Selective eicosanoid-generating capacity of cytoplasmic phospholipase A2 in Pseudomonas aeruginosa-infected epithelial cells

Airway neutrophil infiltration is a pathological hallmark observed in multiple lung diseases including pneumonia and cystic fibrosis. Bacterial pathogens such as Pseudomonas aeruginosa instigate neutrophil recruitment to the air space. Excessive accumulation of neutrophils in the lung often contributes to tissue destruction. Previous studies have unveiled hepoxilin A(3) as the key molecular signal driving neutrophils across epithelial barriers. The eicosanoid hepoxilin A(3) is a potent neutrophil chemoattractant produced by epithelial cells in response to infection with P. aeruginosa. The enzyme phospholipase A(2) liberates arachidonic acid from membrane phospholipids, the rate-limiting step in the synthesis of all eicosanoids, including hepoxilin A(3). Once generated, aracidonic acid is acted upon by multiple cyclooxygenases and lipoxygenases producing an array of functionally diverse eicosanoids. Although there are numerous phospholipase A(2) isoforms capable of generating arachidonic acid, the isoform most often associated with eicosanoid generation is cytoplasmic phospholipase A(2)α. In the current study, we observed that the cytoplasmic phospholipase A(2)α isoform is required for mediating P. aeruginosa-induced production of certain eicosanoids such as prostaglandin E(2). However, we found that neutrophil transepithelial migration induced by P. aeruginosa does not require cytoplasmic phospholipase A(2)α. Furthermore, P. aeruginosa-induced hepoxilin A(3) production persists despite cytoplasmic phospholipase A(2)α suppression and generation of the 12-lipoxygenase metabolite 12-HETE is actually enhanced in this context. These results suggest that alterative phospholipase A(2) isoforms are utilized to synthesize 12-lipoxygenase metabolites. The therapeutic implications of these findings are significant when considering anti-inflammatory therapies based on targeting eicosanoid synthesis pathways.     

A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues

Background  

Analogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker.     

Boltzmann energy-based image analysis demonstrates that extracellular domain size differences explain protein segregation at immune synapses

Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell:cell communication and cell signalling, governing protein interactions and protein aggregation.     

Structural variation in two human genomes mapped at single-nucleotide resolution by whole genome de novo assembly

Here we use whole-genome de novo assembly of second-generation sequencing reads to map structural variation (SV) in an Asian genome and an African genome. Our approach identifies small- and intermediate-size homozygous variants (1-50 kb) including insertions, deletions, inversions and their precise breakpoints, and in contrast to other methods, can resolve complex rearrangements. In total, we identified 277,243 SVs ranging in length from 1-23 kb. Validation using computational and experimental methods suggests that we achieve overall <6% false-positive rate and <10% false-negative rate in genomic regions that can be assembled, which outperforms other methods. Analysis of the SVs in the genomes of 106 individuals sequenced as part of the 1000 Genomes Project suggests that SVs account for a greater fraction of the diversity between individuals than do single-nucleotide polymorphisms (SNPs). These findings demonstrate that whole-genome de novo assembly is a feasible approach to deriving more comprehensive maps of genetic variation.     

Analysis of omega-3 and omega-6 fatty acid-derived lipid metabolite formation in human and mouse blood samples

Mass spectrometry techniques have enabled the identification of different lipid metabolites and mediators derived from omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFA) that are implicated in various biological processes. However, the broad-spectrum assessment of physiologically formed lipid metabolites and mediators in blood samples has not been presented so far. Here lipid mediators and metabolites of the n-6 PUFA arachidonic acid as well as the long-chain n-3 PUFA eicosapentaenoic acids (EPA) and docosahexaenoic acid (DHA) were measured in human blood samples as well as in mouse blood. There were detectable but mostly very low amounts of the assayed compounds in human native plasma samples, whereas in vitro activation of whole blood with the calcium ionophore A23187 led to highly significant increases of metabolite formation, with a predominance of the 12-lipoxygenase (12-LOX) products 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA). A23187 activation also led to significant increases in the formation of 5-LOX products including leukotriene B(4) (LTB(4)), leukotriene B(5) (LTB(5)) as well as of 15-LOX products and prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)). Levels were similar or even higher in A23187-activated mouse blood. The approach presented here thus provides a protocol for the comprehensive and concomitant assessment of the generation capacity of n-3 and n-6 PUFA-derived lipid metabolites as well as thromboxanes and prostaglandins in human and murine blood samples. Further studies will now have to evaluate lipid metabolite generation capacity in different physiological and pathophysiological contexts.     

