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Nuclear Magnetic Resonance (NMR) based metabolomic analysis of whole saliva has provided potential diagnostic biomarkers for numerous human diseases contributing to a better understanding of their mechanisms. However, a comprehensive interpretation of the significance of metabolites in whole, parotid, and submandibular/sublingual saliva subtypes is still missing. Precision and reproducibility of sample preparation is an essential step. Here, we present a simple and efficient protocol for saliva 1 H-NMR metabolic profiling. This procedure has been specifically designed and optimized for the identification and quantification of low concentration metabolites (as low as 1.1 μM) and is suitable for all the saliva subtypes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated., Materials and Methods: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO 2 . Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex ® assay., Results: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-α- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation., Discussion: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

Background: Cardiac implantable electronic device (CIED) implantation rates as well as the clinical and procedural characteristics and outcomes in patients with known active coronavirus disease 2019 (COVID-19) are unknown., Objective: The purpose of this study was to gather information regarding CIED procedures during active COVID-19, performed with personal protective equipment, based on an international survey., Methods: Fifty-three centers from 13 countries across 4 continents provided information on 166 patients with known active COVID-19 who underwent a CIED procedure., Results: The CIED procedure rate in 133,655 hospitalized COVID-19 patients ranged from 0 to 16.2 per 1000 patients (P <.001). Most devices were implanted due to high-degree/complete atrioventricular block (112 [67.5%]) or sick sinus syndrome (31 [18.7%]). Of the 166 patients in the study survey, the 30-day complication rate was 13.9% and the 180-day mortality rate was 9.6%. One patient had a fatal outcome as a direct result of the procedure. Differences in patient and procedural characteristics and outcomes were found between Europe and North America. An older population (76.6 vs 66 years; P <.001) with a nonsignificant higher complication rate (16.5% vs 7.7%; P = .2) was observed in Europe vs North America, whereas higher rates of critically ill patients (33.3% vs 3.3%; P <.001) and mortality (26.9% vs 5%; P = .002) were observed in North America vs Europe., Conclusion: CIED procedure rates during known active COVID-19 disease varied greatly, from 0 to 16.2 per 1000 hospitalized COVID-19 patients worldwide. Patients with active COVID-19 infection who underwent CIED implantation had high complication and mortality rates. Operators should take these risks into consideration before proceeding with CIED implantation in active COVID-19 patients., (Copyright © 2021 Heart Rhythm Society. All rights reserved.)

A novel approach for the formation of anomeric carbon-functionalized furanoside systems was accomplished through the employment of an oxo-rhenium catalyst. The transformation boasts a broad range of nucleophiles including allylsilanes, enol ethers, and aromatics in addition to sulfur, nitrogen, and hydride donors, able to react with an oxocarbenium ion intermediate derived from furanosidic structures. The excellent stereoselectivities observed followed the Woerpel model, ultimately providing 1,3- cis -1,4- trans systems. In the case of electron-rich aromatic nucleophiles, an equilibration occurs at the anomeric center with the selective formation of 1,3- trans -1,4- cis systems. This anomalous result was rationalized through density functional theory calculations. Different oxocarbenium ions such as those derived from dihydroisobenzofuran, pyrrolidine, and oxazolidine heterocycles can also be used as a substrate for the oxo-Re-mediated allylation reaction.

Celestial amplitudes which use conformal primary wave functions rather than plane waves as external states offer a novel opportunity to study properties of amplitudes with manifest conformal covariance and give insight into a potential holographic celestial conformal field theory at the null boundary of asymptotically flat space. Since translation invariance is obscured in the conformal basis, features of amplitudes that heavily rely on it appear to be lost. Among these are the remarkable relations between gauge theory and gravity amplitudes known as the double copy. Nevertheless, properties of amplitudes reflecting fundamental aspects of the perturbative regime of quantum field theory are expected to survive a change of basis. Here we show that there exists a well-defined procedure for a celestial double copy. This requires a generalization of the usual squaring of numerators which entails first promoting them to generalized differential operators acting on external wave functions and then squaring them. We demonstrate this procedure for three- and four-point celestial amplitudes and give an argument for its validity to all multiplicities.

The number of patients with cardiac implantable electronic devices (CIEDs) requiring radiation therapy (RT) for cancer treatment is increasing. The purpose of this study is to estimate the prevalence, possible predictors, and clinical impact of RT-related CIEDs malfunctions. We retrospectively reviewed the medical records of all pacemaker (PM)/implantable cardioverter-defibrillator (ICD) patients who underwent RT in the last 14 years. One hundred and twenty-seven patients who underwent 150 separate RT courses were analysed (99 with a PM and 27 with an ICD). Of note, 21/127 (16.6%) patients were PM-dependent. Neutron-producing RT was used in 37/139 (26.6%) courses, whereas non-neutron-producing RT was used in 102/139 (73.4%) courses. The cumulative dose (Dmax) delivered to the CIED exceeded 5 Gy only in 2/132 (1.5%) cases. Device malfunctions were observed in 3/150 (2%) RT courses, but none was life-threatening or led to a major clinical event and all were resolved by CIED reprogramming. In all cases, the Dmax delivered to the CIED was < 2 Gy. Two malfunctions occurred in the 37 patients treated with neutron-producing RT (5.4%), and 1 malfunction occurred in the 102 patients treated with non-neutron-producing RT (1%) (p = 0.17). Device relocation from the RT field was performed in 2/127 (1.6%) patients. RT in patients with CIED is substantially safe if performed in an appropriately organized environment, with uncommon CIEDs malfunctions and no major clinical events. Neutron-producing energies, rather than Dmax, seem to increase the risk of malfunctions. Device interrogation on a regular basis is advised to promptly manage CIED malfunctions.

The emergency caused by Covid-19 pandemic raised interest in studying lifestyles and comorbidities as important determinants of poor Covid-19 prognosis. Data on tobacco smoking, alcohol consumption and obesity are still limited, while no data are available on the role of e-cigarettes and heated tobacco products (HTP). To clarify the role of tobacco smoking and other lifestyle habits on COVID-19 severity and progression, we designed a longitudinal observational study titled COvid19 and SMOking in ITaly (COSMO-IT). About 30 Italian hospitals in North, Centre and South of Italy joined the study. Its main aims are: 1) to quantify the role of tobacco smoking and smoking cessation on the severity and progression of COVID-19 in hospitalized patients; 2) to compare smoking prevalence and severity of the disease in relation to smoking in hospitalized COVID-19 patients versus patients treated at home; 3) to quantify the association between other lifestyle factors, such as e-cigarette and HTP use, alcohol and obesity and the risk of unfavourable COVID-19 outcomes. Socio-demographic, lifestyle and medical history information will be gathered for around 3000 hospitalized and 700-1000 home-isolated, laboratory-confirmed, COVID-19 patients. Given the current absence of a vaccine against SARS-COV-2 and the lack of a specific treatment for -COVID-19, prevention strategies are of extreme importance. This project, designed to highly contribute to the international scientific debate on the role of avoidable lifestyle habits on COVID-19 severity, will provide valuable epidemiological data in order to support important recommendations to prevent COVID-19 incidence, progression and mortality.

