A new role for the dynamin GTPase in the regulation of fusion pore expansion

Dynamin is a master regulator of membrane fission in endocytosis. However, a function for dynamin immediately upon fusion has also been suspected from a variety of experiments that measured release of granule contents. The role of dynamin guanosine triphosphate hydrolase (GTPase) activity in controlling fusion pore expansion and postfusion granule membrane topology was investigated using polarization optics and total internal reflection fluorescence microscopy (pTIRFM) and amperometry. A dynamin-1 (Dyn1) mutant with increased GTPase activity resulted in transient deformations consistent with rapid fusion pore widening after exocytosis; a Dyn1 mutant with decreased activity slowed fusion pore widening by stabilizing postfusion granule membrane deformations. The experiments indicate that, in addition to its role in endocytosis, GTPase activity of dynamin regulates the rapidity of fusion pore expansion from tens of milliseconds to seconds after fusion. These findings expand the membrane-sculpting repertoire of dynamin to include the regulation of immediate postfusion events in exocytosis that control the rate of release of soluble granule contents.     

Quantitative phosphoproteomics reveals link between Helicobacter pylori infection and RNA splicing modulation in host cells

The Gram-negative, spiral-shaped bacterium Helicobacter pylori is a common human pathogen that causes chronic inflammation of the human gastric mucosa, leading to peptic ulceration and/or gastric cancer. Here, we analyzed changes in the phosphoproteome of gastric epithelial cells (AGS) upon infection with H. pylori using a combination of SILAC, phosphoprotein enrichment, 2-DE, and MALDI TOF/TOF-MS. From a total of 526 spots we identified 391 protein species (143 proteins) and quantified 332 (127 proteins). Nearly, one-third of the identified proteins (40/143) were associated with the spliceosome or RNA splicing. The abundance of 20 proteins was altered by H. pylori infection, in particular, a number of serine arginine-rich (SR) proteins involved in the regulation and control of alternative splicing. Importantly, the combined methodologies enabled the detection of infection-dependent protein species-specific regulation, suggesting functional modulation of individual protein species. These findings reveal unexpected new insights into the mechanisms of host cell manipulation by H. pylori, which are likely associated with gastric pathologies, including gastric cancer.     

Duodenal ileus caused by a calf feeding nipple in a cow

Background  

The aim of this report was to describe duodenal obstruction caused by a rubber foreign body in a cow.     

Regulatory T cells and IL-10 independently counterregulate cytotoxic T lymphocyte responses induced by transcutaneous immunization

Background

The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (T_reg) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses.

Methodology/Principal Findings

TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3⁺ regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10^-/-) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of T_reg in IL-10^-/- mice and the use of B cell deficient JHT^-/- mice, we can exclude T_reg and B cells as source of IL-10 in the setting of TCI.

Conclusion/Significance

We identify T_reg and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting T_reg function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors.     

Comparison of two devices and two breathing patterns for exhaled breath condensate sampling

Introduction

Analysis of exhaled breath condensate (EBC) is a noninvasive method to access the epithelial lining fluid of the lungs. Due to standardization problems the method has not entered clinical practice. The aim of the study was to assess the comparability for two commercially available devices in healthy controls. In addition, we assessed different breathing patterns in healthy controls with protein markers to analyze the source of the EBC.

Methods

EBC was collected from ten subjects using the RTube and ECoScreen Turbo in a randomized crossover design, twice with every device - once in tidal breathing and once in hyperventilation. EBC conductivity, pH, surfactant protein A, Clara cell secretory protein and total protein were assessed. Bland-Altman plots were constructed to display the influence of different devices or breathing patterns and the intra-class correlation coefficient (ICC) was calculated. The volatile organic compound profile was measured using the electronic nose Cyranose 320. For the analysis of these data, the linear discriminant analysis, the Mahalanobis distances and the cross-validation values (CVV) were calculated.

Results

Neither the device nor the breathing pattern significantly altered EBC pH or conductivity. ICCs ranged from 0.61 to 0.92 demonstrating moderate to very good agreement. Protein measurements were greatly influenced by breathing pattern, the device used, and the way in which the results were reported. The electronic nose could distinguish between different breathing patterns and devices, resulting in Mahalanobis distances greater than 2 and CVVs ranging from 64% to 87%.

Conclusion

EBC pH and (to a lesser extent) EBC conductivity are stable parameters that are not influenced by either the device or the breathing patterns. Protein measurements remain uncertain due to problems of standardization. We conclude that the influence of the breathing maneuver translates into the necessity to keep the volume of ventilated air constant in further studies.     

Intestinal Tumorigenesis Initiated by Dedifferentiation and Acquisition of Stem-Cell-like Properties

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Highlights? Epithelial nonstem cells can be reprogrammed into tumor-initiating cells ? Dedifferentiation depends on degree of Wnt signaling ? Wnt signaling is enhanced by NFkB via the coactivator CBP ? Differentiation of stem cells into nonstem cells is bidirectional     

Tomographic Reconstruction and Analysis of a Silver CO2 Reduction Cathode

Tomographic reconstruction has been well established as a valuable tool in the analysis of polymer electrolyte fuel cell (PEMFC) electrodes. While forays have been made into applying it to polymer electrolyte water electrolyzer (PEMWE) electrodes, CO₂ electrolyzer electrodes are still new ground. Here a tomographic analysis of an electrochemical CO₂ reduction gas diffusion electrode by means of focused ion beam scanning electron microscope tomography is presented. The reconstruction shows a porosity of 68%. While most of the porosity is on the nanoscale, a broad tail of micropores is observed in the distribution. The spatial distribution of the pores is nonuniform. The large pores are concentrated in the center of the layer in the through‐plane direction. From the reconstruction, an effective diffusivity factor of 0.5 for the catalyst layer is calculated. The Knudsen number of 0.19 obtained from the later shows that the diffusion is mostly in the bulk regime. Flooding of the catalyst layer is likely to decrease the effective diffusivity factor substantially.