Electrophysiological and behavioural responses of the housefly to “sweet” volatiles of the flowers of Caralluma europaea (Guss.) N.E. Br.

In sapromyiophilous plants, up to date, long range attraction of fly pollinators has been thoroughly investigated and attributed to “fetid” floral compounds, while the “sweet” floral scent fraction has not been specifically investigated and its role has received little attention. The aim of the present study was to verify if terpenoids, which are the main compounds of the floral bouquet of Caralluma europaea, play a role in the attraction of its pollinator Musca domestica. Terpinolene, α-terpinene and linalool, described as the three main volatiles of the flowers of C. europaea, were evaluated in electrophysiological investigations and blends of these compounds as well as the whole fresh flowers were used in behavioural assays. Antennae of housefly adults showed positive dose-dependent responses to all the chemicals tested. Houseflies were attracted by the odour of the fresh flowers and by the reconstructed terpenoid blend at the dose of 100 μg. At the dose of 10 μg, the blend did not produce any attraction. The results of the present study support the hypothesis that terpinolene, α-terpinene and linalool emitted by C. europaea flowers are involved in pollinator attraction and demonstrate the importance of the “sweet” scent in this sapromyiophilous species.     

Determination of Paclitaxel Distribution in Solid Tumors by Nano-Particle Assisted Laser Desorption Ionization Mass Spectrometry Imaging

A sensitive, simple and reproducible protocol for nanoparticle-assisted laser desorption/ionization mass spectrometry imaging technique is described. The use of commercially available TiO₂ nanoparticles abolishes heterogeneous crystallization, matrix background interferences and enhances signal detection, especially in the low mass range. Molecular image normalization was based on internal standard deposition on tissues, allowing direct comparison of drug penetration and distribution between different organs and tissues. The method was applied to analyze the distribution of the anticancer drug paclitaxel, inside normal and neoplastic mouse tissue sections. Spatial resolution was good, with a linear response between different in vivo treatments and molecular imaging intensity using therapeutic drug doses. This technique distinguishes the different intensity of paclitaxel distribution in control organs of mice, such as liver and kidney, in relation to the dose. Animals treated with 30 mg/kg of paclitaxel had half of the concentration of those treated with 60 mg/kg. We investigated the spatial distribution of paclitaxel in human melanoma mouse xenografts, following different dosage schedules and found a more homogeneous drug distribution in tumors of mice given repeated doses (5×8 mg/kg) plus a 60 mg/kg dose than in those assigned only a single 60 mg/kg dose. The protocol can be readily applied to investigate anticancer drug distribution in neoplastic lesions and to develop strategies to optimize and enhance drug penetration through different tumor tissues.     

Biological,Functional and Genetic Characterization of Bone Marrow-Derived Mesenchymal Stromal Cells from Pediatric Patients Affected by Acute Lymphoblastic Leukemia

Alterations in hematopoietic microenvironment of acute lymphoblastic leukemia patients have been claimed to occur, but little is known about the components of marrow stroma in these patients. In this study, we characterized mesenchymal stromal cells (MSCs) isolated from bone marrow (BM) of 45 pediatric patients with acute lymphoblastic leukemia (ALL-MSCs) at diagnosis (day+0) and during chemotherapy treatment (days: +15; +33; +78), the time points being chosen according to the schedule of BM aspirates required by the AIEOP-BFM ALL 2009 treatment protocol. Morphology, proliferative capacity, immunophenotype, differentiation potential, immunomodulatory properties and ability to support long-term hematopoiesis of ALL-MSCs were analysed and compared with those from 41 healthy donors (HD-MSCs). ALL-MSCs were also genetically characterized through array-CGH, conventional karyotyping and FISH analysis. Moreover, we compared ALL-MSCs generated at day+0 with those isolated during chemotherapy. Morphology, immunophenotype, differentiation potential and in vitro life-span did not differ between ALL-MSCs and HD-MSCs. ALL-MSCs showed significantly lower proliferative capacity (p<0.001) and ability to support in vitro hematopoiesis (p = 0.04) as compared with HD-MSCs, while they had similar capacity to inhibit in vitro mitogen-induced T-cell proliferation (p = N.S.). ALL-MSCs showed neither the typical translocations carried by the leukemic clone (when present), nor other genetic abnormalities acquired during ex vivo culture. Our findings indicate that ALL-MSCs display reduced ability to proliferate and to support long-term hematopoiesis in vitro. ALL-MSCs isolated at diagnosis do not differ from those obtained during treatment.     

