Porcupine-mediated lipidation is required for Wnt recognition by Wls

Wnt proteins are members of a conserved family of secreted signaling ligands and play crucial roles during development and in tissue homeostasis. There is increasing evidence that aberrant Wnt production is an underlying cause of dysregulated Wnt signaling, however little is known about this process. One protein known to play a role in secretion is the transmembrane protein Wntless (Wls). However, the mechanism by which Wls promotes Wnt secretion is a riddle. It is not known which Wnt family members require Wls and what the structural requirements are that make some of them reliant on Wls for secretion. Here we present a systematic analysis of all known Drosophila Wnt family members with respect to their dependence on Wls function for secretion. We first show that the glycosylation status of Wg at conserved sites does not determine its dependence on Wls. Moreover, in apparent contrast to murine wls, Drosophila wls is not a target gene of canonical Wnt signaling. We then show that all Wnts, with the exception of WntD, require Wls for secretion. All Wnts, with the exception of WntD, also contain a conserved Serine residue (in Wg S239), which we show to be essential for their functional and physical interaction with Wls. Finally, all Wnts, with the exception of WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Together, these findings indicate that Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls.     

Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity

Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 ? resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit β5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of β5c/X but not β5i, thereby explaining the selectivity of PR-957 for β5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.     

Drosophilid Assemblages at Different Urbanization Levels in the City of Porto Alegre, State of Rio Grande do Sul, Southern Brazil

The present study analyzed the drosophilid assemblages in different levels of urbanization in the city of Porto Alegre, Rio Grande do Sul, Brazil. Collections were carried out in 2008 in three different environments: a highly urbanized area????Jardim Botanico,?? a forested area with intermediary urbanization????Parque Gabriel Knijnik,?? and in a relatively well-preserved forested area, although threatened by the urban growth????Morro Santana.?? In Jardim Botanico, 36 species belonging to four genera were found, with high abundance of exotic species as Drosophila simulans Sturtevant and Zaprionus indianus (Gupta). In Parque Gabriel Knijnik, 33 species that belonged to four genera were found, with higher abundances of native species belonging to the Drosophila tripunctata species group and Drosophila willistoni species subgroup, and lower abundance of exotic species. As for Morro Santana, 32 species and three genera were found, with higher abundances of native groups, low representativeness of exotic species, and absence of Zaprionus indianus. The analysis of the Jaccard index showed higher similarity in the species composition between samples collected in summer and autumn, and between samples collected in winter and spring. On the other hand, the Morisita index differentiated Jardim Botanico from the other two studied sites. Our results show that Morro Santana is an important area of native biodiversity, reinforcing, therefore, the inclusion of this area in the project for the creation of an ecological corridor as proposed by the Ministry of the Environment of Brazil.     

Calcium Influx Rescues Adenylate Cyclase-Hemolysin from Rapid Cell Membrane Removal and Enables Phagocyte Permeabilization by Toxin Pores

Bordetella adenylate cyclase toxin-hemolysin (CyaA) penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC) domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P) toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.     

Acceptance and perceived barriers of implementing a guideline for managing low back in general practice

Background

There is considerable interest today in shared decision-making (SDM), defined as a decision-making process jointly shared by patients and their health care provider. However, the data show that SDM has not been broadly adopted yet. Consequently, the main goal of this proposal is to bring together the resources and the expertise needed to develop an interdisciplinary and international research team on the implementation of SDM in clinical practice using a theory-based dyadic perspective.

Methods

Participants include researchers from Canada, US, UK, and Netherlands, representing medicine, nursing, psychology, community health and epidemiology. In order to develop a collaborative research network that takes advantage of the expertise of the team members, the following research activities are planned: 1) establish networking and on-going communication through internet-based forum, conference calls, and a bi-weekly e-bulletin; 2) hold a two-day workshop with two key experts (one in theoretical underpinnings of behavioral change, and a second in dyadic data analysis), and invite all investigators to present their views on the challenges related to the implementation of SDM in clinical practices; 3) conduct a secondary analyses of existing dyadic datasets to ensure that discussion among team members is grounded in empirical data; 4) build capacity with involvement of graduate students in the workshop and online forum; and 5) elaborate a position paper and an international multi-site study protocol.

Discussion

This study protocol aims to inform researchers, educators, and clinicians interested in improving their understanding of effective strategies to implement shared decision-making in clinical practice using a theory-based dyadic perspective.     

