Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.

Combined effect of heterogeneity, anisotropy and saturation on steady state flow and transport: Structure recognition and numerical simulation

1 Natural soil profiles may be interpreted as an arrangement of parts which are characterized by properties like hydraulic conductivity and water retention function. These parts form a complicated structure. Characterizing the soil structure is fundamental in subsurface hydrology because it has a crucial influence on flow and transport and defines the patterns of many ecological processes. We applied an image analysis method for recognition and classification of visual soil attributes in order to model flow and transport through a man-made soil profile. Modeled and measured saturation-dependent effective parameters were compared. We found that characterizing and describing conductivity patterns in soils with sharp conductivity contrasts is feasible. Differently, solving flow and transport on the basis of these conductivity maps is difficult and, in general, requires special care for representation of small-scale processes.

Mouse models and quantitative trait loci of susceptibility to bleomycin- and radiation-induced pulmonary fibrosis

Radiotherapy involving the thoracic cavity and chemotherapy with the drug bleomycin are both dose limited by the development of pulmonary fibrosis. From evidence that there is variation in the population in susceptibility to pulmonary fibrosis, and animal data, it was hypothesized that individual variation in susceptibility to bleomycin-induced, or radiation-induced, pulmonary fibrosis is, in part, genetically controlled. In this thesis a three generation mouse genetic model of C57BL/6J (fibrosis prone) and C3Hf/Kam (fibrosis resistant) mouse strains and F1 and F2 (F1 intercross) progeny derived from the parental strains was developed to investigate the genetic basis of susceptibility to fibrosis. In the bleomycin studies the mice received 100 mg/kg (125 for females) of bleomycin, via mini osmotic pump. The animals were sacrificed at eight weeks following treatment or when their breathing rate indicated respiratory distress. In the radiation studies the mice were given a single dose of 14 or 16 Gy (Co$\sp{60})$ to the whole thorax and were sacrificed when moribund. The phenotype was defined as the percent of fibrosis area in the left lung as quantified with image analysis of histological sections. Quantitative trait loci (QTL) mapping was used to identify the chromosomal location of genes which contribute to susceptibility to bleomycin-induced pulmonary fibrosis in C57BL/6J mice compared to C3Hf/Kam mice and to determine if the QTL's which influence susceptibility to bleomycin-induced lung fibrosis in these progenitor strains could be implicated in susceptibility to radiation-induced lung fibrosis. For bleomycin, a genome wide scan revealed QTL's on chromosome 17, at the MHC, (LOD = 11.7 for males and 7.2 for females) accounting for approximately 21% of the phenotypic variance, and on chromosome 11 (LOD = 4.9), in male mice only, adding 8% of phenotypic variance. The bleomycin QTL on chromosome 17 was also implicated for susceptibility to radiation-induced fibrosis (LOD = 5.0) and contributes 7% of the phenotypic variance in the radiation study. In conclusion, susceptibility to both bleomycin-induced and radiation-induced pulmonary fibrosis are heritable traits, and are influenced by a genetic factor which maps to a genomic region containing the MHC. ^

In vitro and in vivo activity of human interleukin-8 in dogs

Interleukin-8 (IL-8), a proinflammatory cytokine produced by human monocytes, fibroblasts, and endothelial and epithelial cells, is effective not only on cells and tissues of human beings but also on those of several animal species. We investigated the importance of recombinant human IL-8 for the activation of canine neutrophils in vitro and its potential for inducing inflammation in vivo. Shape change (10(-9)-10(-7) M IL-8) and chemotaxis (10(-10)-10(-6) M IL-8) assays were used to determine the activation of canine neutrophils in vitro. Chemotaxis was induced by IL-8 at doses > 10(-8) M with a maximum response at 10(-6) M. A rapid shape change of comparable intensity was elicited by 10(-9)-10(-7) M IL-8. Thirty minutes after intradermal injection of 10(-9) moles of IL-8, emigration of neutrophils could be observed and became more intense at 60 minutes and 240 minutes, respectively. Zymosan-activated canine plasma, which served as a positive control, induced a rapid, massive, and more diffuse neutrophil accumulation, whereas the reaction after IL-8 was weaker but still significant. The neutrophil accumulation after IL-8 was preferentially located in perivenular areas of the deep dermis. Recombinant human IL-8 is capable of activating canine neutrophils in vitro and is able to generate significant neutrophil accumulation in dog skin. Its activity is lower than that in human, rabbit, and rat systems.

Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs

OBJECTIVE To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. SAMPLE POPULATION Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. PROCEDURE The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). RESULTS All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and non-stimulated cultures. CONCLUSIONS AND CLINICAL RELEVANCE Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub-populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi-potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell-enriched population showed the most pronounced deregulation of migration-associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.

Protean and boundaryless career attitudes: relationships with subjective and objective career success

The protean and boundaryless career attitudes have gained special attention during the past years. Aprotean career attitude implies that aperson strives towards adevelopmental progression and self-fulfillment; aboundaryless career attitude is characterized by ahigh physical and/or psychological mobility. The aim of the present paper is twofold: First, we want to empirically investigate the relationship between protean and boundaryless career attitudes and subjective (i.e., career satisfaction and success in comparison with colleagues) and objective (i.e., promotions and salary) career success. Second, we want to integrate research on protean and boundaryless career attitudes with research on predictors of career success. We analyze the career attitudes-career success relationship with asample of 116 professionals. Our findings suggest that components of the protean career attitude are rather more related with subjective career success and that components of the boundaryless career attitude are more related with objective career success. Conclusions regarding the relevance of career attitudes for career success are drawn.

K-t GRAPPA-accelerated 4D flow MRI of liver hemodynamics: influence of different acceleration factors on qualitative and quantitative assessment of blood flow.

OBJECTIVE We sought to evaluate the feasibility of k-t parallel imaging for accelerated 4D flow MRI in the hepatic vascular system by investigating the impact of different acceleration factors. MATERIALS AND METHODS k-t GRAPPA accelerated 4D flow MRI of the liver vasculature was evaluated in 16 healthy volunteers at 3T with acceleration factors R = 3, R = 5, and R = 8 (2.0 × 2.5 × 2.4 mm(3), TR = 82 ms), and R = 5 (TR = 41 ms); GRAPPA R = 2 was used as the reference standard. Qualitative flow analysis included grading of 3D streamlines and time-resolved particle traces. Quantitative evaluation assessed velocities, net flow, and wall shear stress (WSS). RESULTS Significant scan time savings were realized for all acceleration factors compared to standard GRAPPA R = 2 (21-71 %) (p < 0.001). Quantification of velocities and net flow offered similar results between k-t GRAPPA R = 3 and R = 5 compared to standard GRAPPA R = 2. Significantly increased leakage artifacts and noise were seen between standard GRAPPA R = 2 and k-t GRAPPA R = 8 (p < 0.001) with significant underestimation of peak velocities and WSS of up to 31 % in the hepatic arterial system (p <0.05). WSS was significantly underestimated up to 13 % in all vessels of the portal venous system for k-t GRAPPA R = 5, while significantly higher values were observed for the same acceleration with higher temporal resolution in two veins (p < 0.05). CONCLUSION k-t acceleration of 4D flow MRI is feasible for liver hemodynamic assessment with acceleration factors R = 3 and R = 5 resulting in a scan time reduction of at least 40 % with similar quantitation of liver hemodynamics compared with GRAPPA R = 2.

Detection of the pan neuronal marker PGP9.5 by immuno-histochemistry and quantitative PCR in eutopic endometrium from women with and without endometriosis.

PURPOSE To assess endometrial gene as well as protein expression of neuroendocrine and supposedly endometriosis-associated product PGP9.5 and pain symptoms in women with endometriosis and controls undergoing laparoscopy, using molecular biological and immuno-histochemical approaches in the same patients. METHODS Biopsy of eutopic endometrium from 29 patients by sharp curettage, and preparation of paraffin blocks. Determination of PGP9.5 gene expression and protein abundance using qPCR and immuno-histochemistry. RESULTS qPCR; The PGP9.5 mRNA expression level between women with (N = 16) and without (N = 13) endometriosis was not different, regardless of pain symptoms or menstrual cycle phase. PGP9.5 expression was higher in women who reported pain compared to those who did not; however, this association was not statistically significant. The expression of PGP9.5 mRNA was higher in women with endometriosis and pain during the proliferative than in the secretory phase (P = 0.03). Furthermore, in the first half of the cycle, the abundance of the PGP9.5 transcript was also significantly higher in endometriosis patients compared to those without (P = 0.03). Immuno-histochemistry; Thirteen of the 16 endometriosis patients showed positive PGP9.5 immuno-reactivity in the endometrium, whereas no such signal was observed in women without endometriosis. The absolute number of nerve fibres per mm(2) in women with endometriosis was similar, regardless of the pain symptoms. CONCLUSIONS PGP9.5 mRNA expression is increased in the proliferative phase of endometriotic women with pain. The presence of nerve fibres was demonstrated by a PGP9.5 protein signal in immuno-histochemistry and restricted to patients with endometriosis. Based on these results, however, there did not appear to be a direct association between the gene expression and protein abundance in women with and without endometriosis or those that experienced pain.

Plant strengtheners enhance parasitoid attraction to herbivore-damaged cotton via qualitative and quantitative changes in induced volatiles

BACKGROUND Herbivore-damaged plants release a blend of volatile organic compounds (VOCs) that differs from undamaged plants. These induced changes are known to attract the natural enemies of the herbivores and therefore are expected to be important determinants of the effectiveness of biological control in agriculture. One way of boosting this phenomenon is the application of plant strengtheners, which has been shown to enhance parasitoid attraction in maize. It is unclear whether this is also the case for other important crops. RESULTS The plant strengtheners BTH [benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester] and laminarin were applied to cotton plants, and the effects on volatile releases and the attraction of three hymenopteran parasitoids, Cotesia marginiventris, Campoletis sonorensis and Microplitis rufiventris, were studied. After treated and untreated plants were induced by real or simulated caterpillar feeding, it was found that BTH treatment increased the attraction of the parasitoids, whereas laminarin had no significant effect. BTH treatment selectively increased the release of two homoterpenes and reduced the emission of indole, the latter of which had been shown to interfere with parasitoid attraction in earlier studies. Canonical variate analyses of the data show that the parasitoid responses were dependent on the quality rather than the quantity of volatile emission in this tritrophic interaction. CONCLUSION Overall, these results strengthen the emerging paradigm that induction of plant defences with chemical elicitors such as BTH could provide a sustainable and environmentally friendly strategy for biological control of pests by enhancing the attractiveness of cultivated plants to natural enemies of insect herbivores. © 2014 Society of Chemical Industry

Longitudinal Measurement Invariance Issues Illustrated by Examples of Marital Satisfaction in Later Life and the Structure of Psychopathology Before and After Psychotherapy

Talk at the Symposium "Opportunities and Challenges of Longitudinal Perspectives"