Investigation of de novo totally random biosequences, Part IV: Folding Properties of de novo, totally random RNAs

This work lies within the framework of a broader project aimed at exploring the realm of all possible folded polypeptides; the main question addressed here is whether the corresponding RNAs also assume a folded conformation. We present an investigation on the structural properties of de novo, totally random RNAs by means of the 'RNA Foster' assay. Experimental results show that all RNAs studied are folded at 37 degrees , so that fold seems to be a common feature of RNAs. Random RNAs' fold shows a surprising thermal stability with an average T(m) value at ca. 50 degrees which prompts the idea that thermo-stable structures might not be as rare as they are commonly thought to be. The results are discussed within the general framework of random RNA properties such as those that might have been produced in a prebiotic scenario.     

Searching for markers of Creutzfeldt-Jakob disease in cerebrospinal fluid by two-dimensional mapping

Differential proteomic analysis has been performed on the cerebrospinal fluid (CSF) of six healthy and six patients suffering form sporadic Creutzfeldt-Jakob disease (sCJD), age- and sex-matched, after immuno-subtraction of albumin and immunoglobulins. These maps have revealed 28 polypeptide chains differentially modulated in the sCJD samples, of which 10 appeared to be up-regulated, the remaining 18 being down-regulated. Among those, 13 could be identified upon digestion and MALDI-TOF, MS analysis. In addition, the strong modulation of cystatin C was also confirmed by immunoblot analysis and the highly altered level of the 14-3-3 proteins that escaped detection by 2-D mapping, could be assessed by Western blots and immuno-detection of monomeric and homo- and hetero-dimeric 14-3-3 isotypes. In search for a panel of potential markers for sCJD, we highlight cystatin C, 14-3-3 proteins, transferrin, ubiquitin, Apoliprotein J and perhaps some of the still unidentified, but strongly modulated polypeptide chains detected in the differential map.     

Sarcoptic mange in wild carnivores and its co-occurrence with parasitic helminths in the Western Italian Alps

Between 2001 and 2004, 229 foxes, 36 stone martens and 48 badgers from the western Italian Alps were examined for sarcoptic mange and for gastrointestinal helminths to investigate their prevalence and geographical distribution and to point out the existence of potential interactions among them. Sarcoptic mange was observed in 25.3±2.8% SE of foxes and in 5.6±3.8% SE of stone martens, while no badger was found infected. Helminths belonged to Cestoidea Cyclophillidea (3.0±1.1% SE), Nematoda Trichurida (Capillaria aerophila and Trichuris vulpis: 6.5±1.6% SE; Trichinella britovi: 3.0±1.1% SE), Ascaridida (12.2±2.2% SE) and Strongylida (6.9±1.7% SE). Sarcoptic mange infection and the presence of helminths proved to be associated, with mangy foxes showing significantly higher prevalence of both cestode and nematode (particularly Ascaridida) worms. Moreover, considering three clusters of parasites (S. scabiei, nematodes and cestodes), more foxes than expected hosted simultaneously 2 and 3 taxa. These evidences suggest the existence of some kind of interaction, whose modalities and implications are discussed in this paper.     

Congenital pulmonary lymphangiectasia

Congenital pulmonary lymphangiectasia (PL) is a rare developmental disorder involving the lung, and characterized by pulmonary subpleural, interlobar, perivascular and peribronchial lymphatic dilatation. The prevalence is unknown. PL presents at birth with severe respiratory distress, tachypnea and cyanosis, with a very high mortality rate at or within a few hours of birth. Most reported cases are sporadic and the etiology is not completely understood. It has been suggested that PL lymphatic channels of the fetal lung do not undergo the normal regression process at 20 weeks of gestation. Secondary PL may be caused by a cardiac lesion. The diagnostic approach includes complete family and obstetric history, conventional radiologic studies, ultrasound and magnetic resonance studies, lymphoscintigraphy, lung functionality tests, lung biopsy, bronchoscopy, and pleural effusion examination. During the prenatal period, all causes leading to hydrops fetalis should be considered in the diagnosis of PL. Fetal ultrasound evaluation plays a key role in the antenatal diagnosis of PL. At birth, mechanical ventilation and pleural drainage are nearly always necessary to obtain a favorable outcome of respiratory distress. Home supplemental oxygen therapy and symptomatic treatment of recurrent cough and wheeze are often necessary during childhood, sometimes associated with prolonged pleural drainage. Recent advances in intensive neonatal care have changed the previously nearly fatal outcome of PL at birth. Patients affected by PL who survive infancy, present medical problems which are characteristic of chronic lung disease.     