A decisive function of transforming growth factor-β/Smad signaling in tissue morphogenesis and differentiation of human HaCaT keratinocytes

The mechanism by which transforming growth factor-β (TGFβ) regulates differentiation in human epidermal keratinocytes is still poorly understood. To assess the role of Smad signaling, we engineered human HaCaT keratinocytes either expressing small interfering RNA against Smads2, 3, and 4 or overexpressing Smad7 and verified impaired Smad signaling as decreased Smad phosphorylation, aberrant nuclear translocation, and altered target gene expression. Besides abrogation of TGFβ-dependent growth inhibition in conventional cultures, epidermal morphogenesis and differentiation in organotypic cultures were disturbed, resulting in altered tissue homeostasis with suprabasal proliferation and hyperplasia upon TGFβ treatment. Neutralizing antibodies against TGFβ, similar to blocking the actions of EGF-receptor or keratinocyte growth factor, caused significant growth reduction of Smad7-overexpressing cells, thereby demonstrating that epithelial hyperplasia was attributed to TGFβ-induced "dermis"-derived growth promoting factors. Furthermore impaired Smad signaling not only blocked the epidermal differentiation process or caused epidermal-to-mesenchymal transition but induced a switch to a complex alternative differentiation program, best characterized as mucous/intestinal-type epithelial differentiation. As the same alternative phenotype evolved from both modes of Smad-pathway interference, and reduction of Smad7-overexpression caused reversion to epidermal differentiation, our data suggest that functional TGFβ/Smad signaling, besides regulating epidermal tissue homeostasis, is not only essential for terminal epidermal differentiation but crucial in programming different epithelial differentiation routes.     

PETIR-001, a dual inhibitor of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN), ameliorates experimental autoimmune encephalomyelitis in SJL/J mice

Cellular dipeptidyl peptidase IV (DP IV, CD26) and amino-peptidase N (APN, CD13) play regulatory roles in T cell activation and represent potential targets for treatment of inflammatory disorders. We have developed a novel therapeutic strategy, 'peptidase-targeted Immunoregulation' (PETIR?), which simultaneously targets both cellular DP IV and APN via selective binding sites different from the active sites with a single inhibitor. To prove the therapeutic concept of PETIR? in autoimmunity of the central nervous system (CNS), we evaluated the effect of a single substance, PETIR-001, in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. Administration of PETIR-001 significantly delayed and decreased clinical signs of active EAE, when given in a therapeutic manner intraperitoneally from day 15 to day 24 after induction of EAE. Both the acute phase and the first relapse of EAE were markedly inhibited. Importantly, a similar therapeutic benefit was obtained after oral administration of PETIR-001 from day 12 to day 21 after disease induction. Our results demonstrate that PETIR-001 exhibits a therapeutic effect on EAE in SJL/J mice. Thus, PETIR? represents a novel and efficient therapeutic approach for immunotherapy of CNS inflammation.     

Age correction in dementia--matching to a healthy brain

In recent research, many univariate and multivariate approaches have been proposed to improve automatic classification of various dementia syndromes using imaging data. Some of these methods do not provide the possibility to integrate possible confounding variables like age into the statistical evaluation. A similar problem sometimes exists in clinical studies, as it is not always possible to match different clinical groups to each other in all confounding variables, like for example, early-onset (age<65 years) and late-onset (age≥65) patients with Alzheimer's disease (AD). Here, we propose a simple method to control for possible effects of confounding variables such as age prior to statistical evaluation of magnetic resonance imaging (MRI) data using support vector machine classification (SVM) or voxel-based morphometry (VBM). We compare SVM results for the classification of 80 AD patients and 79 healthy control subjects based on MRI data with and without prior age correction. Additionally, we compare VBM results for the comparison of three different groups of AD patients differing in age with the same group of control subjects obtained without including age as covariate, with age as covariate or with prior age correction using the proposed method. SVM classification using the proposed method resulted in higher between-group classification accuracy compared to uncorrected data. Further, applying the proposed age correction substantially improved univariate detection of disease-related grey matter atrophy using VBM in AD patients differing in age from control subjects. The results suggest that the approach proposed in this work is generally suited to control for confounding variables such as age in SVM or VBM analyses. Accordingly, the approach might improve and extend the application of these methods in clinical neurosciences.