Insights into gliadin supramolecular organization at digestive pH 3.0

Alpha-gliadin is a highly immunogenic protein from wheat, which is associated with many human diseases, like celiac disease and non-celiac gluten sensitivity. Because of that, gliadin solution is subject to intense biomedical research. However, the physicochemical nature of the employed gliadin solution at physiological pH is not understood. Herein, we present a supramolecular evaluation of the alpha-gliadin protein in water at pH 3.0 by dynamic light scattering (DLS), cryo-transmission electron microscopy (cryo-TEM) and small-angle-.X-ray scattering (SAXS). We report that at 0.5 wt% concentration (0.1 mg/ml), gliadin is already a colloidal polydisperse system with an average hydrodynamic radius of 30 ± 10 nm. By cryo-TEM, we detected mainly large clusters. However, it was possible to visualise for the first time prolate oligomers of around 68 nm and 103 nm, minor and major axis, respectively. SAXS experiments support the existence of prolate/rod-like structures. At 1.5 wt% concentration gliadin dimers, small oligomers and large clusters coexist. The radius of gyration (Rg1) of gliadin dimer is 5.72 ± 0.23 nm with a dimer cross-section (Rc) of 1.63 nm, and an average length of around 19 nm, this suggests that gliadin dimers are formed longitudinally. Finally, our alpha-gliadin 3D model, obtained by ab initio prediction and analysed by molecular dynamics (MD), predicts that two surfaces prone to aggregation are exposed to the solvent, at the C-terminus. We hypothesise that this region may be involved in the dimerisation process of alpha-gliadin.Fil: Herrera, Maria Georgina. Universitat Bielefeld; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vazquez, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Sreij, R.. Universitat Bielefeld; AlemaniaFil: Drechsler, M.. University of Bayreuth; AlemaniaFil: Hertle, Y.. Universitat Bielefeld; AlemaniaFil: Hellweg, T.. Universitat Bielefeld; AlemaniaFil: Dodero, Veronica Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universitat Bielefeld; Alemani

Serratia marcescens internalization and replication in human bladder epithelial cells

BACKGROUND: Serratia marcescens, a frequent agent of catheterization-associated bacteriuria, strongly adheres to human bladder epithelial cells in culture. The epithelium normally provides a barrier between lumal organisms and the interstitium; the tight adhesion of bacteria to the epithelial cells can lead to internalization and subsequent lysis. However, internalisation was not shown yet for S. marcescens strains. METHODS: Elektronmicroscopy and the common gentamycin protection assay was used to assess intracellular bacteria. Via site directed mutagenesis, an hemolytic negative isogenic Serratia strain was generated to point out the importance of hemolysin production. RESULTS: We identified an important bacterial factor mediating the internalization of S. marcescens, and lysis of epithelial cells, as the secreted cytolysin ShlA. Microtubule filaments and actin filaments were shown to be involved in internalization. However, cytolysis of eukaryotic cells by ShlA was an interfering factor, and therefore hemolytic-negative mutants were used in subsequent experiments. Isogenic hemolysin-negative mutant strains were still adhesive, but were no longer cytotoxic, did not disrupt the cell culture monolayer, and were no longer internalized by HEp-2 and RT112 bladder epithelial cells under the conditions used for the wild-type strain. After wild-type S. marcescens became intracellular, the infected epithelial cells were lysed by extended vacuolation induced by ShlA. In late stages of vacuolation, highly motile S. marcescens cells were observed in the vacuoles. S. marcescens was also able to replicate in cultured HEp-2 cells, and replication was not dependent on hemolysin production. CONCLUSION: The results reported here showed that the pore-forming toxin ShlA triggers microtubule-dependent invasion and is the main factor inducing lysis of the epithelial cells to release the bacteria, and therefore plays a major role in the development of S. marcescens infections

Electro-optical bunch length monitor for flute: Layout and simulations

A new compact linear accelerator FLUTE is currently under construction at Karlsruhe Institute of Technology (KIT) in collaboration with DESY and PSI. It aims at obtaining femtosecond electron bunches (~1fs - 300 fs) with a wide charge range (1 pC - 3 nC) and requires a precise bunch length diagnostic system. Here we present the layout of a bunch length monitor based on the electro-optic technique of spectral decoding using an Yb-doped fiber laser system (central wavelength 1030 nm) and a GaP crystal. Simulations of the electro-optic signal for different operation modes of FLUTE were performed and main challenges are discussed in this talk. This work is funded by the European Union under contract PITN-GA-2011-28919

