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Nowadays, rapid technological development into acquiring geo-spatial information; joined to the capabilities to process these data in a relative short period of time, allows the generation of detailed 3D textured city models that will become an essential part of the modern city information infrastructure (Spatial Data Infrastructure) and, can be used to integrate various data from different sources for public accessible visualisation and many other applications. One of the main bottlenecks, which at the moment limit the use of these datasets to few experts, is a lack on efficient visualization systems through the web and interoperable frameworks that allow standardising the access to the city models. The work presented in this paper tries to satisfy these two requirements developing a 3D web-based visualization system based on OGC standards and effective visualization concepts. The architectural framework, based on Services Oriented Architecture (SOA) concepts, provides the 3D city data to a web client designed to support the view process in a very effective way. The first part of the work is to design a framework compliant to the 3D Portrayal Service drafted by the of the Open Geospatial Consortium (OGC) 3D standardization working group. The latter is related to the development of an effective web client able to render in an efficient way the 3D city models.

The rapid technological evolution, which is characterizing all the disciplines involved within the wide concept of smart cities, is becoming a key factor to trigger true user-driven innovation. However to fully develop the Smart City concept to a wide geographical target, it is required an infrastructure that allows the integration of heterogeneous geographical information and sensor networks into a common technological ground. In this context 3D city models will play an increasingly important role in our daily lives and become an essential part of the modern city information infrastructure (Spatial Data Infrastructure). The work presented in this paper describes an innovative Services Oriented Architecture software platform aimed at providing smartcities services on top of 3D urban models. 3D city models are the basis of many applications and can became the platform for integrating city information within the Smart-Cites context. In particular the paper will investigate how the efficient visualisation of 3D city models using different levels of detail (LODs) is one of the pivotal technological challenge to support Smart-Cities applications. The goal is to provide to the final user realistic and abstract 3D representations of the urban environment and the possibility to interact with a massive amounts of semantic information contained into the geospatial 3D city model. The proposed solution, using OCG standards and a custom service to provide 3D city models, lets the users to consume the services and interact with the 3D model via Web in a more effective way.

The rapid technological evolution, which is characterizing all the disciplines involved within the wide concept of smart cities, becomes a key factor to trigger true user-driven innovation. In this context 3D city models will play an increasingly important role in our daily lives and become an essential part of the modern city information infrastructure (Spatial Data Infrastructure). The goal of this paper is to introduce the i-SCOPE (interoperable Smart City services through an Open Platform for urban Ecosystems) project methodology and implementations together with key technologies and open standards. Based on interoperable 3D CityGML UIMs, the aim of i-Scope is to deliver an open platform on top of which it possible to develop, within different domains, various "smart city" services. Moreover, in i-SCOPE different issues, transcending the mere technological domain, are being tackled, including aspects dealing with social and environmental issues. Indeed several tasks including citizen awareness, crowd source and voluntary based data collection as well as privacy issue concerning involved people should be considered.

This paper deals with the dynamic analysis of pre-stressed laminated composite plates. Particular emphasis is devoted to the case of in-plane mono-axial, biaxial, shear and combined loadings. Both equivalent single layer and layer-wise plate kinematic description are addressed, according to the hierarchical approach proposed by the Carrera's unified formulation. The different kinematic approaches are compared in order to identify the appropriate modeling for laminated composite plates subjected to combined loadings. The principle of virtual displacement is applied in order to obtain governing equations and the corresponding problem is solved through the finite element method. When possible, assessments/comparisons with exact solutions are proposed. Moreover, the effects of different stacking sequences, boundary conditions, geometries and materials on plate natural frequencies are illustrated.

