Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes. The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Here, the authors study an A. fumigatus enzyme that deacetylates GAG in a metal-dependent manner and constitutes a founding member of a new carbohydrate esterase family.Bio-organic Synthesi

Effects and safety of rituximab in systemic sclerosis: An analysis from the European Scleroderma Trial and Research (EUSTAR) group

Objectives: To assess the effects of Rituximab (RTX) on skin and lung fibrosis in patients with systemic sclerosis (SSc) belonging to the European Scleroderma Trial and Research (EUSTAR) cohort and using a nested case-control design. Methods: Inclusion criteria were fulfilment of American College of Rheumatology classification criteria for SSc, treatment with RTX and availability of follow-up data. RTX-treated patients were matched with control patients from the EUSTAR database not treated with RTX. Matching parameters for skin/lung fibrosis were the modified Rodnan Skin Score (mRSS), forced vital capacity (FVC), follow-up duration, scleroderma subtype, disease duration and immunosuppressive co-treatment. The primary analysis was mRSS change from baseline to follow-up in the RTX group compared with the control group. Secondary analyses included change of FVC and safety measures. Results: 63 patients treated with RTX were included in the analysis. The case-control analysis in patients with severe diffuse SSc showed that mRSS changes were larger in the RTX group versus matched controls (N=25; -24.0±5.2% vs-7.7±4.3%; p=0.03). Moreover, in RTX-treated patients, the mean mRSS was significantly reduced at follow-up compared with baseline (26.6±1.4 vs 20.3±1.8; p=0.0001). In addition, in patients with interstitial lung disease, RTX prevented significantly the further decline of FVC compared with matched controls (N=9; 0.4±4.4% vs-7.7±3.6%; p=0.02). Safety measures showed a good profile consistent with previous studies in autoimmune rheumatic diseases. Conclusions: The comparison of RTX treated versus untreated matched-control SSc patients from the EUSTAR cohort demonstrated improvement of skin fibrosis and prevention of worsening lung fibrosis, supporting the therapeutic concept of B cell inhibition in SSc

Explicit de Sitter Flux Vacua for Global String Models with Chiral Matter

We address the open question of performing an explicit stabilisation of all closed string moduli (including dilaton, complex structure and Kaehler moduli) in fluxed type IIB Calabi-Yau compactifications with chiral matter. Using toric geometry we construct Calabi-Yau manifolds with del Pezzo singularities. D-branes located at such singularities can support the Standard Model gauge group and matter content. In order to control complex structure moduli stabilisation we consider Calabi-Yau manifolds which exhibit a discrete symmetry that reduces the effective number of complex structure moduli. We calculate the corresponding periods in the symplectic basis of invariant three-cycles and find explicit flux vacua for concrete examples. We compute the values of the flux superpotential and the string coupling at these vacua. Starting from these explicit complex structure solutions, we obtain AdS and dS minima where the Kaehler moduli are stabilised by a mixture of D-terms, non-perturbative and perturbative alpha'-corrections as in the LARGE Volume Scenario. In the considered example the visible sector lives at a dP_6 singularity which can be higgsed to the phenomenologically interesting class of models at the dP_3 singularity.Comment: 49 pages, 5 figures; v2: references adde

Structure in 6D and 4D N=1 supergravity theories from F-theory

We explore some aspects of 4D supergravity theories and F-theory vacua that are parallel to structures in the space of 6D theories. The spectrum and topological terms in 4D supergravity theories correspond to topological data of F-theory geometry, just as in six dimensions. In particular, topological axion-curvature squared couplings appear in 4D theories; these couplings are characterized by vectors in the dual to the lattice of axion shift symmetries associated with string charges. These terms are analogous to the Green-Schwarz terms of 6D supergravity theories, though in 4D the terms are not generally linked with anomalies. We outline the correspondence between F-theory topology and data of the corresponding 4D supergravity theories. The correspondence of geometry with structure in the low-energy action illuminates topological aspects of heterotic-F-theory duality in 4D as well as in 6D. The existence of an F-theory realization also places geometrical constraints on the 4D supergravity theory in the large-volume limit.Comment: 63 page

Activation of canonical Wnt signalling is required for TGF-β-mediated fibrosis

The transforming growth factor-β (TGF-β) signalling pathway is a key mediator of fibroblast activation that drives the aberrant synthesis of extracellular matrix in fibrotic diseases. Here we demonstrate a novel link between transforming growth factor-β and the canonical Wnt pathway. TGF-β stimulates canonical Wnt signalling in a p38-dependent manner by decreasing the expression of the Wnt antagonist Dickkopf-1. Tissue samples from human fibrotic diseases show enhanced expression of Wnt proteins and decreased expression of Dickkopf-1. Activation of the canonical Wnt pathway stimulates fibroblasts in vitro and induces fibrosis in vivo. Transgenic overexpression of Dickkopf-1 ameliorates skin fibrosis induced by constitutively active TGF-β receptor type I signalling and also prevents fibrosis in other TGF-β-dependent animal models. These findings demonstrate that canonical Wnt signalling is necessary for TGF-β-mediated fibrosis and highlight a key role for the interaction of both pathways in the pathogenesis of fibrotic diseases

Promoter methylation of Wnt-antagonists in polypoid and nonpolypoid colorectal adenomas.