Iron porphyrin methoxy complexes, of the general formula [Fe(porphyrin)(OCH 3 )], are able to catalyze the reaction of diazo compounds with alkenes to give cyclopropane products with very high efficiency and selectivity. The overall mechanism of these reactions was thoroughly investigated with the aid of a computational approach based on density functional theory calculations. The energy profile for the processes catalyzed by the oxidized [Fe III (Por)(OCH 3 )] (Por = porphine) as well as the reduced [Fe II (Por)(OCH 3 )] - forms of the iron porphyrin was determined. The main reaction step is the same in both of the cases, that is, the one leading to the terminal -carbene intermediate [Fe(Por)(OCH 3 )(CHCO 2 Et)] with simultaneous dinitrogen loss; however, the reduced species performs much better than the oxidized one. Contrarily to the iron(III) profile in which the carbene intermediate is directly obtained from the starting reactant complex, the favored iron(II) process is more intricate. The initially formed reactant adduct between [Fe II (Por)(OCH 3 )] - and ethyl diazoacetate ( EDA ) is converted into a closer reactant adduct, which is in turn converted into the terminal iron porphyrin carbene [Fe(Por)(OCH 3 )(CHCO 2 Et)] - . The two corresponding transition states are almost isoenergetic, thus raising the question of whether the rate-determining step corresponds to dinitrogen loss or to the previous structural and electronic rearrangement. The ethylene addition to the terminal carbene is a downhill process, which, on the open-shell singlet surface, presents a defined but probably short-living diradicaloid intermediate, though other spin-state surfaces do not show this intermediate allowing a direct access to the cyclopropane product. For the crucial stationary points, the more complex catalyst [Fe( 2 )(OCH 3 )], in which a sterically hindered chiral bulk is mounted onto the porphyrin, was investigated. The corresponding computational data disclose the very significant effect of the porphyrin skeleton on the reaction energy profile. Though the geometrical features around the reactive core of the system remain unchanged, the energy barriers become much lower, thus revealing the profound effects that can be exerted by the three-dimensional organic scaffold surrounding the reaction site.

The detection of salivary molecules associated with pathological and physiological alterations has encouraged the search of novel and non-invasive diagnostic biomarkers for oral health evaluation. While genomic, transcriptomic, and proteomic profiles of human saliva have been reported, its metabolic composition is a topic of research: metabolites in submandibular/sublingual saliva have never been analyzed systematically. In this study, samples of whole, parotid, and submandibular/sublingual saliva from 20 healthy donors, without dental or periodontal diseases, were examined by nuclear magnetic resonance. We identified metabolites which are differently distributed within the three saliva subtypes (54 in whole, 49 in parotid, and 36 in submandibular/sublingual saliva). Principal component analysis revealed a distinct cluster for whole saliva and a partial overlap for parotid and submandibular/sublingual metabolites. We found exclusive metabolites for each subtype: 2-hydroxy-3-methylvalerate, 3-methyl-glutarate, 3-phenylpropionate, 4-hydroxyphenylacetate, 4-hydroxyphenyllactate, galactose, and isocaproate in whole saliva; caprylate and glycolate in submandibular/sublingual saliva; arginine in parotid saliva. Salivary metabolites were classified into standard and non-proteinogenic amino acids and amines; simple carbohydrates; organic acids; bacterial-derived metabolites. The identification of a salivary gland-specific metabolic composition in healthy people provides the basis to invigorate the search for salivary biomarkers associated with oral and systemic diseases.

An orthogonal selectivity for distal meta-C-H activation of benzophenone is acheived by overriding the inherent proximal ortho-selectivity through a template assisted metalation approach. This strategy has been successfully utilized in Pd-catalyzed regioselective C-C and C-Si bond formation.

A silver(I) catalyzed regioselective trifluoromethylation of allenes using Langlois's salt (NaOSOCF 3 ) is demonstrated. This transformation enables direct expedient access to α-trifluoromethylated acroleins, which are valuable synthons for a number of pharmaceuticals and agrochemicals containing vinyl-CF 3 moieties. Versatility of this trifluoromethylation method has been established with good yield and excellent regioselectivity. Preliminary experiments and computational studies were carried out to elucidate the mechanistic insight of this protocol., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Signaling mediated by purines is a widespread mechanism of cell-cell communication related to vasomotor responses and the control of platelet function in the vascular system. However, little is known about the involvement of this signaling as well as the role of reactive oxygen species (ROS) in the development of hypothyroidism. Therefore, the present study investigates changes in the purinergic system, including enzyme activities and expression in platelets, and oxidative profiles in patients with post-thyroidectomy hypothyroidism. The nucleoside triphosphate diphosphohydrolase 1 (NTPDase/CD39) expression in patients increased by 40%, and the adenosine triphosphate (ATP) or adenosine diphosphate (ADP) hydrolyzing activity increased by 82% and 70%, respectively. The activities of ecto-5´-nucleotidase and adenosine deaminase (ADA) also significantly enhanced (39% and 52%, respectively), which correlates with a 45% decrease in adenosine concentration. Furthermore, these patients demonstrated an increased production of ROS (42%), thiobarbituric acid reactive substances (TBARS) (115%), carbonyl protein (30%) and a decreased glutathione S-transferase (GST) activity (20%). This study demonstrates that hypothyroidism interferes with adenine nucleoside and nucleotide hydrolysis and this is correlated with oxidative stress, which might be responsible for the increase in ADA activity. This increase causes rapid adenosine deamination, which can generate a decrease in their concentration in the systemic circulation, which can be associated with the development of vascular complications.

