Control of Nitrification by Tree Species in a Common-Garden Experiment

We studied the effect of tree species on nitrification in five young plantations and an old native beech coppice forest at the Breuil experimental site in central France. The potential net nitrification (PNN) of soil was high in beech, Corsican pine, and Douglas fir plantations (high nitrifying stands denoted H) and low in spruce and Nordmann fir plantations as well as in native forest stands (low nitrifying stands denoted L). We hypothesized that tree species would stimulate or inhibit nitrification in transplanted soil cores within a few years after the cores were transplanted between stands. We first initiated a transplant experiment where soil cores were exchanged between all stands. The PNN remained high in soil cores from H transferred to H and low in soil cores from L transferred to L. The PNN increased considerably after 16 months in soil cores transferred from L to H, whereas the transfer of soil cores from H to L decreased the PNN only slightly after 28 months. In a second transplant experiment, forest floor material was exchanged between the Douglas fir (H) and the native forest (L) stand. Six months later, the forest floor from the native forest had increased the PNN of the Douglas fir soil considerably, whereas the forest floor from Douglas fir did not affect the PNN of the soil in the native forest stand. It was concluded that beech, Corsican pine, and Douglas fir rapidly stimulate soil nitrification by either activation of suppressed nitrifier communities and/or colonization by new nitrifier communities. Conversely, the slow and irregular reduction of nitrification in spruce, Nordmann fir, and native forest was probably due to the low and heterogeneously distributed flux of inhibiting substances per volume of soil. Our experiments suggest that the inhibition of nitrification is not tightly connected to forest floor leachates, but that the forest floor both reflects and maintains the major ongoing processes. In the long term, humus build up and the production of inhibiting substances may completely block the nitrification activity.     

Identification and mapping of two powdery mildew resistance genes in <Emphasis Type="Italic">Triticum boeoticum</Emphasis> L.

Powdery mildew (PM) caused by Blumeria graminis f. sp. tritici (Bgt), is one of the important foliar diseases of wheat that can cause serious yield losses. Breeding for cultivars with diverse resources of resistance is the most promising approach for combating this disease. The diploid A genome progenitor species of wheat are an important resource for new variability for disease resistance genes. An accession of Triticum boeoticum (A^bA^b) showed resistance against a number of Bgt isolates, when tested using detached leaf segments. Inheritance studies in a recombinant inbred line population (RIL), developed from crosses of PM resistant T. boeoticum acc. pau5088 with a PM susceptible T. monococcum acc. pau14087, indicated the presence of two powdery mildew resistance genes in T. boeoticum acc. pau5088. Analysis of powdery mildew infection and molecular marker data of the RIL population revealed that both powdery mildew resistance genes are located on the long arm of chromosome 7A. Mapping was conducted using an integrated linkage map of 7A consisting of SSR, RFLP, STS, and DArT markers. These powdery mildew resistance genes are tentatively designated as PmTb7A.1 and PmTb7A.2. The PmTb7A.2 is closely linked to STS markers MAG2185 and MAG1759 derived from RFLP probes which are linked to powdery mildew resistance gene Pm1. This indicated that PmTb7A.2 might be allelic to Pm1. The PmTb7A.1, flanked by a DArT marker wPt4553 and an SSR marker Xcfa2019 in a 4.3 cM interval, maps proximal to PmT7A.2. PmTb7A.1 is putatively a new powdery mildew resistance gene. The powdery mildew resistance genes from T. boeoticum are currently being transferred to cultivated wheat background through marker-assisted backcrossing, using T. durum as bridging species.     

Molecular reclassification of Crohn's disease by cluster analysis of genetic variants

Background

Crohn''s Disease (CD) has a heterogeneous presentation, and is typically classified according to extent and location of disease. The genetic susceptibility to CD is well known and genome-wide association scans (GWAS) and meta-analysis thereof have identified over 30 susceptibility loci. Except for the association between ileal CD and NOD2 mutations, efforts in trying to link CD genetics to clinical subphenotypes have not been very successful. We hypothesized that the large number of confirmed genetic variants enables (better) classification of CD patients.