Social Media Release Increases Dissemination of Original Articles in the Clinical Pain Sciences

A barrier to dissemination of research is that it depends on the end-user searching for or ‘pulling’ relevant knowledge from the literature base. Social media instead ‘pushes’ relevant knowledge straight to the end-user, via blogs and sites such as Facebook and Twitter. That social media is very effective at improving dissemination seems well accepted, but, remarkably, there is no evidence to support this claim. We aimed to quantify the impact of social media release on views and downloads of articles in the clinical pain sciences. Sixteen PLOS ONE articles were blogged and released via Facebook, Twitter, LinkedIn and ResearchBlogging.org on one of two randomly selected dates. The other date served as a control. The primary outcomes were the rate of HTML views and PDF downloads of the article, over a seven-day period. The critical result was an increase in both outcome variables in the week after the blog post and social media release. The mean ± SD rate of HTML views in the week after the social media release was 18±18 per day, whereas the rate during the other three weeks was no more than 6±3 per day. The mean ± SD rate of PDF downloads in the week after the social media release was 4±4 per day, whereas the rate during the other three weeks was less than 1±1 per day (p<0.05 for all comparisons). However, none of the recognized measures of social media reach, engagement or virality related to either outcome variable, nor to citation count one year later (p>0.3 for all). We conclude that social media release of a research article in the clinical pain sciences increases the number of people who view or download that article, but conventional social media metrics are unrelated to the effect.     

Impact of hemodynamics on lumen boundary displacements in abdominal aortic aneurysms by means of dynamic computed tomography and computational fluid dynamics

The aim of the present work is to quantitatively assess the three-dimensional distributions of the displacements experienced during the cardiac cycle by the luminal boundary of abdominal aortic aneurysm (AAA) and to correlate them with the local bulk hemodynamics. Ten patients were acquired by means of time resolved computed tomography, and each patient-specific vascular morphology was reconstructed for all available time frames. The AAA lumen boundary motion was tracked, and the lumen boundary displacements (LBD) computed for each time frame. The intra-aneurysm hemodynamic quantities, specifically wall shear stress (WSS), were evaluated with computational fluid dynamics simulations. Co-localization of LBD and WSS distributions was evaluated by means of Pearson correlation coefficient. A clear anisotropic distribution of LBD was evidenced in both space and time; a combination of AAA lumen boundary inward- and outward-directed motions was assessed. A co-localization between largest outward LBD and high WSS was demonstrated supporting the hypothesis of a mechanistic relationship between anisotropic displacement and hemodynamic forces related to the impingement of the blood on the lumen boundary. The presence of anisotropic displacement of the AAA lumen boundary and their link to hemodynamic forces have been assessed, highlighting a new possible role for hemodynamics in the study of AAA progression.     

Fine tuning of the active site modulates specificity in the interaction of O-acetylserine sulfhydrylase isozymes with serine acetyltransferase

O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of l-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with serine acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein–peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3–P4–P5 for the strength of binding, and P1–P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with K_diss in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents.     

Identification of Citrus sinensis BAC clones containing genes relevant to fruit quality by two-dimensional overgo hybridization

As a complement to whole-genome sequencing efforts, we are comparing targeted genomic regions among sweet orange cultivars to identify coding and conserved noncoding regions, including regulatory elements, responsible for biological features unique to this species. Here, we report the identification of 1,018 bacterial artificial chromosome (BAC) clones containing genes relevant to fruit quality from a Citrus sinensis cv. “Vaniglia” 19.3X BAC library by two-dimensional 9?×?9 overgo hybridization. To design the overgo probes, we used the “C38” expressed sequence tag assembly (http://harvest.ucr.edu/) and OligoSpawn software (http://138.23.178.42). For BAC library screening, we selected 81 overgo probes associated with unigenes that putatively code for enzymes relevant to fruit quality (flavonol, anthocyanin, carotenoid, cellulose, starch, ascorbic acid, aromatic amino acid, and lignin biosynthesis; sucrose catabolism; glycolysis; oxidative/nonoxidative pentose phosphate pathway; fatty acid biosynthesis and oxidation; Krebs cycle). Hybridization probes were pooled and hybridized in groups of intersecting rows and columns to high-density BAC filters, followed by a deconvolution process that established BAC-probe addresses. BAC addresses were obtained for 75 of the 81 overgo probes initially selected, for a total of 1,018 BAC clones, a number consistent with the depth of coverage of the BAC library. BAC end sequencing was carried out, and end-sequence pairs were mapped to their best location in the Citrus clementina genome sequence assembly using the comparative genomic database Phytozome (http://www.phytozome.net/). The BAC clones corresponding to each probe were mapped within the same scaffold as the target gene, demonstrating that the approach we used was successful in isolating the targeted genomic regions.     