The upholding of red blood cells (RBC) quality and the removal of leukocytes are two essential issues in transfusion therapy. Leukodepletion provides optimum results, nonetheless there are cases where irradiation is recommended for some groups of hematological patients such as the ones with chronic graft-vs-host disease, congenital cellular immunodeficiency, and hematopoietic stem cell transplant recipients. The European guidelines suggest irradiation doses from 25 to 50 Gray (Gγ). We evaluated the effect of different prescribed doses (15 to 50 Gγ) of X-ray irradiation on fresh leukodepleted RBCs bags using a novel protocol that provides a controlled irradiation. Biochemical assays integrated with RBCs metabolome profile, assessed by nuclear magnetic resonance spectroscopy, were performed on RBC units supernatant, during 14 days storage. Metabolome analysis evidenced a direct correlation between concentration increase of three metabolites, glycine, glutamine and creatine, and irradiation dose. Higher doses (35 and 50 Gγ) effect on RBC mean corpuscular volume, hemolysis, and ammonia concentration are considerable after 7 and 14 days of storage. Our data show that irradiation with 50 Gγ should be avoided and we suggest that 35 Gγ should be the upper limit. Moreover, we suggest for leukodepleted RBCs units the irradiation with the prescribed dose of 15 Gγ, value at center of bag, and ranging between 13.35-15 Gγ, measured over the entire bag volume, may guarantee the same benefits of a 25 Gγ dose assuring, in addition, a better quality of RBCs.

Background: Time dependent quantification of endogenous metabolites in biological samples (blood, urine, biological tissues extracts) in normal and pathological conditions as well as following therapeutic protocols is well established. In the clinical practice, such a dynamic flux of information allows the physician to identify and appreciate alterations associated to biochemical pathways of specific organs. In the years, many biochemical assays have been developed to detect, selectively, this vast array of molecules., Methods: The Proton Nuclear Magnetic Resonance ( 1 H NMR) spectrum allows the identification and quantification of more than 30 RBC-associated metabolites with minimum manipulation of the sample. To validate the use of 1 H NMR spectroscopy for quality control purposes in transfusion medicine, a series of statistical tools have been employed to analyse and compare accuracy and precision of the 1 H NMR results with respect to the ones obtained by standard biochemical assays., Results: Among the many metabolites that can be detected and quantified by 1 H NMR spectroscopy we selected creatinine and lactate, since they are routinely quantified by standard biochemical assays and because they are characterized by a wide concentration dynamic range. We show that 1D 1 H NMR spectroscopy is an accurate a precise method for metabolite quantification., Conclusion: These results validate the use of 1 H NMR spectroscopy in transfusion medicine as a method to evaluate the quality of RBC packed units and to develop novel and more efficient RBCs storage protocols., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the concentrations of five essential (Fe, Mn, Zn, Cu and Se) and four non-essential/toxic elements (Cr, Cd, Ni and Pb) in 35 different starter infant formulas (0-6 months) sold in Italy. In addition, a safety assessment of these trace elements was carried out, by comparing the estimated daily intake (EDI) with the adequate intake (AI) and the provisional tolerable daily intake (PTDI), with a view to provide information on the metal distribution patterns and health risk to infants arising from the consumption of these products. The concentrations were determined by using inductively coupled plasma mass spectrometry after microwave digestion. The concentrations expressed in geometric mean ± geometric standard deviation of Fe (6.17 ± 1.61 mg/L), Zn (6.21 ± 1.31 mg/L), Cu (416.4 ± 1.21 μg/L), Mn (121.5 ± 1.85 μg/L) and Se (13.27 ± 1.67 μg/L) were within legal limits. In spite of this, the mean EDIs of Fe (4.81 mg/day) and Mn (94.75 μg/day) were many times higher than the recommended AI, especially for Mn. Chromium, Ni, Cd and Pb concentrations were not detectable in 11, 37, 57 and 66% of the samples, respectively. Considering the overall sample, the GM ± GSD of these elements were 4.80 ± 5.35 μg/L for Cr, 1.02 ± 11.65 μg/L for Ni, 0.21 ± 14.83 μg/L for Cd and 0.14 ± 17.13 μg/L for Pb. The mean EDIs were far below the respective PTDI. When the safety assessment was based on the 75° percentile level of each elements, all EDIs remained well below the PTDI, with the exception of Cd, whose EDI approached (74.7%), albeit remaining below the PTDI. In conclusion, our results and the increased awareness on the potential risks of excessive Mn and Fe for infants support that an urgent scientific-based definition of the appropriated levels of fortification in formulas is required. Moreover, regular monitoring of all the stages of production of infant formulas is essential in order to limit toxic metal contamination.

Background: The species Mus musculus experiences an obligate proteinuria: predominant are the Major Urinary Proteins (MUPs), that, collectively known as the major mouse allergen Mus m 1, are among the most important aeroallergens for mouse allergic patients. The production of a soluble and stable hypoallergenic form of Mus m 1 is essential for the development of immunotherapeutic protocols to treat allergic symptoms., Methods: We introduced the substitution C138S in recombinant Mus m 1.0102, an allergenic isoform of Mus m 1. Solubility, conformation, stability and ability to refold after chemical denaturation were investigated with dynamic light scattering, circular dichroism, fluorescence and NMR spectroscopy. An in vitro degranulation assay was used to evaluate the protein allergenic potential, and compare it with Mus m 1.0102 and with an hypoallergenic variant bearing the substitution Y120A., Results: Mus m 1.0102-C138S retains a native-like fold revealing, however, local conformational alterations that influence some of its physical and allergenic properties: it is monodispersed, thermostable up to 56°C, able to reversibly unfold and it exhibits an enhanced allergenicity., Conclusions: The unique free thiol group affects the solution structural stability of the native protein. Because the mutant C138S does not aggregate over time it is a good lead protein to develop diagnostic and therapeutic applications., General Significance: We elucidated the relationship between unfolding reversibility and sulphydryl reactivity. We ascribed the enhanced allergenicity of the mutant C138S to an increased accessibility of its allergenic determinants, an enticing feature to further investigate the structural elements of the allergen-IgE interface., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: The impact on long-term outcomes of implantable cardioverter defibrillators (ICDs) and biventricular defibrillators for cardiac resynchronization (CRT-D) devices in 'real world' patients with heart failure (HF) needs to be assessed in terms of clinical effectiveness., Methods and Results: A registry including consecutive HF patients who underwent a first implant of an ICD (891 patients) or a CRT-D device (709 patients) in 2006-2010 was followed (median 1487 days and 1516 days, respectively), collecting administrative data on survival, all-cause hospitalizations, cardiovascular or HF hospitalizations, and days alive and out of hospital (DAOH). Survival free from death/cardiac transplant was 61.9% and 63.8% at 5 years for ICD and CRT-D patients, respectively. Associated comorbidities (Charlson Comorbidity Index) had a significant impact on death/cardiac transplant, as well as on hospitalizations. The median values of DAOH% were 97.4% for ICD and 97.7% for CRT-D patients, but data were highly skewed, with the lower quartile of DAOH% values including values ranging between 0% and 52.8% for ICD and between 0% and 56.1% for CRT-D patients. Charlson Comorbidity Index was a very strong predictor of DAOH%., Conclusions: Patients who were implanted in 'real world' clinical practice with an ICD or a CRT-D device have, on average, a relatively favourable outcome, with a survival of around 62-64% at 5 years, but with an important burden of hospitalizations. Comorbidities, as evaluated by means of the Charlson Comorbidity Index, have a significant impact on outcomes in terms of mortality/heart transplant, hospitalizations and days spent alive and out of hospital., (© 2016 The Authors European Journal of Heart Failure © 2016 European Society of Cardiology.)