Serratamolide is a hemolytic factor produced by Serratia marcescens

Serratia marcescens is a common contaminant of contact lens cases and lenses. Hemolytic factors of S. marcescens contribute to the virulence of this opportunistic bacterial pathogen. We took advantage of an observed hyper-hemolytic phenotype of crp mutants to investigate mechanisms of hemolysis. A genetic screen revealed that swrW is necessary for the hyper-hemolysis phenotype of crp mutants. The swrW gene is required for biosynthesis of the biosurfactant serratamolide, previously shown to be a broad-spectrum antibiotic and to contribute to swarming motility. Multicopy expression of swrW or mutation of the hexS transcription factor gene, a known inhibitor of swrW expression, led to an increase in hemolysis. Surfactant zones and expression from an swrW-transcriptional reporter were elevated in a crp mutant compared to the wild type. Purified serratamolide was hemolytic to sheep and murine red blood cells and cytotoxic to human airway and corneal limbal epithelial cells in vitro. The swrW gene was found in the majority of contact lens isolates tested. Genetic and biochemical analysis implicate the biosurfactant serratamolide as a hemolysin. This novel hemolysin may contribute to irritation and infections associated with contact lens use. © 2012 Shanks et al

Efficacy and safety of nilotinib in patients with KIT-mutated metastatic or inoperable melanoma: final results from the global, single-arm, phase II TEAM trial

Background: The single-arm, phase II Tasigna Efficacy in Advanced Melanoma (TEAM) trial evaluated the KIT-selective tyrosine kinase inhibitor nilotinib in patients with KIT-mutated advanced melanoma without prior KIT inhibitor treatment. Patients and methods: Forty-two patients with KIT-mutated advanced melanoma were enrolled and treated with nilotinib 400mg twice daily. TEAM originally included a comparator arm of dacarbazine (DTIC)-treated patients;the design was amended to a single-arm trial due to an observed low number of KIT-mutated melanomas. Thirteen patients were randomized to DTIC before the protocol amendment removing this study arm. The primary endpoint was objective response rate (ORR), determined according to Response Evaluation Criteria In Solid Tumors. Results: ORR was 26.2% (n = 11/42;95% CI, 13.9%-42.0%), sufficient to reject the null hypothesis (ORR <= 10%). All observed responses were partial responses (PRs;median response duration, 7.1 months). Twenty patients (47.6%) had stable disease and 10 (23.8%) had progressive disease;1 (2.4%) response was unknown. Ten of the 11 responding patients had exon 11 mutations, four with an L576P mutation. The median progression-free survival and overall survival were 4.2 and 18.0 months, respectively. Three of the 13 patients on DTIC achieved a PR, and another patient had a PR following switch to nilotinib. Conclusion: Nilotinib activity in patients with advanced KIT-mutated melanoma was similar to historical data from imatinib-treated patients. DTIC treatment showed potential activity, although the low patient number limits interpretation. Similar to previously reported results with imatinib, nilotinib showed greater activity among patients with an exon 11 mutation, including L576P, suggesting that nilotinib may be an effective treatment option for patients with specific KIT mutations

Mutations in FRMD7, a newly identified member of the FERM family, cause X-linked idiopathic congenital nystagmus.

Idiopathic congenital nystagmus is characterized by involuntary, periodic, predominantly horizontal oscillations of both eyes. We identified 22 mutations in FRMD7 in 26 families with X-linked idiopathic congenital nystagmus. Screening of 42 singleton cases of idiopathic congenital nystagmus (28 male, 14 females) yielded three mutations (7%). We found restricted expression of FRMD7 in human embryonic brain and developing neural retina, suggesting a specific role in the control of eye movement and gaze stability

Modulation of enhancer looping and differential gene targeting by Epstein-Barr virus transcription factors directs cellular reprogramming

