Role of post translational modifications and novel crosstalk between phosphorylation and O-beta-GlcNAc modifications in human claudin-1, -3 and -4

The precise characterization of post translational modifications (PTMs) is important for the understanding of protein regulatory mechanisms and their role in disease. However, experimental studies on PTMs, especially with multifunctional proteins are difficult to follow and investigate. Bioinformatic tools are therefore helpful in predicting key protein modifications. To study the role of PTMs in claudin proteins, specifically claudin-1, -3 and -4 in the onset or progression of human cancers, we performed an in silico study of various PTMs and investigated their interplay. Given that the activity of claudins is known to be influenced by two types of PTMs, specifically palmitoylation and kinase- dependent phosphorylation, we predicted two conserved regions in the topological domains of claudin-1, -3 and -4 as potential palmitoylation sites. Furthermore, conserved phosphorylation residues, which may be targets for kinases and can alter claudin’s ability to maintain the integrity of tight junctions, were identified. To our knowledge, this is the first report to suggest O-glycosylation of claudin proteins, as well as a potential novel interplay between phosphorylation and O-glycosylation at Yin Yang sites. Thus, our findings may facilitate the production of anti-cancer drugs, and suggest that novel therapeutic strategies should target post translational events.     

Interference in foraging behaviour of European and American house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) by catmint, Nepeta cataria (Lamiaceae)

The European and American house dust mites, Dermatophagoides pteronyssinus and D. farinae, have a huge impact upon human health worldwide due to being the most important indoor trigger of atopic diseases such as asthma, rhinitis and atopic dermatitis. Preceding studies have shown that the behavioural response of house dust mites towards volatile chemicals from food sources can be assessed using a Y-tube olfactometer assay. In the current study, we used this assay to investigate, for the first time, the ability of the essential oil of the catmint plant, Nepeta cataria (Lamiaceae), known to repel other ectoparasites affecting human and animal health, to interfere with the attraction of D. pteronyssinus and D. farinae towards a standard food source (fish flakes). Two distinct chemotypes (A and B), enriched in the iridoid compounds (4aS,7S,7aR)-nepetalactone and (4aS,7S,7aS)-nepetalactone, and the sesquiterpene (E)-(1R,9S)-caryophyllene, were used. Initial assays with a hexane extract of fish flakes (FF extract) confirmed attraction of mites to this positive control (P < 0.001 and P < 0.05 for D. pteronyssinus and D. farinae respectively), but when presented in combination with either N. cataria chemotype, tested across a range of doses (10, 1, 0.1 and 0.01 μg), decreasing attraction of mites to their food source was observed as the dose augmented. Our study shows that N. cataria, enriched in iridoid nepetalactones and (E)-(1R,9S)-caryophyllene, exhibits potent repellent activity for house dust mites, and has the potential for deployment in control programmes based on interference with normal house dust mite behaviour.     

Delayed administration of pituitary adenylate cyclase-activating polypeptide 38 ameliorates renal ischemia/reperfusion injury in mice by modulating Toll-like receptors

We investigated whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) ameliorates kidney injury after ischemia/reperfusion (IR) by modulating Toll-like receptor (TLR)-associated signaling pathways. Male C57BL/6 mice were subjected to bilateral renal ischemia for 45 min. PACAP38, 20 μg in 100 μl of saline, was administered i.p. at 24 and 48 h after IR, and mice were euthanized at 72 h. In IR mice, PACAP38 maintained serum creatinine near control levels (0.81 ± 0.08 vs. 0.69 ± 0.17 mg/dl in controls, p = NS, vs. 1.8 ± 0.03 in saline-treated IR mice, p < 0.01) and significantly reduced the expression of kidney injury biomarkers. PACAP38 significantly reduced the levels of apoptosis and neutrophil infiltration, and protected against tubular damage. With PCR arrays, 59 of 83 TLR-related genes significantly changed their expression after IR. TLR2 increased 162 fold, followed by Fas-associated death domain (37 fold) and TLR6 (24 fold), while ubiquitin-conjugating enzyme E2 variant 1 (UBE2V1) decreased 55 fold. PACAP38 given 24 and 48 h after IR injury significantly reversed these changes in 56 genes, including TLR2, TLR3, TLR4, TLR6, and genes in the NF-κB pathways. The alterations in TLR2, TLR3, TLR6, and UBE2V1 were confirmed by RT-PCR. After IR, PACAP38 also suppressed protein levels of TLR-associated cytokines. PACAP38 reversed the changes in IR-activated TLR-associated NF-κB signaling pathways even when treatment was delayed 24 h. Therefore, PACAP38 could be an effective therapeutic for unexpected IR-mediated renal injury. The prominently IR-induced TLR-related genes identified in this study could be novel drug targets.     