Blood transfusion is a fundamental therapy in numerous pathological conditions. Regrettably, many clinical reports describe adverse transfusion's drawbacks due to red blood cells alterations during storage. Thus, the possibility for a blood bank to ameliorate the quality of the erythrocyte concentrates units is crucial to improve clinical results and reduce transfusion adverse occurrences. Leukodepletion is a pre-storage treatment recognized to better preserve the quality of red blood cells with respect to leukoreduction. Aim of this work is to unravel the biochemical and biophysical basis that sustain the good clinical outcomes associated to the use of leukodepleted erythrocytes units. Erythrocytes concentrates were prepared as leukoreduced (n = 8) and pre-storage leukodepleted (n = 8) and then studied during 6 weeks in blood bank conditions. Overall, the data indicate that leukodepletion not only provide red blood cells with an appropriate amount of nutrients for a longer time but also selects red blood cells characterized by a more resilient plasma membrane fit to prolong their viability. We believe these results will stimulate new ideas to further optimize the current storage protocols.

Background: Blood transfusion is an established therapeutic practice. The characteristics of blood components at different storage times are expected to affect the efficacy of transfusion therapy. Metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy requires little or no sample treatment and allows identification of more than 50 soluble metabolites in a single experiment. The aim of this study was to assess the metabolic behaviour of red blood cells during 42 days of storage in blood bank conditions., Materials and Methods: Red blood cells (RBC), collected from eight healthy male donors, aged 25-50 years, were prepared as prestorage leukoreduced erythrocyte concentrates and stored under standard blood bank conditions. Samples taken at various storage times were separated in two fractions: the supernatant, recovered after centrifugation, and the red blood cell lysate obtained after protein depletion by ultrafiltration. The metabolic profile of the red blood cells was determined from analysis of (1)H-NMR spectra., Results: The red blood cell supernatant was studied to track the consumption of the preservative additives and to detect and quantify up to 30 metabolites excreted by the erythrocytes. The NMR spectra of the RBC lysate provided complementary information on some biochemical pathways and set the basis for building a time-dependent red blood cell metabolic profile., Discussion: We proved the analytical power of (1)H-NMR spectroscopy to study red blood cell metabolism under blood bank conditions. A potential biomarker able to provide information on the level of cellular oxidative stress protection was identified. Our data support the hypothesis that a more detailed knowledge of metabolic modifications during storage opens the way to the development of new and more effective protocols for red blood cell conservation and patient-oriented transfusion therapy.

Background: Consensus guidelines define indications for cardiac resynchronization therapy (CRT), but the variability in implant rates in 'real world' clinical practice, as well as the relationship with the epidemiology of heart failure are not defined., Methods and Results: In Emilia-Romagna, an Italian region with around 4.4 million inhabitants, a registry was instituted to collect data on implanted devices for CRT, with (CRT-D) or without defibrillation (CRT-P) capabilities. Data from all consecutive patients resident in this region who underwent a first implant of a CRT device in years 2006-2010 were collected and standardized (considering each of the nine provinces of the region). The number of CRT implants increased progressively, with a 71% increase in 2010 compared to 2006. Between 84 and 90% of implants were with CRT-D devices. The variability in standardized implant rates among the provinces was substantial and the ratio between the provinces with the highest and the lowest implant rates was always greater than 2. Considering prevalent cases of heart failure in the period 2006-2010, the proportion of patients implanted with CRT per year ranged between 0.23 and 0.30%., Conclusions: The application in 'real world' clinical practice of CRT in heart failure is quite heterogeneous, with substantial variability even among areas belonging to the same region, with the need to make the access to this treatment more equitable. Despite the increased use of CRT, its overall rate of adoption is low, if a population of prevalent heart failure patients is selected on the basis of administrative data on hospitalizations.

Aims: Cardiac resynchronization therapy (CRT) has shown morbidity and mortality benefits in patients with advanced congestive heart failure (HF). Since about one-third of the patients did not appear to respond to CRT, it would seem reasonable to try to identify patients more accurately before implantation. Left atrial (LA) dimension has been proposed as a powerful outcome predictor in patients with heart disease. Accordingly, the aim of this study is to prospectively assess the predictive value of LA for selecting CRT responders., Methods: Fifty two consecutive patients with refractory HF, sinus rhythm and left bundle branch block were enrolled in the study and planned for CRT implantation. Clinical and echocardiographic evaluations were performed before CRT implantation and after 6 months. Three LA volumes indexed to body surface area (iLAV) were computed to evaluate the LA complexity: maximal LAV (iLAVmax), LAV just before atrial systole (iLAVpre), and minimal LAV (iLAVpost). CRT responders were defined as those who presented a reduction of >10% in LVESVi at 6-month follow-up., Results: Responders (63%) and nonresponders (37%) had similar baseline clinical characteristics and pre-implantation LV volumes. However, baseline LA volumes were significantly associated with the extent of LV reverse remodeling: in particular, baseline iLAVmax was remarkably lower in responders than in nonresponders (50.2±14.1 ml/m(2) vs 65.8±15.7 ml/m(2), p=0.001) resulting predictive for CRT response., Conclusion: Patients with small iLAV result as better responders to CRT than larger one. iLAVmax is an independent predictor of LV reverse remodeling and allows to indentify the best candidates for CRT., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Aims: In candidates for cardiac resynchronization therapy (CRT), the choice between pacemaker (CRT-P) and defibrillator (CRT-D) implantation is still debated. We compared the long-term prognosis of patients who received CRT-D or CRT-P according to class IA recommendations of the European Society of Cardiology (ESC) and who were enrolled in a multicentre prospective registry., Methods and Results: A total of 620 heart failure patients underwent successful implantation of a CRT device and were enrolled in the Contak Italian Registry. This analysis included 266 patients who received a CRT-D and 108 who received a CRT-P according to class IA ESC indications. Their survival status was verified after a median follow-up of 55 months. During follow-up, 73 CRT-D and 44 CRT-P patients died (rate 6.6 vs. 10.4%/year; log-rank test, P = 0.020). Patients receiving CRT-P were predominantly older, female, had no history of life-threatening ventricular arrhythmias, and more frequently presented non-ischaemic aetiology of heart failure, longer QRS durations, and worse renal function. However, the only independent predictor of death from any cause was the use of CRT-P (hazard ratio, 1.97; 95% confidence interval, 1.21-3.16; P = 0.007)., Conclusion: The implantation of CRT-D, rather than CRT-P, may be preferable in patients presenting with current class IA ESC indications for CRT. Indeed, CRT-D resulted in greater long-term survival and was independently associated with a better prognosis.

