Quantification of cytosolic interactions identifies Ede1 oligomers as key organizers of endocytosis

Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein–protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein–protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo.     

Ail protein binds ninth type III fibronectin repeat (9FNIII) within central 120-kDa region of fibronectin to facilitate cell binding by Yersinia pestis

The Yersinia pestis adhesin molecule Ail interacts with the extracellular matrix protein fibronectin (Fn) on host cells to facilitate efficient delivery of cytotoxic Yop proteins, a process essential for plague virulence. A number of bacterial pathogens are known to bind to the N-terminal region of Fn, comprising type I Fn (FNI) repeats. Using proteolytically generated Fn fragments and purified recombinant Fn fragments, we demonstrated that Ail binds the centrally located 120-kDa fragment containing type III Fn (FNIII) repeats. A panel of monoclonal antibodies (mAbs) that recognize specific epitopes within the 120-kDa fragment demonstrated that mAb binding to (9)FNIII blocks Ail-mediated bacterial binding to Fn. Epitopes of three mAbs that blocked Ail binding to Fn were mapped to a similar face of (9)FNIII. Antibodies directed against (9)FNIII also inhibited Ail-dependent cell binding activity, thus demonstrating the biological relevance of this Ail binding region on Fn. Bacteria expressing Ail on their surface could also bind a minimal fragment of Fn containing repeats (9-10)FNIII, and this binding was blocked by a mAb specific for (9)FNIII. These data demonstrate that Ail binds to (9)FNIII of Fn and presents Fn to host cells to facilitate cell binding and delivery of Yops (cytotoxins of Y. pestis), a novel interaction, distinct from other bacterial Fn-binding proteins.     

RhoA Ambivalently Controls Prominent Myofibroblast Characteritics by Involving Distinct Signaling Routes

IntroductionRhoA has been shown to be beneficial in cardiac disease models when overexpressed in cardiomyocytes, whereas its role in cardiac fibroblasts (CF) is still poorly understood. During cardiac remodeling CF undergo a transition towards a myofibroblast phenotype thereby showing an increased proliferation and migration rate. Both processes involve the remodeling of the cytoskeleton. Since RhoA is known to be a major regulator of the cytoskeleton, we analyzed its role in CF and its effect on myofibroblast characteristics in 2 D and 3D models.ResultsDownregulation of RhoA was shown to strongly affect the actin cytoskeleton. It decreased the myofibroblast marker α-sm-actin, but increased certain fibrosis-associated factors like TGF-β and collagens. Also, the detailed analysis of CTGF expression demonstrated that the outcome of RhoA signaling strongly depends on the involved stimulus. Furthermore, we show that proliferation of myofibroblasts rely on RhoA and tubulin acetylation. In assays accessing three different types of migration, we demonstrate that RhoA/ROCK/Dia1 are important for 2D migration and the repression of RhoA and Dia1 signaling accelerates 3D migration. Finally, we show that a downregulation of RhoA in CF impacts the viscoelastic and contractile properties of engineered tissues.ConclusionRhoA positively and negatively influences myofibroblast characteristics by differential signaling cascades and depending on environmental conditions. These include gene expression, migration and proliferation. Reduction of RhoA leads to an increased viscoelasticity and a decrease in contractile force in engineered cardiac tissue.     

The Quest for Comparability: Studying the Invariance of the Teachers’ Sense of Self-Efficacy (TSES) Measure across Countries

Teachers’ self-efficacy is an important motivational construct that is positively related to a variety of outcomes for both the teachers and their students. This study addresses challenges associated with the commonly used ‘Teachers’ Sense of Self-Efficacy (TSES)’ measure across countries and provides a synergism between substantive research on teachers’ self-efficacy and the novel methodological approach of exploratory structural equation modeling (ESEM). These challenges include adequately representing the conceptual overlap between the facets of self-efficacy in a measurement model (cross-loadings) and comparing means and factor structures across countries (measurement invariance). On the basis of the OECD Teaching and Learning International Survey (TALIS) 2013 data set comprising 32 countries (N = 164,687), we investigate the effects of cross-loadings in the TSES measurement model on the results of measurement invariance testing and the estimation of relations to external constructs (i.e., working experience, job satisfaction). To further test the robustness of our results, we replicate the 32-countries analyses for three selected sub-groups of countries (i.e., Nordic, East and South-East Asian, and Anglo-Saxon country clusters). For each of the TALIS 2013 participating countries, we found that the factor structure of the self-efficacy measure is better represented by ESEM than by confirmatory factor analysis (CFA) models that do not allow for cross-loadings. For both ESEM and CFA, only metric invariance could be achieved. Nevertheless, invariance levels beyond metric invariance are better achieved with ESEM within selected country clusters. Moreover, the existence of cross-loadings did not affect the relations between the dimensions of teachers’ self-efficacy and external constructs. Overall, this study shows that a conceptual overlap between the facets of self-efficacy exists and can be well-represented by ESEM. We further argue for the cross-cultural generalizability of the corresponding measurement model.     