Abstract Objectives Psychosocial factors have been hypothesized to increase the risk of cancer. This study aims (1) to test whether psychosocial factors (depression, anxiety, recent loss events, subjective social support, relationship status, general distress, and neuroticism) are associated with the incidence of any cancer (any, breast, lung, prostate, colorectal, smoking‐related, and alcohol‐related); (2) to test the interaction between psychosocial factors and factors related to cancer risk (smoking, alcohol use, weight, physical activity, sedentary behavior, sleep, age, sex, education, hormone replacement therapy, and menopausal status) with regard to the incidence of cancer; and (3) to test the mediating role of health behaviors (smoking, alcohol use, weight, physical activity, sedentary behavior, and sleep) in the relationship between psychosocial factors and the incidence of cancer. Methods The psychosocial factors and cancer incidence (PSY‐CA) consortium was established involving experts in the field of (psycho‐)oncology, methodology, and epidemiology. Using data collected in 18 cohorts (N = 617,355), a preplanned two‐stage individual participant data (IPD) meta‐analysis is proposed. Standardized analyses will be conducted on harmonized datasets for each cohort (stage 1), and meta‐analyses will be performed on the risk estimates (stage 2). Conclusion PSY‐CA aims to elucidate the relationship between psychosocial factors and cancer risk by addressing several shortcomings of prior meta‐analyses.

The clinical course of intensive care unit (ICU) patients may be complicated by a large spectrum of lower respiratory tract infections (LRTI), defined by specific epidemiological, clinical and microbiological aspects. A European network for ICU-related respiratory infections (ENIRRIs), supported by the European Respiratory Society, has been recently established, with the aim at studying all respiratory tract infective episodes except community-acquired ones. A multicentre, observational study is in progress, enrolling more than 1000 patients fulfilling the clinical, biochemical and radiological findings consistent with a LRTI. This article describes the methodology of this study. A specific interest is the clinical impact of non-ICU-acquired nosocomial pneumonia requiring ICU admission, non-ventilator-associated LRTIs occurring in the ICU, and ventilator-associated tracheobronchitis. The clinical meaning of microbiologically negative infectious episodes and specific details on antibiotic administration modalities, dosages and duration are also highlighted. Recently released guidelines address many unresolved questions which might be answered by such large-scale observational investigations. In light of the paucity of data regarding such topics, new interesting information is expected to be obtained from our network research activities, contributing to optimisation of care for critically ill patients in the ICU.

G Protein Coupled Receptors (GPCRs) constitute the largest family of signalling proteins responsible for translating extracellular stimuli into intracellular functions. They play crucial roles in numerous physiological processes and are major targets for drug discovery. Dysregulation of GPCRs is implicated in various diseases, making understanding their structural dynamics critical for therapeutic development. Here, we use Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) to explore the structural dynamics of the turkey β1-adrenergic receptor (tβ1AR) bound with nine different ligands, including agonists, partial agonists, and antagonists. We find that these ligands induce distinct dynamic patterns across the receptor, which can be grouped by compound modality. Notably, full agonist binding destabilises the intracellular loop 1 (ICL1), while antagonist binding stabilises it, highlighting ICL1's role in G protein recruitment. Our findings indicate that the conserved L72 residue in ICL1 is crucial for maintaining receptor structural integrity and stabilising the GDP-bound state. Overall, our results provide a platform for determining drug modality and highlight how HDX-MS can be used to dissect receptor ligand interaction properties and GPCR mechanism., (© 2024. The Author(s).)

The concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity. In this study we demonstrate that mechanical stimulation can induce β 2 -adrenoceptor agonist-independent Gs-mediated cAMP signalling that is sensitive to inhibition by inverse agonists such as ICI-118551 and propranolol. The size of the mechano-sensitive response is dependent on the cell surface receptor expression level in HEK293G cells, is still observed in a ligand-binding deficient D113A mutant β 2 -adrenoceptor and can be attenuated by site-directed mutagenesis of the extracellular N-glycosylation sites on the N-terminus and second extracellular loop of the β 2 -adrenoceptor. Similar mechano-sensitive agonist-independent responses are observed in HEK293G cells overexpressing the A 2A -adenosine receptor. These data provide new insights into how agonist-independent constitutive receptor activity can be enhanced by mechanical stimulation and regulated by inverse agonists., (© 2024. The Author(s).)