Background/objectives: Cardiac resynchronization therapy (CRT) improves left ventricular (LV) function in patients with advanced heart failure (HF) and there are some evidences about beneficial effects also on left atrial (LA) dimension and function. The contribution of atrioventricular delay (AVD) optimization on LA changes has not been evaluated. The purpose of the present study was to further investigate the effect of CRT on LA reverse remodelling and to evaluate the contribution of AVD optimization., Methods and Results: From the Cardiology Department of Piacenza Hospital and Modena University Hospital fifty one patients with refractory systolic HF and left bundle branch block were prospectively enrolled before CRT implantation. Patients were 1:1 randomized to either an optimized AVD (AV Opt group) determined by continuous wave Doppler aortic velocity-time integral (VTI) or an empiric AVD of 110 ms (AV Fixed group). Optimal AVD was defined as the AVD that yielded the largest aortic VTI at one of eight tested AV intervals (between 60 and 200 ms). LA volumes and emptying fractions were assessed by two-dimensional echocardiography at baseline and 6 months after CRT. At 6-month follow-up, CRT induced LA reverse remodeling in the whole population (maximal LA volume: 55.8 ± 16.4 ml/m² vs 50.3 ± 18.9 ml/m², p=0.006; pre-systolic LA volume: 47.0 ± 15.2 ml/m² vs 41.4 ± 17.4 ml/m², p=0.003; post-systolic LA volume: 36.4 ± 15.0 ml/m² vs 30.3 ± 18.0 ml/m(2), p=0.001); nevertheless, no substantial difference was observed about LA structural and functional remodeling between both AV Opt group and AV Fixed group., Conclusion: CRT induces LA reverse remodeling that appears independent from AVD optimization., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Introduction: Novel oral anticoagulants have been tested against warfarin for atrial fibrillation, yet no direct comparison is available. We thus aimed to perform pair-wise (direct) and warfarin-adjusted network (i.e. indirect) meta-analyses of novel oral anticoagulants for atrial fibrillation., Methods: Databases were searched for randomized warfarin-controlled trials of novel anticoagulants for non-valvular atrial fibrillation. The primary end-point was long-term stroke/systemic embolism. Odds ratios (95% intervals) were computed with RevMan and WinBUGS., Results: Seven trials (52701 patients) were included, focusing on apixaban, dabigatran, edoxaban and rivaroxaban. Pair-wise meta-analysis showed that after a weighted average of 23 months these novel anticoagulants lead to significant reductions in the risk of stroke/systemic embolism (odds ratio=0.81 [0.71-0.92], I2=23%) and all cause death (odds ratio=0.88 [0.82-0.95], I2=0%) in comparison to warfarin. Network meta-analysis showed that apixaban and dabigatran proved similarly superior to warfarin in preventing stroke/systemic embolism (odds ratio=0.78 [0.62-0.96] for apixaban vs warfarin; odds ratio=0.66 [0.52-0.84] for high-dose dabigatran vs warfarin; odds ratio for apixaban vs high-dose dabigatran=1.17 [0.85-1.63]), but apixaban was associated with fewer major bleedings (odds ratio=0.73 [0.57-0.93]) and drug discontinuations (odds ratio=0.64 [0.52-0.78]) than dabigatran. Rivaroxaban did not reduce stroke/systemic embolism (odds ratio=0.87 [0.71-1.07]) or major bleedings in comparison to warfarin (odds ratio=0.87 [0.71-1.07]) and was associated with more major bleedings in comparison to apixaban (odds ratio=1.52 [1.19-1.92]). Data for edoxaban were inconclusive., Conclusions: Novel oral anticoagulants appear as a very promising treatment option for atrial fibrillation.

Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity., Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation., Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained., Conclusions: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders., (Copyright © 2011 S. Karger AG, Basel.)

Studies have shown that seizures in young animals lead to later cognitive deficits. There is evidence that long-term potentiation (LTP) and long-term depression (LTD) might contribute to the neural basis for learning and memory mechanism and might be modulated by ATP and/or its dephosphorylated product adenosine produced by a cascade of cell-surface transmembrane enzymes, such as E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolases) and ecto-5'-nucleotidase. Thus, we have investigated if hippocampal ecto-nucleotidase activities are altered at different time periods after one episode of seizure induced by kainic acid (KA) in 7 days old rats. We also have evaluated if 90 day-old rats previously submitted to seizure induced by KA at 7 days of age presented cognitive impairment in Y-maze behavior task. Our results have shown memory impairment of adult rats (Postnatal day 90) previously submitted to one single seizure episode in neonatal period (Postnatal day 7), which is accompanied by an increased ATP hydrolysis in hippocampal synaptosomes. The metabolism of ATP evaluated by HPLC confirmed that ATP hydrolysis was faster in adult rats treated with KA in neonatal period than in controls. Surprisingly, the mRNA and protein levels as seen by PCR and Western blot, respectively, were not altered by the KA administration in early age. Since we have found an augmented hydrolysis of ATP and this nucleotide seems to be important to LTP induction, we could assume that impairment of memory and learning observed in adult rats which have experienced a convulsive episode in postnatal period may be a consequence of the increased ATP hydrolysis. These findings correlate the purinergic signaling to the cognitive deficits induced by neonatal seizures and contribute to a better understanding about the mechanisms of seizure-induced memory dysfunction., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Aims: Atrial fibrillation (AFib) induces remodelling of the left atrium (LA). Indexed LA volume (iLAV) as more accurate measure of LA size has not been evaluated as predictor of recurrence of AFib after cardioversion., Methods and Results: We identified 411 adults (mean age 64.1 ± 11.4 years, 34.5% women) who underwent successful cardioversion and with no history of other atrial arrhythmia, stroke, congenital heart disease, valvular dysfunction, surgery, thyroid dysfunction, acute or chronic inflammatory disease, and pacemaker. All echocardiographic data were retrieved from the laboratory database. iLAV was measured off-line using Simpson's method. Clinical characteristics and recurrence of clinical AFib were determined by review of medical records. Patients with scheduled follow-up of at least 6 months were included. About 250 patients (60.8%) developed AFib recurrence after a median (25th-75th percentile) follow-up of 345.0 (210.0-540.0) days. Patients with AFib recurrence had significantly greater iLAV than patients without AFib recurrence (39.7 ± 8.4 vs. 31.4 ± 4.6, P < 0.001). Each mL/m(2) increase in iLAV was associated with a 30% increased risk of AFib recurrence [odds ratio (OR) 1.30, confidence interval (CI) 1.23-1.38, P < 0.001]. In a multivariable model, each mL/m(2) increase in iLAV was independently associated with a 21% increase in the risk of AFib recurrence (OR 1.21, CI 1.11-1.30, P < 0.001). The areas under receiver operating characteristic curves, generated to compare LA diameter and iLAV as predictors of AFib recurrence, were 0.59 ± 0.3 and 0.85 ± 0.2, respectively (P < 0.001)., Conclusion: The present study is the first to show that larger iLAV before cardioversion, as a more accurate measure of LA remodelling than LA diameter, is strongly and independently associated with higher risks of AFib recurrence.

