Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors

LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the β-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways.     

Intra-specific competition and the radial development of wood density, microfibril angle and modulus of elasticity in plantation-grown Eucalyptus nitens

In eucalypt plantations managed for solid-wood products, radial trends in wood density, microfibril angle (MFA) and stiffness (modulus of elasticity, MoE) are properties of potential commercial importance that can be affected by competition from neighbouring trees. In this study, wood properties at breast height (1.3?m) were studied on radial strips prepared from 12-mm pith-to-bark wood cores taken from 20 trees in a 22-year-old Eucalyptus nitens (Deane and Maiden) Maiden thinning trial in north-eastern Tasmania, Australia. Thinning treatments were applied at age 6?years. Trees were sampled from each of the 200, 400?stems ha^?1 and unthinned control treatments. Intra-specific competition for each sampled tree was estimated using the basal area growth of surrounding trees. SilviScan? technology was used to produce radial profiles of wood density, MFA and MoE. Results indicate a reduction in intra-specific competition through thinning of E. nitens plantations at an early age leads to a transient increase in MFA but has no significant effect on wood density or the intra-annual cycle of wood density. The correlation between the level of intra-specific competition and initial change in MFA following thinning, and a significant relationship between tree shape and mean MFA at breast height suggests the change in MFA is a post-thinning response to increased exposure and wind sway.     

Direct fluorimetric sensing of UV-excited analytes in biological and environmental samples using molecularly imprinted polymer nanoparticles and fluorescence polarization

A rapid, robust, sensitive and economic sensing method, based on a molecularly imprinted polymer (MIP) synthetic antibody mimic, and fluorescence polarization analysis, for the direct detection of UV-excited fluorescent analytes in food and environmental samples was developed. Fluoroquinolone (FQ) antibiotics were used as fluorescent model analytes. Water-compatible MIP nanoparticles were synthesized with enrofloxacin (ENRO) as the imprinting template. Fluorescence polarization measurements then allow the direct determination of the amount of ENRO and other structurally related piperazine-based fluoroquinolones that bind to the MIP. No separation step was required since this technique distinguishes in situ analyte molecules bound to the MIP from the free analyte in solution. This assay was successfully applied for the first time to determine FQs in real samples, i.e. tap water and milk, without any prior concentration step, by simply adding a known amount of MIP. No interference by the sample components was observed even though the excitation was in the UV region. In tap water, a low limit of detection of 0.1 nM for ENRO was achieved with 5 μg mL(-1) of MIP. In milk, ENRO and danofloxacin, whose MRLs have been fixed at 0.28 μM and 0.08 μM, respectively, could be selectively measured and distinguished from other families of antibiotics. The procedure is very easy and practical as it consists of simply precipitating the milk proteins with acetonitrile and adding buffer and MIP to the supernatant before reading the polarization values with a spectrofluorimeter.     

Multi-population QTL detection for aerial morphogenetic traits in the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Medicago truncatula, as a model species, is useful to study the genetic control of traits of agronomic interest in legumes species. Aerial morphogenesis is a key component of forage and seed yield. It was measured in four mapping populations originating from five parental lines. Single and multi-population quantitative trait locus (QTL) detections were carried out. A large variation was observed within populations and transgressive segregation was noted. Most traits showed high heritabilities in all seasons. Length of primary branches (LPB, cm) was positively correlated to branch elongation rate (BER, cm day⁻¹) and aerial dry matter (ADM, g). Flowering time (FT, °C day⁻¹) showed negative correlations with length of main stem (LMS, cm) and BER. One hundred and forty-one QTLs for BER, LMS, FT, LPB, diameter of primary branches (DPB), number of primary branches (NPB), number of nodes (NI) and ADM were identified and localized over all eight chromosomes. Single and multi-population analyses showed that the most important regions for aerial morphogenetic traits were chromosomes 1, 2, 7 and 8. Multi-population analysis revealed three regions of major QTLs affecting aerial morphogenetic traits (LPB, LMS, NPB, BER and FT). A region involved in flowering time variation was revealed on chromosome 6 on a single population. These results were used to identify candidate genes that could control variation for aerial morphogenesis traits in this species and in related crop legume species.     

