The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop

Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity

Optimizing the relaxivity of MRI probes at high magnetic field strengths with binuclear GdIIIComplexes

The key criteria to optimize the relaxivity of a Gd(III) contrast agent at high fields (defined as the region 65 1.5 T) can be summarized as follows: (i) the occurrence of a rotational correlation time \u3c4R in the range of ca. 0.2\u20130.5 ns; (ii) the rate of water exchange is not critical, but a \u3c4M < 100 ns is preferred; (iii) a relevant contribution from water molecules in the second sphere of hydration. In addition, the use of macrocycle-based systems ensures the formation of thermodynamically and kinetically stable Gd(III) complexes. Binuclear Gd(III) complexes could potentially meet these requirements. Their efficiency depends primarily on the degree of flexibility of the linker connecting the two monomeric units, the absence of local motions and the presence of contribution from the second sphere water molecules. With the aim to maximize relaxivity (per Gd) over a wide range of magnetic field strengths, two binuclear Gd(III) chelates derived from the well-known macrocyclic systems DOTA-monopropionamide and HPDO3A (Gd2L1 and Gd2L2, respectively) were synthesized through a multistep synthesis. Chemical Exchange Saturation Transfer (CEST) experiments carried out on Eu2L2 at different pH showed the occurrence of a CEST effect at acidic pH that disappears at neutral pH, associated with the deprotonation of the hydroxyl groups. Then, a complete 1H and 17O NMR relaxometric study was carried out in order to evaluate the parameters that govern the relaxivity associated with these complexes. The relaxivities of Gd2L1 and Gd2L2 (20 MHz, 298 K) are 8.7 and 9.5 mM 121 s 121, respectively, +77% and +106% higher than the relaxivity values of the corresponding mononuclear GdDOTAMAP-En and GdHPDO3A complexes. A significant contribution of second sphere water molecules was accounted for the strong relaxivity enhancement of Gd2L2. MR phantom images of the dinuclear complexes compared to GdHPDO3A, recorded at 7 T, confirmed the superiority of Gd2L2. Finally, ab initio (DFT) calculations were performed to obtain information about the solution structure of the dinuclear complexes

From the experience of \u201cPLANT\u201d and \u201cPYRGY\u201d EU Projects onwards: interdisciplinary link of medicinal plant science with history and art

The iconography of medicinal and aromatic plant in art masterpieces can be an interesting contribution to the ethnobotanical knowledge related to the historic period when the artefact was produced. Along herbarium specimens, sometimes misrepresenting the morphological characters, the images inside artistic expressions not meant for scientific purposes, and often related to complex symbolic meanings, are of interest also from the botanical and the pharmaceutical point of view. Within the work of the EU project \u201cPlants in European Masterpieces (PLANT)\u201d we considered 100 species of ethnobotanical interest into 25 paintings and frescoes of XVI century in the Genoese museums or in other museums but having Genoa as a subject. Part of the dissemination activities of the EU project \u201cStrategia d\u2019impresa in settori di nicchia per l\u2019economia agroindustriale del Mediterraneo (PYRGI)\u201dwere devoted to the comparative study of a Genoese collection of pharmacy jars, pharmacy recipes of pharmacopoeias used in the past in Liguria, and aromatic and medicinal plants of the Ligurian area. For both projects a relational database ad hoc developed, having specific tables for all the three main sections of study (art, botany and pharmaceutical and ethnobotanical knowledge), was produced. These databases were the backbones of the PLANT desktop application and of the web site for PYRGI respectively. The hyperlink trees allow the user to navigate among reconstructions of ancient recipes, the botanical information and the related masterpiece. The ongoing research is focused on other paintings of the Genoese collections of the Musei di Strada Nuova. The painting I santi Sebastiano, Giovanni Battista e Francesco; Madonna con il Bambino e Angeli (1502-1503) by Filippino Lippi, is of particular relevance, with 43 entities; 36 of which are herbaceous species. 18 taxa are clearly identifiable, belonging to Pteridophtyes (2 taxa) and Angiosperm Dicotyledons (16 taxa)