(15)N and (1)HN chemical shift data and (15)N relaxation studies have been used to characterise the binding of N-phenyl-naphthylamine (NPN) to mouse major urinary protein (MUP). NPN binds in the beta-barrel cavity of MUP, hydrogen bonding to Tyr120 and making extensive non-bonded contacts with hydrophobic side chains. In contrast to the natural pheromone 2-sec-butyl-4,5-dihydrothiazole, NPN binding gives no change to the overall mobility of the protein backbone of MUP. Comparison with 11 different ligands that bind to MUP shows a range of binding modes involving 16 different residues in the beta-barrel cavity. These finding justify why MUP is able to adapt to allow for many successful binding partners.

AD is characterized by a widespread cognitive impairment and deficit in functional competency to perform activities of daily living (ADL). The longitudinal reliability of cognitive and functional performance indices and the strength of relationship between patients cognitive impairment and their functional competence are still open issues. The aim of this study has been to evaluate, in a selected sample of patients with AD treated with AChEl, the slopes of cognitive impairment and disability. Among 249 AD patients, according to DSM-IV criteria, with presence/absence of associated vascular lesions (AD+VD), eligible for AChEl treatment, we selected subjects with mild to moderate cognitive impairment, without high comorbidity and severe psychiatric disease, with caregiver who resided with, or had frequent contact with the patient. Patients that changed treatment shifting from one AChEl to another, that didn't tolerate inhibitors (drop-out), and that presented behavioral and psychological symptoms of dementia (BPSD) requiring neuroleptic treatment during the study period were excluded from the final analysis. A sample of 99 subjects (30 males, 69 females; mean age of 79.4+/-5.0 years), completing a 15 months follow-up was considered. Cognitive performance remained stable after 15 months of treatment, but disability increased. No difference was found due to the AChEl compound used. The same hold true for the subgroups with presence/absence of a vascular components, whereas subgroup with mild cognitive performance showed a cognitive decline, parallel to the functional one. Our data underline the efficacy of AChEl in the treatment of AD with presence/absence of vascular component. Nevertheless, the judgement on the level of efficacy of AChEl could be biased by the level of reliability of the indices considered.

We describe a case of isthmus-dependant atrial flutter ablation with a superior approach due to an anomalous inferior vena cava (IVC), azygos, and emiazygos continuation. A 56-year-old man was referred to our institution for the treatment of a common atrial flutter. Two years prior, the patient had an attempted electrophysiological study not performed due to the inability to advance the guidewire through IVC. A computed tomography of the abdominal venous system revealed the absence of the subrenal IVC, azygos, and emiazygos continuation. Following double puncture of the left subclavian vein, two catheters were inserted into the coronary sinus and into the right atrium. Programmed atrial stimulation was performed and a sustained isthmus-dependant right atrial tachycardia was obtained. After 11 applications of radiofrequency energy with an inversion technique, sinus rhythm was restored and a repeat atrial programmed stimulation failed to induce any arrhythmia.

Background: There is increasing evidence that cardiac resynchronization therapy (CRT) may trigger an inverse remodeling process leading to decreased left ventricular (LV) volumes in patients with heart failure and wide QRS. However, it is still important to simplify patient selection and achieve a widely applicable parameter to better stratify patients who are candidates for CRT., Methods: Eighteen patients (13 males, 5 females, mean age 67.5 +/- 7.2 years) with advanced heart failure due to ischemic (n = 12) or idiopathic dilated cardiomyopathy (n = 6) and complete left bundle branch block received biventricular pacing. The patients were considered eligible in the presence of echocardiographic evidence of intra- and interventricular asynchrony, defined on the basis of LV electromechanical delay. Investigations were performed before pacemaker implantation (at baseline), the day after, and 3 and 6 months later., Results: Two patients died before the first outpatient examination. There were 15 (83%) responders to reverse remodeling among the remainder. In the overall population, there was a significant and progressive improvement in LV sphericity indexes, ejection fraction, mitral regurgitation area and LV volumes (p < 0.001). The improvement in the interventricular mechanical delay after CRT was significantly correlated with the decrease in LV end-systolic volume (r2 = 0.2558, p = 0.04)., Conclusions: CRT reduces LV volumes in patients with advanced heart failure, complete left bundle branch block and detailed documentation of ventricular asynchrony prior to therapeutic pacing. Broadly applicable Doppler echocardiographic measures may increase the specificity of the long-term response to CRT in terms of LV performance.

HSA at appropriate concentrations shows cytostatic and/or cytotoxic effects on murine Lewis carcinoma cell line C108. The cytostatic effect is mediated by an arrest in the cell cycle machinery, with accumulation of cells in G2-M. The combination of enzymatic assays, cell cycle kinetics studies and immunoprecipitation shows that HSA causes to a certainty an accumulation of cells in the M phase, while a similar effect in G2 has still to be demonstrated. It also inhibits histone H1 kinase activity up to 95% of that of mitotic cells, having as a direct or indirect target the cdc2 complex.

