in alveolar macrophages during the first 48 hours of infection. Tn-seq analysis identified 94 additional genes that are required for survival in the lung at 48 hours post infection. Among them, 42 genes are common to RAW 264.7 macrophages and the lung conditions, including the T4SS and purine synthesis genes. But 52 genes are not identified in RAW 264.7 macrophages, including genes implicated in lipopolysaccharide (LPS) biosynthesis, methionine transport, tryptophan synthesis as well as fatty acid and carbohydrate metabolism. Interestingly, genes implicated in LPS synthesis and β oxidation of fatty acids are no longer required in Interleukin (IL)-17RA^-/- mice and asthmatic mice, respectively. This demonstrates that the immune status determines which genes are required for optimal survival and growth of B. melitensis in vivo.     

Genome-wide analysis of genes targeted by qLTG3-1 controlling low-temperature germinability in rice

The control of seed germination under environmental conditions, where plants will be grown, is important for the adaptability of plants. Low-temperature is one of the most common environmental stress factors that affect plant growth and development and places a major limit on crop productivity in cultivated areas. Previously, qLTG3-1, a major quantitative trait locus controlling low-temperature tolerance at the germination stage in rice (called low-temperature germinability) was identified, which encodes a protein of unknown function. To identify genes targeted by qLTG3-1, a genome-wide expression profiling analysis using the 44 K Rice Oligo microarray was performed. Because the expression of qLTG3-1 was dramatically increased at 1 day after incubation, the expression profiles at this time were compared between Hayamasari, which has a loss-of-function qLTG3-1 allele, and a near isogenic line with a functional allele. A total of 4,587 genes showed significant differences between their expression levels in the two lines. Most of these genes might be involved in the process of seed germination itself, and then a focus was made on qLTG3-1 dependently induced or suppressed genes, defined as ‘qLTG3-1 dependent’ genes. Twenty-nine ‘qLTG3-1 dependent’ genes with diverse functions were categorized, implying that disruption of cellular homeostasis leads to a wide range of metabolic alterations and diverse cross-talk between various signaling pathways. In particular, genes involved in defense responses were up-regulated by qLTG3-1, indicating that qLTG3-1 expression is required for the expression of defense response genes in low-temperature germinability in rice.     

Homology search for genes

MOTIVATION: Life science researchers often require an exhaustive list of protein coding genes similar to a given query gene. To find such genes, homology search tools, such as BLAST or PatternHunter, return a set of high-scoring pairs (HSPs). These HSPs then need to be correlated with existing sequence annotations, or assembled manually into putative gene structures. This process is error-prone and labor-intensive, especially in genomes without reliable gene annotation. RESULTS: We have developed a homology search solution that automates this process, and instead of HSPs returns complete gene structures. We achieve better sensitivity and specificity by adapting a hidden Markov model for gene finding to reflect features of the query gene. Compared to traditional homology search, our novel approach identifies splice sites much more reliably and can even locate exons that were lost in the query gene. On a testing set of 400 mouse query genes, we report 79% exon sensitivity and 80% exon specificity in the human genome based on orthologous genes annotated in NCBI HomoloGene. In the same set, we also found 50 (12%) gene structures with better protein alignment scores than the ones identified in HomoloGene. AVAILABILITY: The Java implementation is available for download from http://www.bioinformatics.uwaterloo.ca/software.     

Genome-wide screenings for circadian clock genes in Drosophila

Sleep and Biological Rhythms - A high-density oligonucleotide probe array (GeneChip) has been used to learn how gene expression is globally regulated by the circadian clock mechanism. Here I review...