IGF-I Receptor-induced Cell-Cell Adhesion of MCF-7 Breast Cancer Cells Requires the Expression of Junction Protein ZO-1

Hyperactivation of the insulin-like growth factor I receptor (IGF-IR) contributes to primary breast cancer development, but the role of the IGF-IR in tumor metastasis is unclear. Here we studied the effects of the IGF-IR on intercellular connections mediated by the major epithelial adhesion protein, E-cadherin (E-cad). We found that IGF-IR overexpression markedly stimulated aggregation in E-cad-positive MCF-7 breast cancer cells, but not in E-cad-negative MDA-MB-231 cells. However, when the IGF-IR and E-cad were co-expressed in MDA-MB-231 cells, cell-cell adhesion was substantially increased. The IGF-IR-dependent cell-cell adhesion of MCF-7 cells was not related to altered expression of E-cad or alpha-, beta-, or gamma-catenins but coincided with the up-regulation of another element of the E-cad complex, zonula occludens-1 (ZO-1). ZO-1 expression (mRNA and protein) was induced by IGF-I and was blocked in MCF-7 cells with a tyrosine kinase-defective IGF-IR mutant. By co-immunoprecipitation, we found that ZO-1 associates with the E-cad complex and the IGF-IR. High levels of ZO-1 coincided with an increased IGF-IR/alpha-catenin/ZO-1-binding and improved ZO-1/actin association, whereas down-regulation of ZO-1 by the expression of an anti-ZO-1 RNA inhibited IGF-IR-dependent cell-cell adhesion. The results suggested that one of the mechanisms by which the activated IGF-IR regulates E-cad-mediated cell-cell adhesion is overexpression of ZO-1 and the resulting stronger connections between the E-cad complex and the actin cytoskeleton. We hypothesize that in E-cad-positive cells, the IGF-IR may produce antimetastatic effects

Long-Term Field Evaluation of Conventional vs. Micropropagated Plants of Chrysanthemum cinerariifolium

Pyrethrum is a perennial herbaceous plant endemic to the eastern coast of the Adriatic Sea, and introduced in large areas of nearly all continents, where it is cultivated for the industrial extraction of pyrethrins. Pyrethrins are a group of six closely related monoterpene esters, widely used as natural insecticides. The world production of natural pyrethrins is lower than the market demand, and a wider introduction of this crop within the Mediterranean agrosystems could be an appealing opportunity for farmers and manufacturers. The availability of adequate amounts of selected plant material to bring into cultivation is, however, one of the major issues. Therefore, the in vitro propagation of elite pyrethrum genotypes could be a suitable alternative to conventional propagation methods. In this paper, we present the results of a 9-year field comparison between pyrethrum plants coming from an in vitro propagation protocol and plants obtained by cutting from the same mother plants. Furthermore, since plantlets derived from in vitro regeneration may experience ploidy changes, we evaluated the stability of the ploidy level of pyrethrum micropropagated plants by flow cytometry (FCM) analysis. FCM screening revealed no differences among the morphotypes and between them and the mother plant. Likewise, the field evaluation of plants gave no significant differences between flower yields in both groups. Hence, micropropagation was confirmed as an easy, efficient and reproducible method to obtain large quantities of selected pyrethrum genotypes

Leptin Enhances, via AP-1, Expression of Aromatase in the MCF-7 Cell Line *

Leptin, a product of adipocytes, is involved in the regulation of body weight and results strongly correlated to body fat content. An excess of fat mass represents a breast cancer risk factor particularly in postmenopausal women, where estrogen production by adipose tissue through its own aromatase activity stimulates tumor progression. Leptin stimulates estrogen production through the increase of aromatase expression and activity in human luteinized granulosa cells and adipose stromal cells. In the present study, we have examined the possible link that exists between leptin and breast cancer, focusing our attention on the direct effect of leptin on aromatase activity, which may enhance estrogen production and induce tumor cell growth stimulation. We have shown that leptin enhances aromatase mRNA expression, aromatase content, and its enzymatic activity in MCF-7. Aromatase expression appears to be regulated by tissue-specific promoter. It has been demonstrated that promoters II and 1.3 are the major promoters that drive aromatase expression in MCF-7. Transient transfection experiments using vector containing human aromatase promoters II and 1.3 sequence fused with luciferase reporter gene demonstrated that leptin is able to activate this promoter. In the presence of either mitogen-activated protein kinase inhibitor PD 98059 or ERK2 dominant negative as well as in the presence of STAT3 dominant negative, the stimulatory effects of leptin on aromatase promoter, enzymatic activity, and aromatase protein content were inhibited. Functional studies of mutagenesis and electrophoretic mobility shift assay revealed that the AP-1 motif is important in determining the up-regulatory effects induced by leptin on aromatase expression in MCF-7