The in vitro effects of hydroxystearic acid on the proliferation of human colon carcinoma cells (HT29) and human embryonic intestine cells (I407) were examined and compared to previous results obtained in murine C108 lung carcinoma cells. The cells were cultured in the presence, or in the absence, of hydroxystearic acid and tested for cell proliferation and viability; the distribution of cells in the cell cycle was evaluated by flow cytometry. Results show that hydroxystearic acid is also an inhibitor of human cell proliferation, and not only of murine C108 cells. Differently from C108 cells, which upon treatment with hydroxystearic acid accumulate in G2-M phases, hydroxystearic acid-treated HT29 cells increase significantly in numbers in G0-G1; I407, embryonic cells used as a control, when treated show only a slight increase in G0-G1.

It was suggested that Doppler study of pulmonary venous flow (PVF) in combination with mitral flow is useful to evaluate ventricular function. In order to investigate the utility of PVF study in patients with total A-V block treated with pacemaker DDD, we performed transesophageal echocardiography pulsed Doppler of PVF and mitral flow in 12 patients. We measured the following parameters in PVF: peak systolic velocities (S1 and S2), peak diastolic velocity (D) reversal atrial flow diastolic deceleration time. In mitral flow we measured: E wave, A wave and deceleration time of E. We compared our results with the same patients with pacemaker inhibited. In patients with total A-V block with pacemaker inhibited we reported one systolic wave of PVF, a larger D wave with a shortened deceleration time. The PVA seems to disappear. Transmitral flow slowed a higher E wave in patients A-V block with pacemaker inhibited. In patients with total A-V block stimulated by DDD pacemaker we restored the atrioventricular sequentialty; therefore electric stimulation determines the onset of S1 peak and the reduction of S2 peak and the systematic presence of the atrial-pulmonary regurgitant wave.

Cardiac evaluation of stroke or transient ischemic attack (TIA) patients includes transthoracic and transesophageal echocardiography to identify potential cardiac source of embolism. In the present study transesophageal echocardiography was performed in 53 consecutive patients with non valvular atrial fibrillation and a previous embolic stroke. All patients were referred to our department because of an episode of atrial fibrillation. They all underwent computed tomography (CT) scan: 31 patients had a positive scan whereas 22 had a negative scan with a positive history of TIA diagnosed from a neurologist during hospitalization. In the group of 31 patients with a positive scan 9 patients with no history of a previous ischemic attack were found. The transesophageal echocardiography was performed in all the study subjects: 7 patients had a thrombus in the left atrial appendage, 2 patients had a thrombus in the right atrium. Spontaneous echo contrast was reported in 15 patients. Evaluating the interatrial septum we observed a patent foramen ovale in 4 patients, a defect in 2 patients and a fossa ovalis aneurysm in 3 patients. The mitral valve analysis showed a leaflet prolapse in 3 patients. The entire thoracic aorta was imaged in each patient: in 12 an abnormal atherosclerotic plaque was found. Transesophageal echocardiography is an important component of the comprehensive evaluation of potential sources of embolism in patients with ischemic cerebral attack and nonrheumatic atrial fibrillation.

The short-term clinical efficacies of furosemide, 25 mg/day, and torasemide, 10 mg/day, have been compared in 2 groups of 12 patients with severe heart failure. In each group there were similar numbers of patients in whom heart failure was due to coronary heart disease, high blood pressure, or idiopathic dilated cardiac myopathy. The design of the study was open and the diuretics were added to baseline treatment with digoxin. Treatment for 8 days with the 2 loop diuretics resulted in similar substantial and significant improvements in symptoms and exercise tolerance as judged by the increase in total body oxygen consumption, and reductions in radiographic evidence of pulmonary congestion and in right heart filling pressure as judged from the jugular venous pressure wave. The results of this short-term comparative study confirm that furosemide and torasemide are equally efficacious in inducing substantial improvements in the clinical status of patients in severe heart failure.

Doppler evaluation of pulmonary venous flow (PVF) and of mitral flow (MF) can be useful to understand left ventricular function. It is well known that left bundle branch block (LBBB) can influence ventricular function. The aim of the present study was to evaluate the role of LBBB induced by ventricular pacing on cardiac performance. We performed transesophageal echocardiography (TEE) in 10 patients affected by sick sinus syndrome (SSS) treated with a permanent pacemaker (VDD modality). We evaluated PVF and MF and compared the pattern from the same patients in sinus rhythm and during cardiac pacing. We observed an increase in the reversal flow in the pulmonary vein during atrial contraction. The diastolic flow of pulmonary vein increased during cardiac pacing. The MF showed an increase in deceleration time. Our results suggest that LBBB is associated with a reduction in ventricular function. This phenomenon induce a higher contribution of atrial contraction to ventricular function.

Lipid peroxidation products have recently been proposed among the possible regulators of tumour cell growth. According to our current working hypothesis, the greatly diminished content of such products in tumour cells might relieve the inhibition of cell growth thus leading to uncontrolled proliferation. Hydroperoxy- and hydroxy derivatives of long chain fatty acids have been identified and determined in normal and tumour cells. Among these, hydroxystearic acid (HSA) has been shown to have a different cytostatic and cytotoxic effect when administered to murine lung carcinoma cells or to human colon tumour cells. It interferes with cell cycle kinetics, blocking the murine cells in G2-M and the human ones in G0-G1. The molecular target of HSA in both cell lines has been shown to be the cdc2 kinase complex. The results so far obtained in tumour as long as in normal highly proliferating cells do not exclude a potential future use of this class of compounds as selective anti tumour drugs.

Whole cell lipids were extracted from the Lewis lung carcinoma in vitro line C108. The fatty acids were derivatized to methylesters in order to identify endogenous oxidized derivatives by gasmass spectroscopy. The presence of 9-hydroxystearic acid and 10-hydroxystearic acid was thus evidenced for the first time in cultured mammalian cells. Moreover a linear correlation was found between the concentration of these products expressed as percentage of total fatty acid methylesters and the cell density in tissue culture flasks. This finding suggests an involvement of hydroxystearic acid in cellular functions related to the cell density in monolayer cultures.

1. The content of oxy-radical scavenging enzymes is decreased in Morris hepatomas in a fashion which is inversely related with the growth rate of the tumour. 2. Hepatoma microsomal membranes are more resistant than normal rat liver membranes to lipid peroxidation induced in vitro by organic hydroperoxides or superoxide radicals. 3. In tumour membranes the most relevant rate-limiting factor of peroxidation is the low availability of polyunsaturated fatty acids (PUFA). Besides lipids, some proteins (particularly cytochrome P-450) act as controlling factors of peroxidation. 4. Tumour microsomes are more ordered and less fluid than liver microsomes. The latter, exposed to superoxide radical attack, exhibit chemical (fatty acid composition) and physical (molecular order) properties that are similar to those of transformed cell membranes. 5. These data indicate an aberration in the oxy-radical metabolism of cancer cells, and a sequence of events is hypothesized that could drive the transformed cell towards uncontrolled proliferation.