Epstein-Barr virus (EBV) epigenetically reprogrammes B-lymphocytes to drive immortalization and facilitate viral persistence. Host-cell transcription is perturbed principally through the actions of EBV EBNA 2, 3A, 3B and 3C, with cellular genes deregulated by specific combinations of these EBNAs through unknown mechanisms. Comparing human genome binding by these viral transcription factors, we discovered that 25% of binding sites were shared by EBNA 2 and the EBNA 3s and were located predominantly in enhancers. Moreover, 80% of potential EBNA 3A, 3B or 3C target genes were also targeted by EBNA 2, implicating extensive interplay between EBNA 2 and 3 proteins in cellular reprogramming. Investigating shared enhancer sites neighbouring two new targets (WEE1 and CTBP2) we discovered that EBNA 3 proteins repress transcription by modulating enhancer-promoter loop formation to establish repressive chromatin hubs or prevent assembly of active hubs. Re-ChIP analysis revealed that EBNA 2 and 3 proteins do not bind simultaneously at shared sites but compete for binding thereby modulating enhancer-promoter interactions. At an EBNA 3-only intergenic enhancer site between ADAM28 and ADAMDEC1 EBNA 3C was also able to independently direct epigenetic repression of both genes through enhancer-promoter looping. Significantly, studying shared or unique EBNA 3 binding sites at WEE1, CTBP2, ITGAL (LFA-1 alpha chain), BCL2L11 (Bim) and the ADAMs, we also discovered that different sets of EBNA 3 proteins bind regulatory elements in a gene and cell-type specific manner. Binding profiles correlated with the effects of individual EBNA 3 proteins on the expression of these genes, providing a molecular basis for the targeting of different sets of cellular genes by the EBNA 3s. Our results therefore highlight the influence of the genomic and cellular context in determining the specificity of gene deregulation by EBV and provide a paradigm for host-cell reprogramming through modulation of enhancer-promoter interactions by viral transcription factors

Anatomical constraints to C4 evolution: light harvesting capacity in the bundle sheath.

In C4 photosynthesis CO2 assimilation and reduction are typically coordinated across mesophyll (M) and bundle sheath (BS) cells, respectively. This system consequently requires sufficient light to reach BS to generate enough ATP to allow ribulose-1,5-bisphosphate (RuBP) regeneration in BS. Leaf anatomy influences BS light penetration and therefore constrains C4 cycle functionality. Using an absorption scattering model (coded in Excel, and freely downloadable) we simulate light penetration profiles and rates of ATP production in BS across the C3 , C3 -C4 and C4 anatomical continua. We present a trade-off for light absorption between BS pigment concentration and space allocation. C3 BS anatomy limits light absorption and benefits little from high pigment concentrations. Unpigmented BS extensions increase BS light penetration. C4 and C3 -C4 anatomies have the potential to generate sufficient ATP in the BS, whereas typical C3 anatomy does not, except some C3 taxa closely related to C4 groups. Insufficient volume of BS, relative to M, will hamper a C4 cycle via insufficient BS light absorption. Thus, BS ATP production and RuBP regeneration, coupled with increased BS investments, allow greater operational plasticity. We propose that larger BS in C3 lineages may be co-opted for C3 -C4 and C4 biochemistry requirements

CNTF Mediates Neurotrophic Factor Secretion and Fluid Absorption in Human Retinal Pigment Epithelium

Ciliary neurotrophic factor (CNTF) protects photoreceptors and regulates their phototransduction machinery, but little is known about CNTF's effects on retinal pigment epithelial (RPE) physiology. Therefore, we determined the expression and localization of CNTF receptors and the physiological consequence of their activation in primary cultures of human fetal RPE (hfRPE). Cultured hfRPE express CNTF, CT1, and OsM and their receptors, including CNTFRα, LIFRβ, gp130, and OsMRβ, all localized mainly at the apical membrane. Exogenous CNTF, CT1, or OsM induces STAT3 phosphorylation, and OsM also induces the phosphorylation of ERK1/2 (p44/42 MAP kinase). CNTF increases RPE survivability, but not rates of phagocytosis. CNTF increases secretion of NT3 to the apical bath and decreases that of VEGF, IL8, and TGFβ2. It also significantly increases fluid absorption (JV) across intact monolayers of hfRPE by activating CFTR chloride channels at the basolateral membrane. CNTF induces profound changes in RPE cell biology, biochemistry, and physiology, including the increase in cell survival, polarized secretion of cytokines/neurotrophic factors, and the increase in steady-state fluid absorption mediated by JAK/STAT3 signaling. In vivo, these changes, taken together, could serve to regulate the microenvironment around the distal retinal/RPE/Bruch's membrane complex and provide protection against neurodegenerative disease