Adhesion G protein-coupled receptors (aGPCRs) are a class of structurally and functionally highly intriguing cell surface receptors with essential functions in health and disease. Thus, they display a vastly unexploited pharmacological potential. Our current understanding of the physiological functions and signaling mechanisms of aGPCRs form the basis for elucidating further molecular aspects. Combining these with novel tools and methodologies from different fields tailored for studying these unusual receptors yields a powerful potential for pushing aGPCR research from singular approaches toward building up an in-depth knowledge that will facilitate its translation to applied science. In this review, we summarize the state-of-the-art knowledge on aGPCRs in respect to structure-function relations, physiology, and clinical aspects, as well as the latest advances in the field. We highlight the upcoming most pressing topics in aGPCR research and identify strategies to tackle them. Furthermore, we discuss approaches how to promote, stimulate, and translate research on aGPCRs 'from bench to bedside' in the future., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Adenosine is a local mediator that regulates changes in the cardiovascular system via activation of four G protein-coupled receptors (A 1 , A 2A , A 2B , A 3 ). Here, we have investigated the effect of A 2A and A 2B -selective agonists on vasodilatation in three distinct vascular beds of the rat cardiovascular system. NanoBRET ligand binding studies were used to confirm receptor selectivity. The regional hemodynamic effects of adenosine A 2A and A 2B selective agonists were investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal artery, mesenteric artery, and the descending abdominal aorta. Cardiovascular responses were measured following intravenous infusion (3 min for each dose) of the A 2A -selective agonist CGS 21680 (0.1, 0.3, 1 µg kg -1 min -1 ) or the A 2B -selective agonist BAY 60-6583 (4,13.3, 40 µg kg -1 min -1 ) following predosing with the A 2A -selective antagonist SCH 58261 (0.1 or 1 mg kg -1 min -1 ), the A 2B /A 2A antagonist PSB 1115 (10 mg kg -1 min -1 ) or vehicle. The A 2A -selective agonist CGS 21680 produced a striking increase in heart rate (HR) and hindquarters vascular conductance (VC) that was accompanied by a significant decrease in mean arterial pressure (MAP) in conscious rats. In marked contrast, the A 2B -selective agonist BAY 60-6583 significantly increased HR and VC in the renal and mesenteric vascular beds, but not in the hindquarters. Taken together, these data indicate that A 2A and A 2B receptors are regionally selective in their regulation of vascular tone. These results suggest that the development of A 2B receptor agonists to induce vasodilatation in the kidney may provide a good therapeutic approach for the treatment of acute kidney injury., (© 2022 Federation of American Societies for Experimental Biology.)

Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity., Competing Interests: None declared., (Thieme. All rights reserved.)

The adenosine A 1 receptor (A 1 AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A 1 AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A 1 AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A 1 AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A 1 AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A 1 AR molecular pharmacology and signaling in living cells.

Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β 2 -adrenoceptor (β 2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β 2 AR (pK D >7.0) with good selectivity over the β 1 AR (pK D <6.0). The most potent and selective ligands being 8c (ICI 118,551-Gly-Ala-BODIPY-FL-X; β 2 AR pK D 7.48), 9c (ICI 118,551-βAla-βAla-BODIPY-FL-X; β 2 AR pK D 7.48), 12a (ICI 118,551-PEG-BODIPY-X-630/650; β 2 AR pK D 7.56), and 12b (ICI 118,551-PEG-BODIPY-FL; β 2 AR pK D 7.42). 9a (ICI 118,551-βAla-βAla-BODIPY-X-630/650) had the highest affinity at recombinant β 2 ARs (pK D 7.57), but also exhibited significant binding affinity to the β 1 AR (pK D 6.69). Nevertheless, among the red fluorescent ligands, 9a had the best imaging characteristics in recombinant HEK293 T cells and labeling was mostly confined to the cell surface. In contrast, 12a showed the highest propensity to label intracellular β 2 ARs in HEK293 T cell expressing exogenous β 2 ARs. This suggests that a combination of the polyethylene glycol (PEG) linker and the BODIPY-X-630/650 makes this ICI 118,551 derivative particularly susceptible to crossing the cell membrane to access the intracellular β 2 ARs. We have also used these ligands in combination with CRISPR/Cas9 genome-edited HEK293 T cells to undertake for the first time real-time ligand binding to native HEK293 T β 2 ARs at low native receptor expression levels. These studies provided quantitative data on ligand-binding characteristics but also allowed real-time visualization of the ligand-binding interactions in genome-edited cells using NanoBRET luminescence imaging., (© 2021 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and major targets for FDA-approved drugs. The ability to quantify GPCR expression and ligand binding characteristics in different cell types and tissues is therefore important for drug discovery. The advent of genome editing along with developments in fluorescent ligand design offers exciting new possibilities to probe GPCRs in their native environment. This review provides an overview of the recent technical advances employed to study the localisation and ligand binding characteristics of genome-edited and endogenously expressed GPCRs., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15-kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognizes the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C terminus to the 11-amino-acid HiBiT tag (VUN400-HiBiT) which complements LgBiT protein, forming a full-length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect allosteric interactions with CXCR4. These data demonstrate that the high specificity offered by extracellular targeted nanobodies can be utilized to probe receptor pharmacology., Competing Interests: Declaration of Interests R.H. is CSO of QVQ Holding B.V., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Among class A G protein-coupled receptors (GPCR), the human adenosine A 2A receptor (hA 2A AR) remains an attractive drug target. However, translation of A 2A AR ligands into the clinic has proved challenging and an improved understanding of A 2A AR pharmacology could promote development of more efficacious therapies. Subtype-selective fluorescent probes would allow detailed real-time pharmacological investigations both in vitro and in vivo. In the present study, two families of fluorescent probes were designed around the known hA 2A AR selective antagonist preladenant (SCH 420814). Both families of fluorescent antagonists retained affinity at the hA 2A AR, selectivity over all other adenosine receptor subtypes and allowed clear visualization of specific receptor localization through confocal imaging. Furthermore, the Alexa Fluor 647-labeled conjugate allowed measurement of ligand binding affinities of unlabeled hA 2A AR antagonists using a bioluminescence resonance energy transfer (NanoBRET) assay. The fluorescent ligands developed here can therefore be applied to a range of fluorescence-based techniques to further interrogate hA 2A AR pharmacology and signaling.

In recent years, several novel aspects of GPCR pharmacology have been described, which are thought to play a role in determining the in vivo efficacy of a compound. Fluorescent ligands have been used to study many of these, which have also required the development of new experimental approaches. Fluorescent ligands offer the potential to use the same fluorescent probe to perform a broad range of experiments, from single-molecule microscopy to in vivo BRET. This review provides an overview of the in vitro use of fluorescent ligands in further understanding emerging pharmacological paradigms within the GPCR field, including ligand-binding kinetics, allosterism and intracellular signalling, along with the use of fluorescent ligands to study physiologically relevant therapeutic agents., (© 2019 The British Pharmacological Society.)

Receptor internalization in response to prolonged agonist treatment is an important regulator of G protein-coupled receptor (GPCR) function. The adenosine A 1 receptor (A 1 AR) is one of the adenosine receptor family of GPCRs, and evidence for its agonist-induced internalization is equivocal. The recently developed NanoBiT technology uses split NanoLuc Luciferase to monitor changes in protein interactions. We have modified the human A 1 AR on the N-terminus with the small high-affinity HiBiT tag. In the presence of the large NanoLuc subunit (LgBiT), complementation occurs, reconstituting a full-length functional NanoLuc Luciferase. Here, we have used complemented luminescence to monitor the internalization of the A 1 AR in living HEK293 cells. Agonist treatment resulted in a robust decrease in cell-surface luminescence, indicating an increase in A 1 AR internalization. These responses were inhibited by the A 1 AR-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), with an antagonist affinity that closely matched that measured using ligand binding with a fluorescent A 1 receptor antagonist (CA200645). The agonist potencies for inducing A 1 AR internalization were very similar to the affinities previously determined by ligand binding, suggesting little or no amplification of the internalization response. By complementing the HiBiT tag to exogenous purified LgBiT, it was also possible to perform NanoBRET ligand-binding experiments using HiBiT-A 1 AR. This study demonstrates the use of NanoBiT technology to monitor internalization of the A 1 AR and offers the potential to combine these experiments with NanoBRET ligand-binding assays.