Methodology/Principal Findings

To look for genetic-based subgroups, genotyping results of 46 SNPs identified from CD GWAS were analyzed by Latent Class Analysis (LCA) in CD patients and in healthy controls. Six genetic-based subgroups were identified in CD patients, which were significantly different from the five subgroups found in healthy controls. The identified CD-specific clusters are therefore likely to contribute to disease behavior. We then looked at whether we could relate the genetic-based subgroups to the currently used clinical parameters. Although modest differences in prevalence of disease location and behavior could be observed among the CD clusters, Random Forest analysis showed that patients could not be allocated to one of the 6 genetic-based subgroups based on the typically used clinical parameters alone. This points to a poor relationship between the genetic-based subgroups and the used clinical subphenotypes.

Conclusions/Significance

This approach serves as a first step to reclassify Crohn''s disease. The used technique can be applied to other common complex diseases as well, and will help to complete patient characterization, in order to evolve towards personalized medicine.     

Excess type I interferon signaling in the mouse seminiferous tubules leads to germ cell loss and sterility

Type I (α and β) interferons (IFNs) elicit antiproliferative and antiviral activities via the surface receptor IFNAR. Serendipitous observations in transgenic mice in 1988 strongly suggested that IFNα/β overexpression in the testis disrupts spermatogenesis. Here, we compare a new mouse strain transgenic for IFNβ (Tg10) and a sister strain lacking the IFNAR1 subunit of IFNAR (Tg10-Ifnar1(-/-)), both strains expressing the transgene in the testis. The main source of IFNβ RNA was the spermatid population. Importantly, the Tg10 mice, but not the double mutant Tg10-Ifnar1(-/-), showed altered spermatogenesis. The first IFNAR-dependent histological alteration was a higher apoptosis index in all germ cell categories apart from non-dividing spermatogonia. This occurred 3 weeks after the onset of IFNβ production at postnatal day 20 and in the absence of somatic cell defects in terms of cell number, expression of specific cell markers, and hormonal activities. Several known interferon-stimulated genes were up-regulated in Tg10 Sertoli cells and prepachytene germ cells but not in pachytene spermatocytes and spermatids. In concordance with this, pachytene spermatocytes and spermatids isolated from wild-type testes did not display measurable amounts of IFNAR1 and phosphorylated STAT1 upon IFNβ challenge in vitro, suggesting hyporesponsiveness of these cell types to IFN. At day 60, Tg10 males were sterile, and Sertoli cells showed increased amounts of anti-Mullerian hormone and decreased production of inhibin B, both probably attributable to the massive germ cell loss. Type I interferon signaling may lead to idiopathic infertilities by affecting the interplay between germ cells and Sertoli cells.     

The molecular subtype classification is a determinant of sentinel node positivity in early breast carcinoma

Introduction

Several authors have underscored a strong relation between the molecular subtypes and the axillary status of breast cancer patients. The aim of our work was to decipher the interaction between this classification and the probability of a positive sentinel node biopsy.

Materials and Methods

Our dataset consisted of a total number of 2654 early-stage breast cancer patients. Patients treated at first by conservative breast surgery plus sentinel node biopsies were selected. A multivariate logistic regression model was trained and validated. Interaction covariate between ER and HER2 markers was a forced input of this model. The performance of the multivariate model in the training and the two validation sets was analyzed in terms of discrimination and calibration. Probability of axillary metastasis was detailed for each molecular subtype.

Results

The interaction covariate between ER and HER2 status was a stronger predictor (p = 0.0031) of positive sentinel node biopsy than the ER status by itself (p = 0.016). A multivariate model to determine the probability of sentinel node positivity was defined with the following variables; tumour size, lympho-vascular invasion, molecular subtypes and age at diagnosis. This model showed similar results in terms of discrimination (AUC = 0.72/0.73/0.72) and calibration (HL p = 0.28/0.05/0.11) in the training and validation sets. The interaction between molecular subtypes, tumour size and sentinel nodes status was approximated.

Discussion

We showed that biologically-driven analyses are able to build new models with higher performance in terms of breast cancer axillary status prediction. The molecular subtype classification strongly interacts with the axillary and distant metastasis process.     

