Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

Effects of sex, age, and visits on receipt of preventive healthcare services: a secondary analysis of national data

BACKGROUND: Sex and age may exert a combined influence on receipt of preventive services with differences due to number of ambulatory care visits. METHODS: We used nationally representative data to determine weighted percentages and adjusted odds ratios of men and women stratified by age group who received selected preventive services. The presence of interaction between sex and age group was tested using adjusted models and retested after adding number of visits. RESULTS: Men were less likely than women to have received blood pressure screening (aOR 0.44;0.40–0.50), cholesterol screening (aOR 0.72;0.65–0.79), tobacco cessation counseling (aOR 0.66;0.55–0.78), and checkups (aOR 0.53;0.49–0.57). In younger age groups, men were particularly less likely than women to have received these services. In adjusted models, this observed interaction between sex and age group persisted only for blood pressure measurement (p = .016) and routine checkups (p < .001). When adjusting for number of visits, the interaction of age on receipt of blood pressure checks was mitigated but men were still overall less likely to receive the service. CONCLUSION: Men are significantly less likely than women to receive certain preventive services, and younger men even more so. Some of this discrepancy is secondary to a difference in number of ambulatory care visits

A novel mutation of the calcium sensing receptor gene is associated with chronic pancreatitis in a family with heterozygous SPINK1 mutations

BACKGROUND: The role of mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene in chronic pancreatitis is still a matter of debate. Active SPINK1 is thought to antagonize activated trypsin. Cases of SPINK1 mutations, especially N34S, have been reported in a subset of patients with idiopathic chronic pancreatitis. However, the inheritance pattern is still unknown. Some cases with N34S heterozygosity have been reported with and without evidence for CP indicating neither an autosomal recessive nor dominant trait. Therefore SPINK1 mutations have been postulated to act as a disease modifier requiring additional mutations in a more complex genetic model. Familial hypocalciuric hypercalcemia (FHH) caused by heterozygous inactivating mutations in the calcium sensing receptor (CASR) gene is considered a benign disorder with elevated plasma calcium levels. Although hypercalcemia represents a risk factor for pancreatitis, increased rates of pancreatitis in patients with FHH have not been reported thus far. METHODS: We studied a family with a FHH-related hypercalcemia and chronic pancreatitis. DNA samples were analysed for mutations within the cationic trypsinogen (N29I, R122H) and SPINK1 (N34S) gene using melting curve analysis. Mutations within CASR gene were identified by DNA sequencing. RESULTS: A N34S SPINK1 mutation was found in all screened family members. However, only two family members developed chronic pancreatitis. These patients also had FHH caused by a novel, sporadic mutation in the CASR gene (518T>C) leading to an amino acid exchange (leucine->proline) in the extracellular domain of the CASR protein. CONCLUSION: Mutations in the calcium sensing receptor gene might represent a novel as yet unidentified predisposing factor which may lead to an increased susceptibility for chronic pancreatitis. Moreover, this family analysis supports the hypothesis that SPINK1 mutations act as disease modifier and suggests an even more complex genetic model in SPINK1 related chronic pancreatitis

Low bone mineral density is related to male gender and decreased functional capacity in early spondylarthropathies

The objective of this study was to determine the prevalence and risk factors of low bone mineral density (BMD) in patients with spondylarthropathies (SpA) at an early stage of disease. In this cross-sectional study, the BMD of lumbar spine and hips was measured in 130 consecutive early SpA patients. The outcome measure BMD was defined as (1) osteoporosis, (2) osteopenia, and (3) normal bone density. Logistic regression analyses were used to investigate relations between the following variables: age, gender, disease duration, diagnosis, HLA-B27, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Bath Ankylosing Spondylitis Metrology Index (BASMI), extra-spinal manifestations and medication, with outcome measure low BMD (osteopenia and/or osteoporosis). The SpA population had a median time since diagnosis of 6.6 months and a disease duration of 6.3 years. In total, 9% of the early SpA patients had osteoporosis, 38% osteopenia, and 53% normal BMD. On univariate analyses, male gender, diagnosis of ankylosing spondylitis, increased CRP, high BASFI, and high BASMI were significantly associated with low BMD. Factors showing a relation with low BMD in the multivariate model were male gender (OR 4.18, 95% confidence interval (CI) 1.73–10.09), high BASMI (OR 1.54, 95% CI 1.14–2.07), and high BASFI (OR 1.18, 95% CI 1.00–1.39). In early SpA patients, a high frequency (47%) of low BMD in femur as well as in lumbar spine was found. Low BMD was associated with male gender and decreased functional capacity. These findings emphasize the need for more alertness for osteoporosis and osteopenia in spondylarthropathy patients at an early stage of the disease

Current European Labyrinthula zosterae Are Not Virulent and Modulate Seagrass (Zostera marina) Defense Gene Expression

Pro- and eukaryotic microbes associated with multi-cellular organisms are receiving increasing attention as a driving factor in ecosystems. Endophytes in plants can change host performance by altering nutrient uptake, secondary metabolite production or defense mechanisms. Recent studies detected widespread prevalence of Labyrinthula zosterae in European Zostera marina meadows, a protist that allegedly caused a massive amphi-Atlantic seagrass die-off event in the 1930's, while showing only limited virulence today. As a limiting factor for pathogenicity, we investigated genotype×genotype interactions of host and pathogen from different regions (10–100 km-scale) through reciprocal infection. Although the endophyte rapidly infected Z. marina, we found little evidence that Z. marina was negatively impacted by L. zosterae. Instead Z. marina showed enhanced leaf growth and kept endophyte abundance low. Moreover, we found almost no interaction of protist×eelgrass-origin on different parameters of L. zosterae virulence/Z. marina performance, and also no increase in mortality after experimental infection. In a target gene approach, we identified a significant down-regulation in the expression of 6/11 genes from the defense cascade of Z. marina after real-time quantitative PCR, revealing strong immune modulation of the host's defense by a potential parasite for the first time in a marine plant. Nevertheless, one gene involved in phenol synthesis was strongly up-regulated, indicating that Z. marina plants were probably able to control the level of infection. There was no change in expression in a general stress indicator gene (HSP70). Mean L. zosterae abundances decreased below 10% after 16 days of experimental runtime. We conclude that under non-stress conditions L. zosterae infection in the study region is not associated with substantial virulence