Construct validity of the Short Form-36 Health Survey and its relationship with BMI in obese outpatients

Objective: To investigate the construct validity of the Short Form‐36 (SF‐36) Health Survey questionnaire in obese patients. Research Methods and Procedures: Our series consisted of 1735 obese patients (age, 44.7 ± 11.0 years; 1346 women) consecutively enrolled in the QUOVADIS study, an observational multicenter study of obese treatment‐seeking outpatients. The construct validity of the SF‐36 was assessed by main component analysis. Age‐, gender‐, and education‐adjusted general linear models were used to investigate the relationship between BMI and SF‐36 domains or factors identified by main component analysis. Results: BMI was significantly associated with poor health‐related quality of life in all eight SF‐36 domains, and the strongest association was observed with physical activity. Main components analysis generated a six‐factor solution explaining 59% of the observed variance. BMI was strongly associated with factors based on the loading of items regarding the physical activity domain and factors based on role‐physical and role‐emotional items or general health and bodily pain items. In contrast, mental health‐, vitality‐, and social functioning‐based factors were not related to BMI. Discussion: In obese treatment‐seeking outpatients, the clustering of SF‐36 items in main components is not significantly different from the domain‐based approach generally used, thus confirming the robustness of such a generic questionnaire in this specific condition. However, the peculiar clustering of some SF‐36 items and their relationship with BMI suggest that the health‐related quality of life profile of subjects belonging to that population may be better described with alternative aggregations of the SF‐36 items or with disease‐tailored questionnaires.     

Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 microM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca(2+) ([Ca(2+)](i)). LMS in mercury-treated cells that had been pre-incubated with a specific Ca(2+)-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca(2+)-signaling pathway. Exposure to 20 microM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca(2+)](i), suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations <400 microM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca(2+)](I), therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.     

Nanoporous surfaces as harvesting agents for mass spectrometric analysis of peptides in human plasma

Silica-based nanoporous surfaces have been developed in order to capture low molecular weight peptides from human plasma. Harvested peptides were subjected to mass spectrometric analysis by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of detecting and assessing the bound molecules. Peptide profiles consisting of about 70 peaks in the range 800-10,000 m/z were generated. The method could allow detection of small peptides at ng/mL concentration levels, either in standard solutions or in plasma. The same molecular cutoff effect was observed for mixtures of standard proteins and peptides incubated with silicon-based nanoporous surfaces.     

Preparation and characterization of Eudragit Retard nanosuspensions for the ocular delivery of cloricromene

The purpose of this study was to improve the stability of cloricromene (AD6) in ophthalmic formulations and its drug availability at the ocular level. To this end, AD6-loaded polymeric nanoparticle suspensions were made using inert polymer resins (Eudragit RS100 and RL100). We modified the quasi-emulsion solvent diffusion technique by varying some formulation parameters (the drug-to-polymer ratio, the total drug and polymer amount, and the stirring speed). The chemical stability of AD6 in the nanosuspensions was assessed by preparing some formulations using (unbuffered) isotonic saline or a pH 7 phosphate buffer solution as the dispersing medium. The formulations were stored at 4°C, and the rate of degradation of AD6 was followed by high performance liquid chromatography (HPLC). The obtained nanosuspensions showed mean sizes and a positive surface charge (ζ-potential) that make them suitable for an ophthalmic application; these properties were maintained upon storage at 4°C for several months. In vitro dissolution tests confirmed a modified release of the drug from the polymer matrixes. Nanosuspensions prepared with saline solution and no or lower amounts of surfactant (Tween 80) showed an enhanced stability of the ester drug for several months, with respect to an AD6 aqueous solution. Based on the tecnological results, AD6-loaded Eudragit Retard nanoparticle suspensions appear to, offer promise as a means to improving the shelf life and bioavailability of this drug after ophthalmic application. Published: March 24, 2006     

Performance of serum-supplemented and serum-free media in IFNγ Elispot Assays for human T cells

The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories’ own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNγ-secretion.