Fluorescent antagonists offer the ability to interrogate G protein-coupled receptor pharmacology. With resonance energy transfer techniques, fluorescent antagonists can be implemented to monitor receptor-ligand interactions using assays originally designed for radiolabeled probes. The fluorescent nature of these antagonists also enables the localization and distribution of the receptors to be visualized in living cells. Here, we describe the generation of modified purinergic receptors with the NanoLuc luciferase or SNAP-tag, using the P1 adenosine A 3 receptor as an example. We also describe the procedure of characterizing a novel fluorescent purinergic antagonist using ligand-mediated bioluminescence resonance energy transfer assays and confocal microscopy.

Background and Purpose: Adenosine is a local mediator that regulates a number of physiological and pathological processes via activation of adenosine A 1 -receptors. The activity of adenosine can be regulated at the level of its target receptor via drugs that bind to an allosteric site on the A 1 -receptor. Here, we have investigated the species and probe dependence of two allosteric modulators on the binding characteristics of fluorescent and nonfluorescent A 1 -receptor agonists., Experimental Approach: A Nano-luciferase (Nluc) BRET (NanoBRET) methodology was used. This used N-terminal Nluc-tagged A 1 -receptors expressed in HEK293T cells in conjunction with both fluorescent A 1 -receptor agonists (adenosine and NECA analogues) and a fluorescent antagonist CA200645., Key Results: PD 81,723 and VCP171 elicited positive allosteric effects on the binding affinity of orthosteric agonists at both the rat and human A 1 -receptors that showed clear probe dependence. Thus, the allosteric effect on the highly selective partial agonist capadenoson was much less marked than for the full agonists NECA, adenosine, and CCPA in both species. VCP171 and, to a lesser extent, PD 81,723, also increased the specific binding of three fluorescent A 1 -receptor agonists in a species-dependent manner that involved increases in B max and pK D ., Conclusions and Implications: These results demonstrate the power of the NanoBRET ligand-binding approach to study the effect of allosteric ligands on the binding of fluorescent agonists to the adenosine A 1 -receptor in intact living cells. Furthermore, our studies suggest that VCP171 and PD 81,723 may switch a proportion of A 1 -receptors to an active agonist conformation (R*)., (© 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey β 1 -adrenoceptor (β 1 AR-m23 StaR), on β 1 -ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tβ 1 -AR, but not its human homologue. Specific binding of mAb3 to tβ 1 -AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [ 3 H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey β 1 -receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [ 3 H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tβ 1 -AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [ 3 H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey β 1 -adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of β 1 -AR conformational states and oligomeric complexes., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Previous research has indicated that allosteric interactions across the dimer interface of β 1 -adrenoceptors may be responsible for a secondary low affinity binding conformation. Here we have investigated the potential for probe dependence, in the determination of antagonist pK i values at the human β 1 -adenoceptor, which may result from such allosterism interactions. Three fluorescent β 1 -adrenoceptor ligands were used to investigate this using bioluminescence energy transfer (BRET) between the receptor-bound fluorescent ligand and the N-terminal NanoLuc tag of a human β 1 -adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high-affinity-specific binding to the NanoLuc- β 1 -adrenoceptor with each of the three fluorescent ligands yielding K D values of 87.1 ± 10 nmol/L ( n  = 8), 38.1 ± 12 nmol/L ( n  = 7), 13.4 ± 2 nmol/L ( n  = 14) for propranolol-Peg8-BY630, propranolol- β (Ala-Ala)-BY630 and CGP-12177-TMR, respectively. Parallel radioligand-binding studies with 3 H-CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc- β 1 -adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein -1 ). Following a 1 h incubation with fluorescent ligands and β 1 -adrenoceptor competing antagonists, there were significant differences ( P  < 0.001) in the pK i values obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and 3 H-CGP 12177. However, increasing the incubation time to 2 h removed these significant differences. The data obtained show that the NanoBRET assay can be applied successfully to study ligand-receptor interactions at the human β 1 -adrenoceptor. However, the study also emphasizes the importance of ensuring that both the fluorescent and competing ligands are in true equilibrium before interpretations regarding probe dependence can be made.

