Molecular Reclassification of Crohn’s Disease: A Cautionary Note on Population Stratification

Complex human diseases commonly differ in their phenotypic characteristics, e.g., Crohn’s disease (CD) patients are heterogeneous with regard to disease location and disease extent. The genetic susceptibility to Crohn’s disease is widely acknowledged and has been demonstrated by identification of over 100 CD associated genetic loci. However, relating CD subphenotypes to disease susceptible loci has proven to be a difficult task. In this paper we discuss the use of cluster analysis on genetic markers to identify genetic-based subgroups while taking into account possible confounding by population stratification. We show that it is highly relevant to consider the confounding nature of population stratification in order to avoid that detected clusters are strongly related to population groups instead of disease-specific groups. Therefore, we explain the use of principal components to correct for population stratification while clustering affected individuals into genetic-based subgroups. The principal components are obtained using 30 ancestry informative markers (AIM), and the first two PCs are determined to discriminate between continental origins of the affected individuals. Genotypes on 51 CD associated single nucleotide polymorphisms (SNPs) are used to perform latent class analysis, hierarchical and Partitioning Around Medoids (PAM) cluster analysis within a sample of affected individuals with and without the use of principal components to adjust for population stratification. It is seen that without correction for population stratification clusters seem to be influenced by population stratification while with correction clusters are unrelated to continental origin of individuals.     

Environmental Particulate (PM2.5) Augments Stiffness-Induced Alveolar Epithelial Cell Mechanoactivation of Transforming Growth Factor Beta

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.     

Effets sur le spermatozoïde humain du Diuron (3-(3,4-dichlorophényl)-1,1-diméthyl-urée) et de l’un de ses produits de transformation, la 3,4-dichloroaniline (3,4-DCA) (Etude préliminaire)

Diuron belongs to the family of halogenophenylureas, one of the main groups of herbicides used for more than 40 years. These herbicides absorb sunlight and can be photochemically transformed in the environment (herbicides are transformed on the soil surface exposed to sunlight) or biotransformed by microorganisms present in soil or in water. The metabolites (chlorohydroxyphenylurea, chlorophenylaniline, respectively) are more toxic than the parent compound, as demonstrated by a bioluminescence inhibition assay performed with a marine bacterium (Vibrio fischeri toxicity test). The lipophilicity of these pesticides makes the cell membrane a target for their action, especially the spermatozoa cell membrane. The aim of this study is to use human spermatozoa to evaluate the effect of this urea pesticide and its biotransformed product on the spermatozoa membrane. We investigated the structural and functional effects of these environmental pollutants on spermatozoa. Three million spermatozoa purified on a 95/47.5% Percoll gradient were suspended in 250 μl of modified Earle’s medium (without phenol red) supplemented with 7.5% of human decomplemented serum. Pesticides (Diuron or 3,4-dichloroaniline (3,4-DCA)) were added at a final concentration of 0.1; 1 and 5 mM. Samples were incubated at room temperature for 24 hours. We show that both Diuron and 3,4-DCA decrease motility and vitality of spermatozoa incubated with the highest concentration of pesticides. Our preliminary results show that the effects are more rapid and more intense with the biotransformed product (3,4-DCA) than with Diuron. Addition of herbicide to human spermatozoa increases membrane fluidity, assessed by measuring the fluorescence polarisation anisotropy with a fluorescent probe: 1,6-diphenyl-1,3,5-hexatriene (DPH). Changes in membrane fluidity may be a primary toxic effect of these herbicides. These results suggest that human spermatozoa may constitute a valuable indicator of the toxic effects of pesticides.     

An evaluation of fMLP pocket dimensions and features using formyltetrapeptides.

The formyltetrapeptides for-Met-Leu-Leu-Phe-OMe 1, for-Met-Leu-Aib-Phe-OMe 2, for-Met-Leu-Ac6c-Phe-OMe 3, for-Met-Leu-Pro-Phe-OMe 4, for-Met-Pro-Pro-Phe-OMe 5, for-Met-Aib-Aib-Phe-OMe 6, for-Met-Pro-Aib-Phe-OMe 7 and for-Met-Aib-Pro-Phe-OMe 8 were synthesized and biologically tested on human neutrophils in an attempt to evaluate the specific receptor pocket dimensions and features. Our results indicate that the shift in the Phe residue to the fourth position in these compounds strongly reduces chemotactic response, but is efficacious in triggering superoxide anion production and lysozyme release (order of potency 3 > 2 > 1 > 4 > 6 > 8 > 5 > 7). The potency of the two latter responses correlates well with the affinity data obtained in binding experiments.     