Potential association of Helicobacter cinaedi with atrial arrhythmias and atherosclerosis

Helicobacter cinaedi has been increasingly recognized as an emerging pathogen. Reports of recurrent bacteremia and isolation of H. cinaedi organisms from a patient with myopericarditis led us to postulate that H. cinaedi is associated with chronic inflammatory cardiovascular diseases such as atrial arrhythmias and atherosclerosis. To assess any association of H. cinaedi with atrial arrhythmias, a retrospective case-control study of patients attending Kumamoto University Hospital from 2005 to 2009 was performed. The arrhythmia status of these patients was determined from their electrocardiography and electrophysiological studies. Multiple logistic regression analysis was used to identify independent risk factors. In a comparison of case patients (n= 132) with control subjects (n= 137), H. cinaedi seropositivity was identified as an independent risk factor for atrial arrhythmia (odds ratio, 5.13; 95% confidence interval, 3.0-8.7; P < 0.001). There were no significant differences, however, between these two groups with respect to anti-H. pylori IgG concentrations, anti-Chlamydophila pneumoniae IgG concentrations, and other studied variables. IgG concentrations against H. cinaedi and H. pylori were inversely correlated, which suggests cross-immunity between these two bacteria. Also, to explore any association of H. cinaedi with atherosclerosis, immunohistochemical analysis of atherosclerotic aortic tissues collected post mortem from nine patients was performed. Immunohistochemistry of atherosclerotic aortic tissues from all nine patients detected H. cinaedi antigens inside CD68(+) macrophages. These findings provide the first evidence, to our knowledge, of a possible association of H. cinaedi with atrial arrhythmias and atherosclerosis.     

Intraperitoneal injection of saline modulates hippocampal brain receptor complex levels but does not impair performance in the Morris Water Maze

The involvement of the hippocampus in pain has been demonstrated but key players, i.e. the major brain receptors have not been shown to be modulated by pain. It was therefore the aim of the study to show the concerted action and pattern of brain receptor complex levels in a non-invasive model of moderate pain. C57BL/6J mice were divided into four groups of 14 animals each: trained injected, trained non-injected, yoked injected and yoked non-injected. Animals were tested in the open field and the elevated plus maze for behavioural evaluation and cognitive functions were tested using the Morris Water Maze. Hippocampi were taken 6 h following sacrification. Membrane proteins were prepared by ultracentrifugation and run on blue native gels to keep the native state, blotted to membranes and western blotting was carried out using the primary antibodies against serotonin receptor 5HT1A, muscarinic acetylcholine receptor M1 (mAChR-M1), nicotinic acetylcholine receptor alpha7 (nAChR-alpha7), glutamate (AMPA) receptor (GluR1) and neurokinin receptor 1 (NK-1). There was no difference between performance in behaviour or in the MWM between groups. Brain receptor level changes involved all receptors given above. Pain affected mAChR-M1, GluR1 and NK-1 complex levels when yoked-injected were compared with yoked non-injected animals. Memory mechanisms affected mAChR-M1 complex levels when trained non-injected animals were compared with yoked non-injected controls. Taken together, the neurochemical basis for testing receptor agonists/antagonists on the role of pain and the hippocampus was generated that may be useful for interpretations of the role of this complex area in moderate pain.     

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 μg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 μg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 μg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.     

3D-QSAR studies of triazolopyrimidine derivatives of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> dihydroorotate dehydrogenase inhibitors using a combination of molecular dynamics,docking, and genetic algorithm-based methods

A series of 35 triazolopyrimidine analogues reported as Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors were optimized using quantum mechanics methods, and their binding conformations were studied by docking and 3D quantitative structure–activity relationship studies. Genetic algorithm-based criteria was adopted for selection of training and test sets while maintaining structural diversity of training and test sets, which is also very crucial for model development and validation. Both the comparative molecular field analyses ( q_\textLOO² = 0.841 q_{\text{LOO}}^2 = 0.{841} , r_\textncv² = 0.99 r_{\text{ncv}}^2 = 0.{99} ) and comparative molecular similarity indices analyses ( q_\textLOO² = 0.757 q_{\text{LOO}}^2 = 0.{757} , r_\textncv² = 0.943 r_{\text{ncv}}^2 = 0.{943} ) show excellent correlation and high predictive power. Furthermore, molecular dynamics simulations were performed to explore the binding mode of the two of the most active compounds of the series, 10 and 14. Harmonization in the two simulation results validate the analysis and therefore applicability of docking parameters based on crystallographic conformation of compound 14 bound to receptor molecule. This work provides useful information about the inhibition mechanism of this class of molecules and will assist in the design of more potent inhibitors of PfDHODH.     

Virtual Fossils: a New Resource for Science Communication in Paleontology

Computer-aided 3-D reconstruction of fossils, or virtual paleontology, is an increasingly common and powerful technique. It is now regularly used for research in paleontology, yet to date has impacted little on public outreach and science communication; however, it is ideally suited for these purposes, being increasingly cheap and available, dynamic and exciting, and applicable to a range of topics. Here, we provide an introduction to the field, and a case study of its use for a public engagement event. The steps involved in creating such an educational resource are outlined, and include computed tomography scanning, digital visualization, and 3-D printing of fossils. We emphasize the value of virtual fossils for science communication; they allow for diverse learning styles in a variety of topics. In the future, we hope that virtual paleontology will become a mainstay of communicating the history of life, thereby promoting accurate understanding of evolution.