TRPV1 channels are critical brain inflammation detectors and neuropathic pain biomarkers in mice

The capsaicin receptor TRPV1 has been widely characterized in the sensory system as a key component of pain and inflammation. A large amount of evidence shows that TRPV1 is also functional in the brain although its role is still debated. Here we report that TRPV1 is highly expressed in microglial cells rather than neurons of the anterior cingulate cortex and other brain areas. We found that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding. Conversely, in the cortex of mice suffering from neuropathic pain, TRPV1 is also present in neurons affecting their intrinsic electrical properties and synaptic strength. Altogether, these findings identify brain TRPV1 as potential detector of harmful stimuli and a key player of microglia to neuron communication

Adolescents' Appraisal of Homophobic Epithets: The Role of Individual and Situational Factors

Homophobic epithets have become commonly-used insults among adolescents. However, evidence suggests that there are differences in how these homophobic epithets are evaluated based on beliefs held by the observer, and by the context in which they are used. In order to examine this, Italian high school students were asked to rate the offensiveness of homophic epithets, as well as to consider how they or others would react to homophobic epithets across various situations. Homophobic beliefs and beliefs about the social acceptability of homophobic epithets were also examined. It was found that greater perceived social acceptability of homophobic epithets was related to dismissive reactions to their use, whereas homophobic beliefs were predictive of negative emotional reactions, but in varying ways depending on the specific context. The results indicate that homophobic epithets may not always be perceived as homophobic by adolescents, and that attempts to alter the social acceptability of these insults may be an effective manner of reducing their use

Outcomes of three years of teamwork on critical limb ischemia in patients with diabetes and foot lesions

To evaluate the outcomes of a multidisciplinary team working on diabetic foot (DF) patients with critical limb ischemia (CLI) in a specialized center, the authors retrospectively traced all the patients admitted in their department in 3 consecutive years with a diagnosis of CLI. From January 2006 to December 2008, 245 consecutive DF patients with CLI according the TransAtlantic interSociety Consensus II criteria were included in the study. Treatment strategy was decided by a team of diabetologists, inteventional radiologists, and vascular surgeons. Technical and clinical success, mortality, and ulcer recurrence were evaluated at 6 months and at a mean follow-up of 19.5 +/- 13.4 months. Percutaneous transluminal angioplasty (PTA) was performed in 189 (77%) patients, whereas medical treatment, open surgical revascularization (OSR), and primary amputation were performed in 44 (18.3%), 11 (4.3%), and 1 (0.5%) patients, respectively. Revascularization was successful in 227/233 (97.4%) patients. At follow-up, the overall clinical success rate was 60.4%; it was significantly (P = .001) higher after revascularization (75.9%) compared with medical treatment (48.3%). During follow-up, surgical interventions in the foot were 1.5 +/- 0.4 in those treated with PTA, 1.6 +/- 0.5 in those treated with OSR, and 0.3 +/- 0.8 in those receiving medical therapy (P &lt; .05 compared with the others). Ulcer recurrence occurred in 29 (11.8%) patients: 4 (1.6%) in PTA, 2 (0.8%) in OSR, and 23 (9.4%) in the medical therapy group (P &lt; .05). Major amputation rate was 9.3%, being significantly (P = .04) lower after revascularization (5.2%) compared with medical therapy alone (13.8%). Cumulative mortality rate was 10.6%. In conclusion, this study confirms the positive role of a PTA-first approach for revascularizing the complex cases of DF with CLI in a teamwork management strategy