The role of commensal bacteria in the regulation of sensitization to food allergens

The prevalence of life-threatening anaphylactic responses to food is rising at an alarming rate. The emerging role of the gut microbiota in regulating food allergen sensitization may help explain this trend. The mechanisms by which commensal bacteria influence sensitization to dietary antigens are only beginning to be explored. We have found that a population of mucosa-associated commensal anaerobes prevents food allergen sensitization by promoting an IL-22-dependent barrier protective immune response that limits the access of food allergens to the systemic circulation. This early response is followed by an adaptive immune response mediated in part by an expansion of Foxp3⁺ Tregs that fortifies the tolerogenic milieu needed to maintain non-responsiveness to food. Bacterial metabolites, such as short-chain fatty acids, may contribute to the process through their ability to promote Foxp3⁺ Treg differentiation. This work suggests that environmentally induced alterations of the gut microbiota offset the regulatory signals conferred by protective bacterial species to promote aberrant responses to food. Our research presents exciting new possibilities for preventing and treating food allergies based on interventions that modulate the composition of the gut microbiota.     

Cytopathogenesis of Sendai Virus in Well-Differentiated Primary Pediatric Bronchial Epithelial Cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.Sendai virus (SeV) is a nonsegmented negative-strand RNA virus of the Paramyxoviridae family. Recombinant SeV (rSeV) has been extensively studied as a vector for vaccines, cancer immunotherapy, and gene therapy (14, 22, 34, 41, 43). SeV is virulent in rodents, but despite extensive antigenic and genetic similarity to human parainfluenza virus type 1 (hPIV1), it is not known to cause disease in humans (33). Interest in rSeV as a vector is exemplified by the fact that (i) its genome can easily be manipulated to stably express heterologous genes (9), (ii) it does not undergo homologous recombination, (iii) cell transduction is independent of the cell cycle, (iv) there is no DNA phase during replication and hence no possibility of cell transformation, and (v) its cell or tissue tropisms and replication competency may be modulated by reverse genetics and appropriate rescue systems (5, 8).Much of the research on rSeV as a vector involves monolayer cells and animal models and employs both replication-competent and transmission-incompetent viruses. In view of its respiratory tract tropism, particular attention was paid to its use as a gene therapy vector for lung diseases such as cystic fibrosis (CF) (2, 13, 14, 43). Indeed, early studies demonstrated its capacity to efficiently overcome a series of extra- and intracellular barriers in the respiratory tract, such as the glycocalyx, mucus layer, mucociliary clearance, and cell membranes (13, 14, 43). However, in vivo studies demonstrated that rSeV-mediated gene transduction was transient (lasting ∼7 days) and that repeated administration was inefficient (16). The reasons for this transient transduction remain unclear.In contrast, the capacity to efficiently and transiently transduce host cells is of considerable interest from a vaccine vector viewpoint. Indeed, promising rSeV-vectored vaccine candidates have been described for other respiratory viruses, such as respiratory syncytial virus (RSV), hPIV1, hPIV2, hPIV3, and systemic viruses, such as HIV-1 (22, 40, 44). Despite its considerable promise as a viral vector, little is known about how rSeV interacts with human airway epithelial cells (HAE).To address this, we established an ex vivo/in vitro model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs) in air-liquid interface (ALI) cultures. The pediatric origin of the primary bronchial cells allowed us to investigate SeV-host interactions in a pediatric context. The need for CF gene therapy or respiratory virus vaccines for infants or children emphasizes the relevance of this ex vivo/in vitro pediatric model. Using rSeV expressing enhanced GFP (rSeV/eGFP), we comprehensively investigated the consequences of SeV infection in these cultures, including the types of cells infected, virus growth kinetics, cytopathic effects (CPE), and inflammatory responses. Our data provided novel insights into the interaction of SeV with pediatric airway epithelium and the limitations and/or advantages of its use as a vector.     

Agonist-dependent Endocytosis of γ-Aminobutyric Acid Type A (GABAA) Receptors Revealed by a γ2(R43Q) Epilepsy Mutation

GABA-gated chloride channels (GABA_ARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABA_ARs on the cell surface to better understand the trafficking of GABA_ARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABA_AR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABA_ARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABA_AR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABA_ARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.