Background: Multi-element interventions for first-episode psychosis (FEP) are promising, but have mostly been conducted in non-epidemiologically representative samples, thereby raising the risk of underestimating the complexities involved in treating FEP in 'real-world' services., Methods/design: The Psychosis early Intervention and Assessment of Needs and Outcome (PIANO) trial is part of a larger research program (Genetics, Endophenotypes and Treatment: Understanding early Psychosis - GET UP) which aims to compare, at 9 months, the effectiveness of a multi-component psychosocial intervention versus treatment as usual (TAU) in a large epidemiologically based cohort of patients with FEP and their family members recruited from all public community mental health centers (CMHCs) located in two entire regions of Italy (Veneto and Emilia Romagna), and in the cities of Florence, Milan and Bolzano. The GET UP PIANO trial has a pragmatic cluster randomized controlled design. The randomized units (clusters) are the CMHCs, and the units of observation are the centers' patients and their family members. Patients in the experimental group will receive TAU plus: 1) cognitive behavioral therapy sessions, 2) psycho-educational sessions for family members, and 3) case management. Patient enrollment will take place over a 1-year period. Several psychopathological, psychological, functioning, and service use variables will be assessed at baseline and follow-up. The primary outcomes are: 1) change from baseline to follow-up in positive and negative symptoms' severity and subjective appraisal; 2) relapse occurrences between baseline and follow-up, that is, episodes resulting in admission and/or any case-note records of re-emergence of positive psychotic symptoms. The expected number of recruited patients is about 400, and that of relatives about 300. Owing to the implementation of the intervention at the CMHC level, the blinding of patients, clinicians, and raters is not possible, but every effort will be made to preserve the independency of the raters. We expect that this study will generate evidence on the best treatments for FEP, and will identify barriers that may hinder its feasibility in 'real-world' clinical settings, patient/family conditions that may render this intervention ineffective or inappropriate, and clinical, psychological, environmental, and service organization predictors of treatment effectiveness, compliance, and service satisfaction.

Reexpansion of a pulmonary atelectasis is often difficult, even after removing possible causes of bronchial obstruction. Chest ultrasounds, inexpensive and readily available at the patient bedside, may offer valuable support to guide recruitment maneuvers. We report the case of a 57-year-old woman that developed a complete collapse of the left lung seven days after undergoing an intestinal resection for perforation. A mucous plug occluding the main bronchus was removed with bronchoscopy, but persistent hypoxemia required mechanical ventilation; 24 hours later, an attempt to wean the patient from the ventilator failed. Chest X-rays revealed the persistence of a partial collapse of the left inferior lobe associated with a pleural effusion. A chest ultrasound confirmed the presence of a lung consolidation and of a modest pleural effusion. Manual reexpansion was then attempted, and ventilatory pressures as well as the duration of forced inspirations were based on real-time ultrasound images. Complete reexpansion was achieved within a few minutes and confirmed by chest X-ray. The patient was weaned from mechanical ventilation on the same day and discharged from ICU three days later.