Cellular stress response,sirtuins and UCP proteins in Alzheimer disease: role of vitagenes

Alzheimer’s Disease (AD) is a neurodegenerative disorder affecting up to one third of individuals reaching the age of 80. Different integrated responses exist in the brain to detect oxidative stress which is controlled by several genes termed Vitagenes. Vitagenes encode for cytoprotective heat shock proteins (Hsp), as well as thioredoxin, sirtuins and uncouple proteins (UCPs). In the present study we evaluate stress response mechanisms in plasma and lymphocytes of AD patients, as compared to controls, in order to provide evidence of an imbalance of oxidant/antioxidant mechanisms and oxidative damage in AD patients and the possible protective role of vitagenes.We found that the levels of Sirt-1 and Sirt-2 in AD lymphocytes were significantly higher than in control subjects. Interestingly, analysis of plasma showed in AD patients increased expression of Trx, a finding associated with reduced expression of UCP1, as compared to control group.This finding can open up new neuroprotective strategies, as molecules inducing this defense mechanisms can represent a therapeutic target to minimize the deleterious consequences associated to oxidative stress, such as in brain aging and neurodegenerative disorders.     

Molecular Characterization of the Multiple Interactions of SpsD,a Surface Protein from Staphylococcus pseudintermedius,with Host Extracellular Matrix Proteins

Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes. A ligand screen and Western immunoblotting revealed that the N-terminal A domain of SpsD bound fibrinogen, fibronectin, elastin and cytokeratin 10. SpsD also interfered with thrombin-induced fibrinogen coagulation and blocked ADP-induced platelet aggregation. The binding site for SpsD was mapped to residues 395–411 in the fibrinogen γ-chain, while binding sites in fibronectin were localized to the N- and C-terminal regions. SpsD also bound to glycine- and serine-rich omega loops within the C-terminal tail region of cytokeratin 10. Ligand binding studies using SpsD variants lacking the C-terminal segment or containing an amino-acid substitution in the putative ligand binding site provided insights into interaction mechanism of SpsD with the different ligands. Together these data demonstrate the multi-ligand binding properties of SpsD and illustrate some interesting differences in the variety of ligands bound by SpsD and related proteins from S. aureus.     

Modulatory Effect of Gliadin Peptide 10-mer on Epithelial Intestinal CACO-2 Cell Inflammatory Response

Celiac Disease (CD) is a chronic inflammatory enteropathy, triggered in genetically susceptible individuals by dietary gluten. Gluten is able to elicit proliferation of specific T cells and secretion of inflammatory cytokines in the small intestine. In this study we investigated the possibility that p10-mer, a decapeptide from durum wheat (QQPQDAVQPF), which was previously shown to prevent the activation of celiac peripheral lymphocytes, may exert an inhibitory effect on peptic-tryptic digested gliadin (PT-Gly)-stimulated intestinal carcinoma CACO-2 cells. In these cells, incubated with PT-Gly or p31-43 α-gliadin derived peptide in the presence or in the absence of p10-mer, IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation were measured by immunoblotting, Cyclooxigenase 2 (COX-2) activity by PGE-2 release assay, and production of cytokines in the cell supernatants by ELISA. Our results showed that pre-treatment of CACO-2 cells with p10-mer significantly inhibited IRAK1 activation and NF-kB, ERK1/2 and p38 MAPK phosphorylation, as well as COX-2 activity (i.e. PGE-2 release) and production of the IL-6 and IL-8 pro-inflammatory cytokines, induced by gliadin peptides. These findings demonstrate the inhibitory effect of the p10-mer peptide on inflammatory response in CACO-2 cells. The results of the present study show that this p10-mer peptide can modulate "in vitro" the inflammatory response induced by gliadin peptides, allowing to move towards new therapeutic strategies. Turning off the inflammatory response, may in fact represent a key target in the immunotherapy of celiac disease.