Adventitious Root Formation in Veronica Spp.

Species of the genus Veronica differ in habitat preferences,growth form and in adventitious root production. The annualspecies rarely or never produce adventitious roots in intactplants in the field but some, for example V. persica and V.arvensis will root vigorously from single node stem segmentsin culture. Others, such as V. agrestis require the presenceof IAA for substantial levels of root formation to occur incultured stem segments. Veronica hederifolia cuttings rarelyproduce roots. Stem cuttings of the perennial species, in general,rooted more vigorously than those of annual plants. Both V.fihiformis and V. serpyllifolia root very strongly. The position of root production from the stem cuttings differedfrom species to species. Roots arose either from the node, theregion of the base or at some intermediate point. Veronica arvensis,V. chamaedrys and V. persica rooted mainly from the basal regionwhereas V. filiformis rooted mainly from the node. Veronicaserpyllifolia cuttings rooted at both of these locations. Veronica filiformis, a perennial species that is infertile inBritain, produces root primordia in intact plants at nodes whichare close to the shoot apex. Thus, even very young stem segmentshave ‘preformed’ root primordia. For this reason,detached stem segments of V. filiformis root very rapidly andthis probably has been of great significance in its successfulinvasion and spread in lawns and short turf areas. Veronica spp., adventitious roots, indol-3-ylacetic acid, root primordia, vegetative reproduction     

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

A New Species of Acrodipsas Sands (Lepidoptera: Lycaenidae) from Inland New South Wales and Southern Queensland

Acrodipsas mortoni sp.n. from inland New South Wales and southern Queensland is described, figured, contrasted with the related A. arcana (Miller and Edwards) and assigned to the illidgei species-group.     

Propionate and butyrate dependent bacterial sulfate reduction at extremely haloalkaline conditions and description of Desulfobotulus alkaliphilus sp. nov.

Evidence on the utilization of simple fatty acids by sulfate-reducing bacteria (SRB) at extremely haloalkaline conditions are practically absent, except for a single case of syntrophy by Desulfonatronum on acetate. Our experiments with sediments from soda lakes of Kulunda Steppe (Altai, Russia) showed sulfide production with sulfate as electron acceptor and propionate and butyrate (but not acetate) as an electron donor at a pH 10–10.5 and a salinity 70–180 g l^?1. With propionate as substrate, a highly enriched sulfidogenic culture was obtained in which the main component was identified as a novel representative of the family Syntrophobacteraceae. With butyrate as substrate, a pure SRB culture was isolated which oxidized butyrate and some higher fatty acids incompletely to acetate. The strain represents the first haloalkaliphilic representative of the family Desulfobacteraceae and is described as Desulfobotulus alkaliphilus sp. nov.     

Subcellular localization of tissue polypeptide antigen and cytokeratins in epithelial cells (salivary ad mammary glands). Combined use of the cryoultramicrotomy and the protein A-gold technique

Epithelial cells from various sites and at various stages of differentiation reveal distinct cytokeratin polypeptide patterns. WE have localized these heterogeneous elements at the subcellular level in human salivary glands and in a solid tumor of the breast using a monoclonal and a polyclonal antibody against cytokeratin, and an antibody against tissue polypeptide antigen (TPA) which seems to be related to some cytokeratins. Labeling by the cytokeratin antibodies was more intense in squamous and duct cells than in acinar cells. The TPA:B1 antibody reacted predominantly with duct cells and to a lesser extent with acinar and squamous cells. A precise evaluation of the labeling pattern and a well-preserved cell structure appeared to be important factors in obtaining more detailed information about intermediate filament proteins. The cryoultramicrotomy and the protein A-gold technique are suitable for these studies.     

Identification of an enzymatic activity that hydrolyzes protein-bound ADP-ribose in skeletal muscle

An enzymatic activity present in high-speed supernatant fluids of rat skeletal muscle was found that catalyzes the release of ADP-ribose from ADP-ribosylated-modified lysozyme. The nature of the product was proved by chromatographic studies and proton nuclear magnetic resonance spectroscopy. The enzyme activity is stimulated by Mg2+, dithioerythritol, and flouride. These results and those published earlier (Soman, G., Mickelson, J.R., Louis, C.F., and Graves, D.J. (1984) Biochem. Biophys. Res. Commun. 120, 973-980) show that ADP-ribosylation is a reversible process in skeletal muscle.