The relationship between steroid binding and protein subunit interactions of rabbit sex steroid binding protein (rSBP) has been studied by steady-state and time-resolved fluorescence spectroscopy. The high-affinity (Ka approximately 10(8) M-1 at 4 degrees C), fluorescent estrogen d-1,3,5(10),6,8-estrapentaene-3,17 beta-diol [dihydroequilenin (DHE)] was used as a fluorescent probe of the steroid-binding site. Perturbation of the binding site with guanidinium chloride (Gdm.Cl) was monitored by changes in the steady-state fluorescence anisotropy of DHE as well as by changes in fluorescence quenching of DHE with acrylamide. The results of acrylamide quenching at 11 degrees C show that, while between 0 and 1 M Gdm.Cl the steroid-binding site is completely shielded from bulk solvent, there is decreased DHE binding. To study the subunit-subunit interactions, rSBP was covalently labeled with dansyl chloride in the presence of saturating 5 alpha-dihydrotestosterone (DHT), which yielded a dansyl-conjugated protein that retained full steroid-binding activity. The protein subunit perturbation was monitored by changes in the steady-state fluorescence anisotropy of the dansyl group. At 11 degrees C, the dansyl anisotropy perturbation, reflecting changes in global and segmental motions of the dimer protein, occurs at concentrations of Gdm.Cl above 1 M. The Gdm.Cl titration in the presence of steroids with equilibrium association constants less than 10(8) M-1 shows a plateau near 3 M Gdm.Cl at 11 degrees C; at this Gdm.Cl concentration, no DHE is bound. No plateau is observed at 21 degrees C. At higher Gdm.Cl concentrations, the dansyl fluorescence anisotropy decreases further and shows no steroid dependence. Recovery of steroid-binding activity (assayed by saturation binding with [3H]DHT), under renaturation conditions, is dependent on both steroid concentration and affinity. Both unlabeled and dansyl-labeled protein recovery the same amount of activity, and according to fluorescence anisotropy, dansyl-labeled rSBP re-forms a dimer upon dilution below 1 M or removal of Gdm.Cl. From the steroid requirement for recovery of steroid-binding activity, it appears that a conformational template is required for the dimeric protein to re-form a steroid-binding site with native-like properties.

Reasons why nurses left a general Hospital were analyzed through a questionnaire sent to all nurses who abandoned their position over the last 5 years (218). 50.6% of these nurses do not live in Lombardy region. 127 (57.7%) answered the questionnaire. 35% of whom answered the questionnaire would get back to work in the hospital. The main reason for hospital leave is related to family reasons (children and parents) for registered nurses and to shift work state enrolled nurses. Low salary is the main problem for 21-30 years old nurses. According to nurses who answered the questionnaire, more efficient recruitment and retention strategies should include higher salaries, riqualification of the profession in general and improvement of working conditions and organization.

Fluorescence studies are reported on gramicidin A' incorporated into lysophosphatidylcholine phospholipid structures. The shift in the emission maximum during incorporation and the quenching of fluorescence by I- and by acrylamide of the incorporated state obtained after prolonged heating are consistent with the presence of the channel state comprised of two single-stranded beta 6 -helices associated head-to-head (formyl end-to-formyl end). The quantum yield for the incorporated state, when gramicidin A' is within the lipid matrix, is very low and indicates the occurrence of intermolecular Trp-Trp interactions. Possible interactions between channels within the lipid matrix are discussed utilizing Trp-Trp contacts.

The effect of the insertion of coenzyme Q10 and some of its shorter chain homologues in membrane models (Reverse Micelles, Small Unilamellar Vesicles and Liposomes) has been studied by NMR and IR spectroscopies. By 1H-NMR we have found that the stretched conformation of the isoprenoid side-chain observed in solution is maintained in membrane models. Interaction between the quinonoid moiety of the Q's and the phosphatidic groups of the phospholipids has been evidenced by 31P-NMR. A large effect of this interaction on the water microdynamics and distribution around the charged groups of the phospholipids has been observed by measurements of 1H and 2H relaxation times and by infrared spectra. The 13C-NMR spectra of the backbone of the acyl chains of phospholipids does not seem to be influenced to a noticeable extent by the presence of the Q's.

Heat derived gramicidin A'/L-alpha-lysophosphatidylcholine complexes were separated on a sucrose gradient to form two fractions: Fraction A which had an approximately constant Gramicidin A' to phospholipid ratio of 8 to 10 lipid molecules per Gramicidin A' molecule and Fraction B which had a larger but variable ratio. Fluorescence and circular dichroism studies confirmed Fraction A to be a lipid-incorporated channel state. Electron microscopic studies, using uranyl acetate negative staining, showed fraction A to be a membranous state with the formation of bilayer vesicles, that is, the interaction of peptide and phospholipid micelles causes the lipid to reorganize into a bilayer structure. Freeze-fracture replicas of the channel incorporated state demonstrated the presence of a supramolecular organization of particles exhibiting a tendency to form rows with a 50-60 A periodicity along the row and with 70-80 A distance between rows. An idealized working model for the incorporated state is presented.

Proton NMR and 13C-NMR studies on the configuration of CoQn homologues show that the polyisoprenoid side-chain is in the all-trans configuration and confers a higher degree of rigidity to the quinones with respect to the acyl-chains of the phospholipids within the membrane bilayer. The quinonoid ring appears to be specifically involved in the redox function of the coenzyme while the side-chain length only affects the lipophilicity of the molecule. LIS data show that the ring strongly interacts with metals as a consequence of the high pi-electron density on the carbonyls that is somewhat larger on the carbonyl oxygen to alpha to the isoprenoid chain.

In the present paper we report a comparative study of physical properties and biochemical composition of isolated melanosomal membranes extracted from bovine eyes and from an equine spleen melanoma. Some biophysical characteristics of such membranes were obtained by steady-state and time resolved fluorescence spectroscopy using DPH as fluorescent probe. By these methods we have measured both static fluorescence polarization and fluorescence lifetimes and from the experimental data we have calculated the rotational correlation times by Perrin's equation. Since dynamic and static parameters, such as fluidity and molecular order, can be determined by these methods, the results are discussed on the basis of the recent theories of the role of the biochemical composition in the molecular structure and properties of membranes.