Analysis of chromosome aberrations in bone marrow cells of rats after treatment with isoniazid

Summary 100 mg INH/kg body weight was administered to male Wistar rats five times, at 24 h intervals, either intraperitoneally or intravenously. In both experimental series the yield of chromosome aberrations in bone marrow cells was not increased as compared to controls.     

Altered expression of Alzheimer's disease-related genes in the cerebellum of autistic patients: a model for disrupted brain connectome and therapy

Autism and Alzheimer''s disease (AD) are, respectively, neurodevelopmental and degenerative diseases with an increasing epidemiological burden. The AD-associated amyloid-β precursor protein-α has been shown to be elevated in severe autism, leading to the ‘anabolic hypothesis'' of its etiology. Here we performed a focused microarray analysis of genes belonging to NOTCH and WNT signaling cascades, as well as genes related to AD and apoptosis pathways in cerebellar samples from autistic individuals, to provide further evidence for pathological relevance of these cascades for autism. By using the limma package from R and false discovery rate, we demonstrated that 31% (116 out of 374) of the genes belonging to these pathways displayed significant changes in expression (corrected P-values <0.05), with mitochondria-related genes being the most downregulated. We also found upregulation of GRIN1, the channel-forming subunit of NMDA glutamate receptors, and MAP3K1, known activator of the JNK and ERK pathways with anti-apoptotic effect. Expression of PSEN2 (presinilin 2) and APBB1 (or F65) were significantly lower when compared with control samples. Based on these results, we propose a model of NMDA glutamate receptor-mediated ERK activation of α-secretase activity and mitochondrial adaptation to apoptosis that may explain the early brain overgrowth and disruption of synaptic plasticity and connectome in autism. Finally, systems pharmacology analyses of the model that integrates all these genes together (NOWADA) highlighted magnesium (Mg²⁺) and rapamycin as most efficient drugs to target this network model in silico. Their potential therapeutic application, in the context of autism, is therefore discussed.     

Mapping‐by‐sequencing in complex polyploid genomes using genic sequence capture: a case study to map yellow rust resistance in hexaploid wheat

Previously we extended the utility of mapping‐by‐sequencing by combining it with sequence capture and mapping sequence data to pseudo‐chromosomes that were organized using wheat–Brachypodium synteny. This, with a bespoke haplotyping algorithm, enabled us to map the flowering time locus in the diploid wheat Triticum monococcum L. identifying a set of deleted genes (Gardiner et al., 2014). Here, we develop this combination of gene enrichment and sliding window mapping‐by‐synteny analysis to map the Yr6 locus for yellow stripe rust resistance in hexaploid wheat. A 110 MB NimbleGen capture probe set was used to enrich and sequence a doubled haploid mapping population of hexaploid wheat derived from an Avalon and Cadenza cross. The Yr6 locus was identified by mapping to the POPSEQ chromosomal pseudomolecules using a bespoke pipeline and algorithm (Chapman et al., 2015). Furthermore the same locus was identified using newly developed pseudo‐chromosome sequences as a mapping reference that are based on the genic sequence used for sequence enrichment. The pseudo‐chromosomes allow us to demonstrate the application of mapping‐by‐sequencing to even poorly defined polyploidy genomes where chromosomes are incomplete and sub‐genome assemblies are collapsed. This analysis uniquely enabled us to: compare wheat genome annotations; identify the Yr6 locus – defining a smaller genic region than was previously possible; associate the interval with one wheat sub‐genome and increase the density of SNP markers associated. Finally, we built the pipeline in iPlant, making it a user‐friendly community resource for phenotype mapping.     

Microbial modification of host long-distance dispersal capacity

Background  

Dispersal plays a key role in shaping biological and ecological processes such as the distribution of spatially-structured populations or the pace and scale of invasion. Here we have studied the relationship between long-distance dispersal behaviour of a pest-controlling money spider,Erigone atra, and the distribution of maternally acquired endosymbionts within the wider meta-population. This spider persists in heterogeneous environments because of its ability to recolonise areas through active long-distance airborne dispersal using silk as a sail, in a process termed 'ballooning'.     

Internalization-dependent recognition of Mycobacterium avium ssp. paratuberculosis by intestinal epithelial cells

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal–oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-κB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection.