Soluble epoxide hydrolase limits mechanical hyperalgesia during inflammation.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.BACKGROUND: Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation. RESULTS: In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH(-/-) mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH(-/-) mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice. CONCLUSION: Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects.Peer Reviewe

Dysregulation of Rho GTPases in the αPix/Arhgef6 mouse model of X-linked intellectual disability is paralleled by impaired structural and synaptic plasticity and cognitive deficits

Mutations in the ARHGEF6 gene, encoding the guanine nucleotide exchange factor αPIX/Cool-2 for the Rho GTPases Rac1 and Cdc42, cause X-linked intellectual disability (ID) in humans. We show here that αPix/Arhgef6 is primarily expressed in neuropil regions of the hippocampus. To study the role of αPix/Arhgef6 in neuronal development and plasticity and gain insight into the pathogenic mechanisms underlying ID, we generated αPix/Arhgef6-deficient mice. Gross brain structure in these mice appeared to be normal; however, analysis of Golgi-Cox-stained pyramidal neurons revealed an increase in both dendritic length and spine density in the hippocampus, accompanied by an overall loss in spine synapses. Early-phase long-term potentiation was reduced and long-term depression was increased in the CA1 hippocampal area of αPix/Arhgef6-deficient animals. Knockout animals exhibited impaired spatial and complex learning and less behavioral control in mildly stressful situations, suggesting that this model mimics the human ID phenotype. The structural and electrophysiological alterations in the hippocampus were accompanied by a significant reduction in active Rac1 and Cdc42, but not RhoA. In conclusion, we suggest that imbalance in activity of different Rho GTPases may underlie altered neuronal connectivity and impaired synaptic function and cognition in αPix/Arhgef6 knockout mic

Giardia trophozoite-secreted proteins and their effects on intestinal epithelia

Giardia is a major cause of diarrheal disease worldwide. It is a flagellated cyst-forming enteric pathogen that inhabits the lumen of the small intestine. Two genetically distinct lineages (assemblages A and B) are of public health relevance and are often associated with water-borne outbreaks. Yet, the mechanism of pathogenesis and virulence in Giardia is poorly understood. A soluble component derived from healthy, viable and human infective Giardia trophozoites was shown to be able to mediate profound changes in the physiology of human derived enteric cells, consistent with the production of secreted virulence factors by the parasite. Quantitative proteomic analysis was successfully applied to the whole parasite and supernatants derived from the parasite in order to ascertain which parasite proteins are secreted. The genome of Giardia is believed to contain open reading frames which could encode as many as 6,000 proteins although hitherto there was only direct evidence for expression of a few hundred of these. Approximately 1,600 proteins were identified from each assemblage, the vast majority of which being common to both lineages. To look for actual enrichment in the supernatant, the ratio of proteins in the supernatant was compared with the pellet. This defined a far smaller group of putatively secreted proteins enriched comprising a high proportion encoded by genes annotated to have signal peptides, known virulence factors such as the Cathepsin B cysteine proteases and Variable Surface Proteins, scavenging proteins such as an extracellular nuclease and a high proportion of hitherto hypothetical proteins and proteins of unknown function. Further analysis of the genes encoding these proteins indicated that they were highly variable and likely to be under positive selection pressure, confirming their probable role in host-pathogen interactions and their potential as markers for discriminating virulent strains. Based on the proteomic analysis, a new model of pathogenic mechanism for Giardia-induced damage to enteric epithelium in which extracellular nuclease, Cathepsin B and Tenascin may have a concerted action was proposed and may have important implications in the understanding of Giardia pathogenesis

Spatial statistics from hyperplexed immunofluorescence images: to elucidate tumor microenvironment, to characterize intratumor heterogeneity, and to predict metastatic potential

The composition of the tumor microenvironment (TME)–the malignant, immune, and stromal cells implicated in tumor biology as well as the extracellular matrix and noncellular elements–and the spatial relationships between its constituents are important diagnostic biomarkers for cancer progression, proliferation, and therapeutic response. In this thesis, we develop methods to quantify spatial intratumor heterogeneity (ITH). We apply a novel pattern recognition framework to phenotype cells, encode spatial information, and calculate pairwise association statistics between cell phenotypes in the tumor using pointwise mutual information. These association statistics are summarized in a heterogeneity map, used to compare and contrast cancer subtypes and identify interaction motifs that may underlie signaling pathways and functional heterogeneity. Additionally, we test the prognostic power of spatial protein expression and association profiles for predicting clinical cancer staging and recurrence, using multivariate modeling techniques. By demonstrating the relationship between spatial ITH and outcome, we advocate this method as a novel source of information for cancer diagnostics. To this end, we have released an open-source analysis and visualization platform, THRIVE (Tumor Heterogeneity Research Image Visualization Environment), to segment and quantify multiplexed imaging samples, and assess underlying heterogeneity of those samples. The quantification of spatial ITH will uncover key spatial interactions, which contribute to disease proliferation and progression, and may confer metastatic potential in the primary neoplasm

Toponomics method for the automated quantification of membrane protein translocation

Abstract Background Intra-cellular and inter-cellular protein translocation can be observed by microscopic imaging of tissue sections prepared immunohistochemically. A manual densitometric analysis is time-consuming, subjective and error-prone. An automated quantification is faster, more reproducible, and should yield results comparable to manual evaluation. The automated method presented here was developed on rat liver tissue sections to study the translocation of bile salt transport proteins in hepatocytes. For validation, the cholestatic liver state was compared to the normal biological state. Results An automated quantification method was developed to analyze the translocation of membrane proteins and evaluated in comparison to an established manual method. Firstly, regions of interest (membrane fragments) are identified in confocal microscopy images. Further, densitometric intensity profiles are extracted orthogonally to membrane fragments, following the direction from the plasma membrane to cytoplasm. Finally, several different quantitative descriptors were derived from the densitometric profiles and were compared regarding their statistical significance with respect to the transport protein distribution. Stable performance, robustness and reproducibility were tested using several independent experimental datasets. A fully automated workflow for the information extraction and statistical evaluation has been developed and produces robust results. Conclusions New descriptors for the intensity distribution profiles were found to be more discriminative, i.e. more significant, than those used in previous research publications for the translocation quantification. The slow manual calculation can be substituted by the fast and unbiased automated method.</p