Objective: In patients affected by intra-abdominal hypertension, bladder or gastric pressure measurement may be usefully integrated by ultrasounds in order to detect early hemodynamic impairment. The purpose of this study was to search for changes in abdominal vein size and flow induced by intra-abdominal hypertension., Design: Physiologic study., Setting: Postoperative intensive care unit of a university hospital., Subjects: Sixteen healthy volunteers., Interventions: Four echographic assessments of vessel sizes and blood velocities were randomly performed in the following settings: 1) baseline, 2) intra-abdominal hypertension simulated by a tight pelvic stabilizer around the waist, 3) noninvasive ventilation with a facial mask, and 4) intra-abdominal hypertension plus noninvasive ventilation., Measurements and Main Results: The model of intra-abdominal hypertension was validated in eight subjects by measuring gastric pressure. During intra-abdominal hypertension, 1) the inferior vena cava was compressed (significant decrease of both anteroposterior and lateral diameters) and deformed (decreased anteroposterior/lateral diameter ratio), and deformation, but not compression, was attenuated by noninvasive ventilation associated with intra-abdominal hypertension; 2) the portal vein was also compressed (decreased diameter); and 3) blood velocities did not change significantly in the inferior vena cava, portal vein, right suprahepatic vein, or right external iliac vein. In the receiver operating characteristic curve analysis, an inferior vena cava section area (normalized for body surface) of lower than 1 cm²/m² discriminated between intra-abdominal hypertension presence and absence with a sensitivity of 65.6% and a specificity of 87.5% (p = .0001). Noninvasive ventilation alone did not significantly affect vein sizes and velocities. The resistive index, calculated by pulse wave Doppler signal from segmental branches of the right renal artery, increased slightly, but significantly, during intra-abdominal hypertension alone, suggesting an increase of intrarenal pressure., Conclusions: Simulated intra-abdominal hypertension was associated with decreased inferior vena cava section area and increased resistive index in renal arteries. Further studies are now needed to investigate whether these changes may be of value to integrate bladder or gastric pressure measurement in clinical practice.

Introduction: The phenomenon of body art, such as tattoos and piercings, has ancient roots, rediscovered in Western society during the '70s. The aim of this research is to investigate the prevalence and the characteristics of tattoos and piercings among Italian adolescents of high school in Padua, with particular attention to family context and temperament., Methods: Some questionnaires about the presence or the wish of tattoos/piercings, smoke and alcohol use, familiar conflicts, and some temperamental features, such as novelty seeking, harm avoidance and reward dependence, have been administered to a sample of 829 students., Results: Tattoo and piercing's prevalence among adolescents was respectively 4% and 24%; 2.5% of the sample had both. Respectively 62% and 35% of the subjects expressed the desire of having a tattoo or piercing. A significant association has been found between tattoo/piercing's presence and smoke and alcohol use (p < 0.001). Subjects with tattoos and piercings, are more likely to have familiar conflicts and minor perceived support and they have higher scores on the novelty seeking scale. Those who wish a tattoo/piercing showed higher reward dependence., Conclusions: This study confirms that tattoos/piercings are common among young people and it stresses the relevance of familiar and temperamental features, and the association between tattoos/piercings and some maladaptive behaviors, like smoke and alcohol use.

Medical management of victims of chemical incidents includes supportive therapy, decontamination and antidote administration. Chemical weapons of mass destruction are available to many countries and are a possible alternative to conventional weapons for terrorist groups. During the last 5 years, some Italian institutions have made big efforts to establish a national system of antidote stockpiling and distribution. Little or no efforts have been addressed to other aspects of the medical management of patients exposed to chemical agents, such as decontamination, personal protective equipment, and specific supportive therapy. Although antidotes are indispensable instruments for some poisonings, as nerve agent and botulin intoxication, antidote stockpiling cannot be considered the only objective of a comprehensive medical preparedness for chemical emergencies. This paper addresses the medical priority when approaching victims of chemical emergencies. The priority actually is to establish a chain of chemical survival in which antidote administration is one out of several links.

Objective: Few studies have explored the relationship between stature and eating disorders (ED). We aimed to investigate the connection between height and risk for ED in a cohort of female subjects., Method: The sample was composed of 1,031 subjects with ED and 832 controls. All participants belonged to the same birth cohort and were living in the same geographical area., Results: ED subjects were, on average, shorter than control subjects, independently from the age of onset. In early-onset anorexia nervosa only, age of onset and lowest body mass index were significant predictors of height. In the whole sample, a lower height was associated with an increased risk of having an ED, even after controlling for possible confounding variables., Conclusion: The association between EDs and low stature is statistically significant. Further studies are necessary to understand which genetic and/or environmental factors might explain this association., ((c) 2007 by Wiley Periodicals, Inc.)