Use of radiolabelled choline as a pharmacodynamic marker for the signal transduction inhibitor geldanamycin

There is an urgent need to develop non-invasive pharmacodynamic endpoints for the evaluation of new molecular therapeutics that inhibit signal transduction. We hypothesised that, when labelled appropriately, changes in choline kinetics could be used to assess geldanamycin pharmacodynamics, which involves inhibition of the HSP90 molecular chaperone→Raf1→Mitogenic Extracellular Kinase→Extracellular Signal-Regulated Kinase 1 and 2 signal transduction pathway. Towards identifying a potential pharmacodynamic marker response, we have studied radiolabelled choline metabolism in HT29 human colon carcinoma cells following treatment with geldanamycin. We studied the effects of geldanamycin, on net cellular accumulation of (methyl-14C)choline and (methyl-14C)phosphocholine production. In parallel experiments, the effects of geldanamycin on extracellular signal-regulated kinase 1 and 2 phosphorylation and cell viability were also assessed. Additional validation studies were carried out with the mitogenic extracellular kinase inhibitor U0126 as a positive control; a cyclin-dependent kinase-2 inhibitor roscovitine and the phosphatidylinositol 3-kinase inhibitor LY294002 as negative controls. Hemicholinium-3, an inhibitor of choline transport and choline kinase activity was included as an additional control. In exponentially growing HT29 cells, geldanamycin inhibited extracellular signal-regulated kinase 1 and 2 phosphorylation in a concentration- and time-dependent manner. These changes were associated with a reduction in (methyl-14C)choline uptake, (methyl-14C) phosphocholine production and cell viability. Brief exposure to U0126, suppressed phosphocholine production to the same extent as Hemicholinium-3. In contrast to geldanamycin and U0126, which act upstream of extracellular signal-regulated kinase 1 and 2, roscovitine and LY294002 failed to suppress phosphocholine production. Our results suggest that when labelled with carbon-11 isotope, (methyl-11C)choline may be a useful pharmacodynamic marker for the non-invasive evaluation of geldanamycin analogues

Whole-genome analysis of introgressive hybridization and characterization of the bovine legacy of Mongolian yaks

The yak is remarkable for its adaptation to high altitude and occupies a central place in the economies of the mountainous regions of Asia. At lower elevations, it is common to hybridize yaks with cattle to combine the yak’s hardiness with the productivity of cattle. Hybrid males are sterile, however, preventing the establishment of stable hybrid populations, but not a limited introgression after backcrossing several generations of female hybrids to male yaks. Here we inferred bovine haplotypes in the genomes of 76 Mongolian yaks using high-density SNP genotyping and whole-genome sequencing. These yaks inherited ~1.3% of their genome from bovine ancestors after nearly continuous admixture over at least the last 1,500 years. The introgressed regions are enriched in genes involved in nervous system development and function, and particularly in glutamate metabolism and neurotransmission. We also identified a novel mutation associated with a polled (hornless) phenotype originating from Mongolian Turano cattle. Our results suggest that introgressive hybridization contributed to the improvement of yak management and breeding

Depletion of Murine Intestinal Microbiota: Effects on Gut Mucosa and Epithelial Gene Expression

Background Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. Methodology/Principal Findings Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. Conclusion We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice

Aging of the mammalian gastrointestinal tract: a complex organ system

Gastrointestinal disorders are a major cause of morbidity in the elderly population. The gastrointestinal tract is the most complex organ system; its diverse cells perform a range of functions essential to life, not only secretion, digestion, absorption and excretion, but also, very importantly, defence. The gastrointestinal tract acts not only as a barrier to harmful materials and pathogens but also contains the vast number of beneficial bacterial populations that make up the microbiota. Communication between the cells of the gastrointestinal tract and the central nervous and endocrine systems modifies behaviour; the organisms of the microbiota also contribute to this brain–gut–enteric microbiota axis. Age-related physiological changes in the gut are not only common, but also variable, and likely to be influenced by external factors as well as intrinsic aging of the cells involved. The cellular and molecular changes exhibited by the aging gut cells also vary. Aging intestinal smooth muscle cells exhibit a number of changes in the signalling pathways that regulate contraction. There is some evidence for age-associated degeneration of neurons and glia of the enteric nervous system, although enteric neuronal losses are likely not to be nearly as extensive as previously believed. Aging enteric neurons have been shown to exhibit a senescence-associated phenotype. Epithelial stem cells exhibit increased mitochondrial mutation in aging that affects their progeny in the mucosal epithelium. Changes to the microbiota and intestinal immune system during aging are likely to contribute to wider aging of the organism and are increasingly important areas of analysis. How changes of the different cell types of the gut during aging affect the numerous cellular interactions that are essential for normal gut functions will be important areas for future aging research

Neonatal Colonisation Expands a Specific Intestinal Antigen-Presenting Cell Subset Prior to CD4 T-Cell Expansion, without Altering T-Cell Repertoire

Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα+) antigen-presenting cell subset, whilst SIRPα−CD11R1+ antigen-presenting cells (APCs) are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα+ antigen-presenting cells as orchestrators of early-life mucosal immune development