Crosstalk between Nitrite,Myoglobin and Reactive Oxygen Species to Regulate Vasodilation under Hypoxia

The systemic response to decreasing oxygen levels is hypoxic vasodilation. While this mechanism has been known for more than a century, the underlying cellular events have remained incompletely understood. Nitrite signaling is critically involved in vessel relaxation under hypoxia. This can be attributed to the presence of myoglobin in the vessel wall together with other potential nitrite reductases, which generate nitric oxide, one of the most potent vasodilatory signaling molecules. Questions remain relating to the precise concentration of nitrite and the exact dose-response relations between nitrite and myoglobin under hypoxia. It is furthermore unclear whether regulatory mechanisms exist which balance this interaction. Nitrite tissue levels were similar across all species investigated. We then investigated the exact fractional myoglobin desaturation in an ex vivo approach when gassing with 1% oxygen. Within a short time frame myoglobin desaturated to 58±12%. Given that myoglobin significantly contributes to nitrite reduction under hypoxia, dose-response experiments using physiological to pharmacological nitrite concentrations were conducted. Along all concentrations, abrogation of myoglobin in mice impaired vasodilation. As reactive oxygen species may counteract the vasodilatory response, we used superoxide dismutase and its mimic tempol as well as catalase and ebselen to reduce the levels of reactive oxygen species during hypoxic vasodilation. Incubation of tempol in conjunction with catalase alone and catalase/ebselen increased the vasodilatory response to nitrite. Our study shows that modest hypoxia leads to a significant nitrite-dependent vessel relaxation. This requires the presence of vascular myoglobin for both physiological and pharmacological nitrite levels. Reactive oxygen species, in turn, modulate this vasodilation response.     

NOMENCLATURAL NOTE ON A THECADINIUM SPECIES (DINOPHYCEAE,GONYAULACALES), WHICH WAS DESCRIBED AS NEW INDEPENDENTLY THREE TIMES WITHIN TWO MONTHS1

Three Thecadinium species, independently described as new in three separate publications, are actually regarded as conspecific. The combined plate formula is Po 3′ 1a 6″ 5‐7/8c 5s 6″′ 2″″. The size range of the species is 38–65 l m in length and 23–42 lm in depth. It has one or two strongly lobed chloroplasts. The correct name of the species is Thecadinium yashimaense Yoshimatsu, Toriumi et Dodge 2004. Thecadinium mucosum Hoppenrath et Taylor 2004 and Thecadinium foveolatum Bolch 2004 are taxonomical synonyms. This note clarifies the plate tabulation and other features of the species.     

Percutaneous Mitral Valve Repair in Mitral Regurgitation Reduces Cell-Free Hemoglobin and Improves Endothelial Function

Background and Objective

Endothelial dysfunction is predictive for cardiovascular events and may be caused by decreased bioavailability of nitric oxide (NO). NO is scavenged by cell-free hemoglobin with reduction of bioavailable NO up to 70% subsequently deteriorating vascular function. While patients with mitral regurgitation (MR) suffer from an impaired prognosis, mechanisms relating to coexistent vascular dysfunctions have not been described yet. Therapy of MR using a percutaneous mitral valve repair (PMVR) approach has been shown to lead to significant clinical benefits. We here sought to investigate the role of endothelial function in MR and the potential impact of PMVR.

Methods and Results

Twenty-seven patients with moderate-to-severe MR treated with the MitraClip® device were enrolled in an open-label single-center observational study. Patients underwent clinical assessment, conventional echocardiography, and determination of endothelial function by measuring flow-mediated dilation (FMD) of the brachial artery using high-resolution ultrasound at baseline and at 3-month follow-up. Patients with MR demonstrated decompartmentalized hemoglobin and reduced endothelial function (cell-free plasma hemoglobin in heme 28.9±3.8 μM, FMD 3.9±0.9%). Three months post-procedure, PMVR improved ejection fraction (from 41±3% to 46±3%, p = 0.03) and NYHA functional class (from 3.0±0.1 to 1.9±1.7, p<0.001). PMVR was associated with a decrease in cell free plasma hemoglobin (22.3±2.4 μM, p = 0.02) and improved endothelial functions (FMD 4.8±1.0%, p<0.0001).

Conclusion

We demonstrate here that plasma from patients with MR contains significant amounts of cell-free hemoglobin, which is accompanied by endothelial dysfunction. PMVR therapy is associated with an improved hemoglobin decompartmentalization and vascular function.     

Effects of aluminium in hard, acid water on metabolic rate, blood gas tensions and ionic status in the rainbow trout

Rainbow trout, Salmo gairdneri , cannulated in the dorsal aorta, were exposed to 2 mgl⁻¹ aluminium (added as Al₂(SO₄)₃, 18H₂O) at pH = 5.0 in hard water of very high calcium concentrations (3.3 mmol 1⁻¹). No changes were observed in the concentration of the major plasma ions. The fish, however, became hypoxic, as seen from a fall in the dorsal aortic oxygen tension from around 100 mmHg to 30–40 mmHg, and a simultaneous increase in carbon dioxide tension. Moreover, exposure resulted in a 15% increase in standard oxygen uptake. The ventilation frequency doubled and swelling of the erythrocytes was observed. The results are discussed in the light of some earlier data on the effects of acid/aluminium exposure in water of low calcium concentration, and reveal an important role for calcium in the physiological responses to low pH and aluminium.     

Incorporation of tritiated thymidine by marine bacterial isolates when undergoing a starvation survival response

Bacterial DNA synthesis, as measured by the incorporation of [methyl-³H] thymidine, was examined during conditions of decreasing biomass and non-growth of three heterotrophic marine bacteria. High rates of [³H] thymidine incorporation were recorded during the initial phase of starvation and two strains exhibited a net increase in DNA during the first few hours of starvation. The decreased rate of [³H] thymidine incorporation with the time of starvation, was in agreement with the decrease in the percentage of the total population that showed uptake of labelled thymidine, as seen by a combined autoradiography-epifluorescence technique. It is suggested that new rounds of replications were initiated after cells had been starved for times that well exceeded the time for replication of genomes during growing The initial increase in cell numbers upon transfer of growing cells to a starvation regime was inhibited by nalidixic acid, suggesting that DNA synthesis, rather than an excess of nuclear bodies, allow for the fragmentation process in these strains.