BACKGROUND: Nonpolypoid adenomas are a subgroup of colorectal adenomas that have been associated with a more aggressive clinical behaviour compared to their polypoid counterparts. A substantial proportion of nonpolypoid and polypoid adenomas lack APC mutations, APC methylation or chromosomal loss of the APC locus on chromosome 5q, suggesting the involvement of other Wnt-pathway genes. The present study investigated promoter methylation of several Wnt-pathway antagonists in both nonpolypoid and polypoid adenomas. METHODS: Quantitative methylation-specific PCR (qMSP) was used to evaluate methylation of four Wnt-antagonists, SFRP2, WIF-1, DKK3 and SOX17 in 18 normal colorectal mucosa samples, 9 colorectal cancer cell lines, 18 carcinomas, 44 nonpolypoid and 44 polypoid adenomas. Results were integrated with previously obtained data on APC mutation, methylation and chromosome 5q status from the same samples. RESULTS: Increased methylation of all genes was found in the majority of cell lines, adenomas and carcinomas compared to normal controls. WIF-1 and DKK3 showed a significantly lower level of methylation in nonpolypoid compared to polypoid adenomas (p < 0.01). Combining both adenoma types, a positive trend between APC mutation and both WIF-1 and DKK3 methylation was observed (p < 0.05). CONCLUSIONS: Methylation of Wnt-pathway antagonists represents an additional mechanism of constitutive Wnt-pathway activation in colorectal adenomas. Current results further substantiate the existence of partially alternative Wnt-pathway disruption mechanisms in nonpolypoid compared to polypoid adenomas, in line with previous observations

Promoter methylation of Wnt-antagonists in polypoid and nonpolypoid colorectal adenomas

BACKGROUND: Nonpolypoid adenomas are a subgroup of colorectal adenomas that have been associated with a more aggressive clinical behaviour compared to their polypoid counterparts. A substantial proportion of nonpolypoid and polypoid adenomas lack APC mutations, APC methylation or chromosomal loss of the APC locus on chromosome 5q, suggesting the involvement of other Wnt-pathway genes. The present study investigated promoter methylation of several Wnt-pathway antagonists in both nonpolypoid and polypoid adenomas. METHODS: Quantitative methylation-specific PCR (qMSP) was used to evaluate methylation of four Wnt-antagonists, SFRP2, WIF-1, DKK3 and SOX17 in 18 normal colorectal mucosa samples, 9 colorectal cancer cell lines, 18 carcinomas, 44 nonpolypoid and 44 polypoid adenomas. Results were integrated with previously obtained data on APC mutation, methylation and chromosome 5q status from the same samples. RESULTS: Increased methylation of all genes was found in the majority of cell lines, adenomas and carcinomas compared to normal controls. WIF-1 and DKK3 showed a significantly lower level of methylation in nonpolypoid compared to polypoid adenomas (p < 0.01). Combining both adenoma types, a positive trend between APC mutation and both WIF-1 and DKK3 methylation was observed (p < 0.05). CONCLUSIONS: Methylation of Wnt-pathway antagonists represents an additional mechanism of constitutive Wnt-pathway activation in colorectal adenomas. Current results further substantiate the existence of partially alternative Wnt-pathway disruption mechanisms in nonpolypoid compared to polypoid adenomas, in line with previous observations

A Common and Unstable Copy Number Variant Is Associated with Differences in Glo1 Expression and Anxiety-Like Behavior

Glyoxalase 1 (Glo1) has been implicated in anxiety-like behavior in mice and in multiple psychiatric diseases in humans. We used mouse Affymetrix exon arrays to detect copy number variants (CNV) among inbred mouse strains and thereby identified a ∼475 kb tandem duplication on chromosome 17 that includes Glo1 (30,174,390–30,651,226 Mb; mouse genome build 36). We developed a PCR-based strategy and used it to detect this duplication in 23 of 71 inbred strains tested, and in various outbred and wild-caught mice. Presence of the duplication is associated with a cis-acting expression QTL for Glo1 (LOD>30) in BXD recombinant inbred strains. However, evidence for an eQTL for Glo1 was not obtained when we analyzed single SNPs or 3-SNP haplotypes in a panel of 27 inbred strains. We conclude that association analysis in the inbred strain panel failed to detect an eQTL because the duplication was present on multiple highly divergent haplotypes. Furthermore, we suggest that non-allelic homologous recombination has led to multiple reversions to the non-duplicated state among inbred strains. We show associations between multiple duplication-containing haplotypes, Glo1 expression and anxiety-like behavior in both inbred strain panels and outbred CD-1 mice. Our findings provide a molecular basis for differential expression of Glo1 and further implicate Glo1 in anxiety-like behavior. More broadly, these results identify problems with commonly employed tests for association in inbred strains when CNVs are present. Finally, these data provide an example of biologically significant phenotypic variability in model organisms that can be attributed to CNVs

Monocyte Derived Microvesicles Deliver a Cell Death Message via Encapsulated Caspase-1

Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1β and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death