A randomized controlled trial of the GnRH antagonist ganirelix in Chinese normal responders: high efficacy and pregnancy rates

Gonadotropin-releasing hormone (GnRH) antagonists for controlled ovarian stimulation (COS) were only recently introduced into China. The efficacy and safety of the GnRH antagonist ganirelix was assessed in a multicenter, controlled, open-label study, in which Chinese women were randomized to either ganirelix (n = 113) or a long GnRH agonist protocol of triptorelin (n = 120). The primary end point was the amount of recombinant follicle-stimulating hormone (rFSH) required to meet the human chorionic gonadotropin criterion (three follicles ≥17 mm). The amount of rFSH needed was significantly lower for ganirelix (1272 IU) vs. triptorelin (1416 IU; P< 0.001). Ongoing pregnancy rates per started cycle were 39.8% (ganirelix) and 39.2% (triptorelin). Although both treatments were well tolerated, cancellation due to risk of ovarian hyperstimulation syndrome (OHSS) was less frequent with ganirelix (1.8%) than triptorelin (7.5%) (P = 0.06). Less rFSH was needed in the ganirelix protocol than the long GnRH agonist protocol, with fewer reported cases of OHSS and similar pregnancy rates.     

Treatment with the arginase inhibitor Nw-hydroxy-nor-L-arginine restores endothelial function in rat adjuvant-induced arthritis

Introduction

Endothelial dysfunction (ED) participates to atherogenesis associated to rheumatoid arthritis. We recently reported increased arginase activity/expression in vessels from adjuvant-induced arthritis (AIA) rats. In the present study, we investigated the effects of a curative treatment with the arginase inhibitor N_w-hydroxy-nor-L-arginine (nor-NOHA) on vascular dysfunction in AIA rats.

Methods

AIA rats were treated with nor-NOHA (40 mg/kg/d, ip) for 21 days after the onset of arthritis. A group of untreated AIA rats and a group of healthy rats served as controls. ED was assessed by the vasodilatory effect of acetylcholine (Ach) on aortic rings. The role of superoxide anions, prostanoids, endothelium-derived hyperpolarizing factor (EDHF) and nitric oxide synthase (NOS) pathway was studied. Plasma levels of IL-6 and vascular endothelial growth factor (VEGF) were determined by ELISA kits. Arthritis severity was estimated by a clinical, radiological and histological analysis.

Results

Nor-NOHA treatment fully restored the aortic response to Ach to that of healthy controls. The results showed that this beneficial effect is mediated by an increase in NOS activity and EDHF and reduced superoxide anion production as well as a decrease in the activity of cyclooxygenase (COX)-2, thromboxane and prostacyclins synthases. In addition, nor-NOHA decreased IL-6 and VEGF plasma levels in AIA rats. By contrast, the treatment did not modify arthritis severity in AIA rats.

Conclusions

The treatment with an arginase inhibitor has a potent effect on ED in AIA independently of the severity of the disease. Our results suggest that this new pharmacological approach has the potential as a novel add-on therapy in the treatment of RA.     

Diversity in the Protein N-Glycosylation Pathways Within the Campylobacter Genus

The foodborne bacterial pathogen, Campylobacter jejuni, possesses an N-linked protein glycosylation (pgl) pathway involved in adding conserved heptasaccharides to asparagine-containing motifs of >60 proteins, and releasing the same glycan into its periplasm as free oligosaccharides. In this study, comparative genomics of all 30 fully sequenced Campylobacter taxa revealed conserved pgl gene clusters in all but one species. Structural, phylogenetic and immunological studies showed that the N-glycosylation systems can be divided into two major groups. Group I includes all thermotolerant taxa, capable of growth at the higher body temperatures of birds, and produce the C. jejuni-like glycans. Within group I, the niche-adapted C. lari subgroup contain the smallest genomes among the epsilonproteobacteria, and are unable to glucosylate their pgl pathway glycans potentially reminiscent of the glucosyltransferase regression observed in the O-glycosylation system of Neisseria species. The nonthermotolerant Campylobacters, which inhabit a variety of hosts and niches, comprise group II and produce an unexpected diversity of N-glycan structures varying in length and composition. This includes the human gut commensal, C. hominis, which produces at least four different N-glycan structures, akin to the surface carbohydrate diversity observed in the well-studied commensal, Bacteroides. Both group I and II glycans are immunogenic and cell surface exposed, making these structures attractive targets for vaccine design and diagnostics.In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide variety of essential cell-cell and cell-matrix processes ranging from immune recognition to cancer development. The first general protein glycosylation (pgl)¹ pathway was discovered in the epsilonproteobacterium Campylobacter jejuni (1). The organism transfers a conserved heptasaccharide en bloc to asparagine residues within the sequon D/E- X₁-N-X₂-S/T (X₁, X₂ ≠ P) of >60 glycoproteins (2–4). Furthermore, the pathway can be functionally transferred into Escherichia coli, and the oligosaccharyltransferase (OTase), PglB, is capable of adding foreign sugars to acceptor proteins (5–7). C. jejuni PglB also possesses hydrolase activity, influenced by the cellular growth phase and osmotic environment, releasing free oligosaccharides (fOS) into the periplasmic space in a 10:1 ratio relative to the amount of heptasaccharide N-linked to protein (8, 9).The C. jejuni N-linked heptasaccharide is conserved in structure in both C. jejuni and C. coli, the two most commonly isolated pathogenic Campylobacter species and major causes of human enteritis worldwide (10, 11). All campylobacters, but one, possess conserved pgl genes required for N-linked protein glycosylation ((12) and this study). This post-translational modification in C. jejuni influences DNA uptake, chicken and mouse colonization, epithelial cell adherence and invasion, recognition by human sera, and binding to the macrophage galactose lectin (MGL) receptor on dendritic cells (2, 13–17). Several Campylobacter species have now been recognized as emerging pathogens and causative agents of human gastroenteritis (e.g. C. upsaliensis and C. hyointestinalis), gingivitis, periodontitis, and human abortions (e.g. C. rectus, C. concisus, C. gracilis, C. showae, and C. upsaliensis) and inflammatory bowel disease in children (e.g. C. concisus) (18). Other species cause venereal disease and infertility in cattle (C. fetus subsp. venerealis; Cfv) or abortions in sheep (C. fetus subsp. fetus; Cff) (19).In this study, we used phylogenetic, immunological, structural and glycoproteomic studies to compare the N-glycosylation systems of 29 Campylobacter species and identified unexpected variations. Thus, although the pathway is a common feature within this genus, variability in the N-glycans and fOS at the species level suggests that each species possess a unique array of glycosyltransferases, which correlate with their phylogenetic relatedness.     

Heterogeneity of ERα and ErbB2 Status in Cell Lines and Circulating Tumor Cells of Metastatic Breast Cancer Patients

Hormone therapy and anti-ErbB2 therapies are prescribed according to the hormone receptor [estrogen receptor α (ERα)/progesterone receptor] and ErbB2 status of the initial tumor, but it appears that circulating tumor cells (CTCs) and, consequently, the metastatic cells may have a different receptor status. As an attempt to meet the crucial need for identification of the subpopulation of patients that will benefit from more individualized therapies, rapidly evolving therapies should allow a profiling of the tumors and/or of the CTCs. We established a triple fluorescence staining using eight cell lines to visualize the CTCs (cytokeratin detection) and then to define their individual ERα and ErbB2 status. Afterward, we used this method for blood samples from 26 metastatic breast cancer patients. We identified major differences of ERα levels between the cell lines and even within one cell line. For the metastatic patients, we detected and characterized CTCs in 38.5% of the patients with a total of 92 CTCs. We could demonstrate that at least 69.6% of the CTCs exhibit an ERα and/or ErbB2 status different from the status of the primary tumor and that the CTCs from only 30% of the patients had no change of receptor status. Strikingly, heterogeneities of the status, aggregation, and size clearly appear within the CTCs. The data we generated outline the importance of a profiling not only of tumors but also of CTCs to establish individualized treatments. CTCs may then appear as new prognosis and treatment marker for both metastatic and adjuvant breast cancers.     

The role of spinal manipulation in addressing disordered sensorimotor integration and altered motor control

This review provides an overview of some of the growing body of research on the effects of spinal manipulation on sensory processing, motor output, functional performance and sensorimotor integration. It describes a body of work using somatosensory evoked potentials (SEPs), transcranial magnetic nerve stimulation, and electromyographic techniques to demonstrate neurophysiological changes following spinal manipulation. This work contributes to the understanding of how an initial episode(s) of back or neck pain may lead to ongoing changes in input from the spine which over time lead to altered sensorimotor integration of input from the spine and limbs.