Italian Poison Centers answer to approximately 100,000 calls per year. Potentially, this activity is a huge source of data for toxicovigilance and for syndromic surveillance. During the last decade, surveillance systems for early detection of outbreaks have drawn the attention of public health institutions due to the threat of terrorism and high-profile disease outbreaks. Poisoning surveillance needs the ongoing, systematic collection, analysis, interpretation, and dissemination of harmonised data about poisonings from all Poison Centers for use in public health action to reduce morbidity and mortality and to improve health. The entity-relationship model for a Poison Center relational database is extremely complex and not studied in detail. For this reason, not harmonised data collection happens among Italian Poison Centers. Entities are recognizable concepts, either concrete or abstract, such as patients and poisons, or events which have relevance to the database, such as calls. Connectivity and cardinality of relationships are complex as well. A one-to-many relationship exist between calls and patients: for one instance of entity calls, there are zero, one, or many instances of entity patients. At the same time, a one-to-many relationship exist between patients and poisons: for one instance of entity patients, there are zero, one, or many instances of entity poisons. This paper shows a relational model for a poison center database which allows the harmonised data collection of poison centers calls.

In Europe, public and scientific concerns about the environmental and food safety of GM (Genetically Modified) crops overshadow the potential benefits offered by crop biotechnology to improve food quality. One of the concerns regarding the use of GM food in human and animal nutrition is the effect that newly introduced sequences may have on the organism. In this paper, we assess the potential transfer of diet-derived DNA to animal tissues after consumption of GM plants. Blood, spleen, liver, kidney and muscle tissues from piglets fed for 35 days with diets containing either GM (MON810) or a conventional maize were investigated for the presence of plant DNA. Only fragments of specific maize genes (Zein, Sh-2) could be detected with different frequencies in all the examined tissues except muscle. A small fragment of the Cry1A(b) transgene was detected in blood, liver, spleen and kidney of the animals raised with the transgenic feed. The intact Cry1A(b) gene or its minimal functional unit were never detected. Statistical analysis of the results showed no difference in recovery of positives for the presence of plant DNA between animals raised with the transgenic feed and animals raised with the conventional feed, indicating that DNA transfer may occur independently from the source and the type of the gene. From the data obtained, we consider it unlikely that the occurrence of genetic transfer associated with GM plants is higher than that from conventional plants.

The concentration of 9 environmental chemical pollutants in the general population was measured in blood and urine. For the 9 different pollutants, the blood samples tested varied from 88 for acetone to 431 for benzene. Urine samples varied from 48 for styrene to 213 for n-hexane. Six of these agents (benzene, toluene, styrene, n-hexane, acetone and carbon disulphide) were present in all or almost all (100-94%) blood samples. The three chlorides (chloroform, trichloroethylene and tetrachloroethylene) were present only in 60-85% of samples. After acetone, with blood concentrations in microgram/1 (mean 840 microgram/l), the highest mean blood levels were those of toluene (1097 ng/l), chloroform (955 ng/l) and n-hexane (642 ng/l). Trichloroethylene and free carbon disulphide showed similar values (458 and 438 ng/l, respectively). Finally, benzene, styrene and tetrachloroethylene showed the lowest values (262, 217 and 149 ng/l, respectively). There was generally a significant difference between rural and urban workers in terms of blood benzene (200 ng/l vs 264 ng/l), trichloroethylene (180 ng/l vs 763 ng/l) and tetrachloroethylene (62 ng/l vs 263 ng/l). In a group of subjects potentially exposed to industrial solvents, classed as chemical workers, blood benzene, toluene, chloroform and n-hexane were significantly higher than in rural and urban workers. Smokers showed a significantly higher blood concentration than non-smokers for benzene (381 ng/l vs 205 ng/1), toluene (1431 ng/l vs 977 ng/l), and n-hexane (838 ng/l vs 532 ng/l). All or almost all urine samples (100-92%) contained all the compounds except trichloroethylene and tetrachloroethylene, present in 79% and 76% of samples, respectively (table 2). Urinary concentrations of all compounds did not differ significantly between rural and urban workers. Benzene and toluene were significantly higher in in urine of smokers than of non-smokers. Chloroform and n-hexane showed significantly higher urinary than blood values. Excluding acetone, with urinary and blood concentrations in pg/l, chloroform, toluene and n-hexane showed the highest mean concentrations both in blood and in urine.