Rossana Rossi

Reliable typing of systemic amyloidoses through proteomic analysis of subcutaneous adipose tissue

Considering the important advances in treating specific types of systemic amyloidoses, unequivocal typing of amyloid deposits is now essential. Subcutaneous abdominal fat aspiration is the easiest, most common diagnostic procedure. We developed a novel, automated approach, based on Multidimensional Protein Identification Technology, for typing amyloidosis. Fat aspirates were obtained from patients with the most common systemic amyloidoses (ALλ, ALκ, transthyretin, and reactive amyloidosis), with Congo red score more than or equal to 3+, and nonaffected controls. Peptides from extracted and digested proteins were analyzed by Multidimensional Protein Identification Technology. On semiquantitative differential analysis (patients vs controls) of mass spectrometry data, specific proteins up-represented in patients were identified and used as deposit biomarkers. An algorithm was developed to classify patients according to type and abundance of amyloidogenic proteins in samples; in all cases, proteomic characterization was concordant with fibril identification by immunoelectron microscopy and consistent with clinical presentation. Our approach allows reliable amyloid classification using readily available fat aspirates.

Proteomics of bronchial biopsies: Galectin-3 as a predictive biomarker of airway remodelling modulation in omalizumab-treated severe asthma patients

Asthma is a chronic inflammatory disease. Reticular basement membrane (RBM) thickening is considered feature of airway remodelling (AR) particularly in severe asthma (SA). Omalizumab, mAb to IgE is effective in SA and can modulate AR. Herein we describe protein profiles of bronchial biopsies to detect biomarkers of anti-IgE effects on AR and to explain potential mechanisms/pathways. We defined the bronchial biopsy protein profiles, before and after treatment. Unsupervised clustering of baseline proteomes resulted in very good agreement with the morphometric analysis of AR. Protein profiles of omalizumab responders (ORs) were significantly different from those of non-omalizumab responders (NORs). The major differences between ORs and NORs lied to smooth muscle and extra cellular matrix proteins. Notably, an IgE-binding protein (galectin-3) was reliable, stable and predictive biomarker of AR modulation. Omalizumab down-regulated bronchial smooth muscle proteins in SA. These findings suggest that omalizumab may exert disease-modifying effects on remodelling components.

Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry of bilobalide in plasma and brain of rats after oral administration of its phospholipidic complex

Standardized extracts of Ginkgo biloba L. leaves are widely used in clinical practice for the symptomatic treatment of mild to moderate dementia syndromes, cerebral insufficiency and for the enhancement of cognitive function. The main active components present in G. biloba extracts are flavonol-glycosides and terpene-lactones. In recent investigations, the sesquiterpene trilactone bilobalide has been described to exert an interesting neuroprotective effect when administered systemically to experimental animals. Oral administration of terpene-lactones either as standardized extracts or purified products is characterized by a low bioavailability. While preparing phospholipidic complex of G. biloba extracts or bilobalide, plasma levels of terpenes and sesquiterpene increase. In the present study, phospholipidic complex of bilobalide (IDN 5604) has been administered orally to rats and bilobalide levels have been determined in plasma and brain by means of a validated method based on liquid chromatography coupled to atmospheric pressure chemical ionization ion trap mass spectrometry (LC/APCI-ITMS). Due to its sensitivity (about 3pmol/ml) and specificity, LC/APCI-ITMS method proved to be a very powerful tool for pharmacokinetic studies of Ginkgo terpene-lactones. The results of the present study clearly confirm the improvement of oral bioavailability of bilobalide administered as phospholipidic complex and, for the first time, demonstrate the detection of significative amounts of bilobalide in brain. This last finding agrees with the neuroprotective activity observed for bilobalide.

Decreased expression of the Augmenter of Liver Regeneration results in increased apoptosis and oxidative damage in human-derived glioma cells

The mammalian growth factor erv1-like (GFER) gene encodes a sulfhydryl oxidase enzyme, named Augmenter of Liver Regeneration (ALR). Recently it has been demonstrated that ALR supports cell proliferation acting as an anti-apoptotic factor. This effect is determined by ALR ability to support the anti-apoptotic gene expression and to preserve cellular normoxic conditions. We recently demonstrated that the addition of recombinant ALR (rALR) in the culture medium of H2O2-treated neuroblastoma cells reduces the lethal effects induced by the hydrogen peroxide. Similar data have been reported in the regenerating liver tissue from partially hepatectomized rats treated with rALR. The purpose of the present study was to evaluate the effect of the GFER inhibition, via the degradation of the complementary mRNA by the specific siRNA, on the behaviour of the apoptosis (apoptotic gene and caspase expression and apoptotic cell number) and of the oxidative stress-induced parameters (reactive oxygen species (ROS), clusterin expression and mitochondrial integrity) in T98G glioma cells. The results revealed a reduction of (i) ALR, (ii) clusterin and (iii) bcl-2 and an increase of (iv) caspase-9, activated caspase-3, ROS, apoptotic cell number and mitochondrial degeneration. These data confirm the anti-apoptotic role of ALR and its anti-oxidative properties, and shed some light on the molecular pathways through which ALR modulates its biological effects.

Biomarker discovery in asthma and COPD by proteomic approaches.

Asthma and chronic obstructive pulmonary disease (COPD) are multifactorial respiratory diseases, characterized by reversible and irreversible airway obstruction, respectively. Even if the primary causes of these diseases remain unknown, inflammation is a central feature that leads to progressive and permanent pulmonary tissue damage (airway remodeling) up to the total loss of lung function. Therefore, the elucidation of the inflammation mechanisms and the characterization of the biological pathways, involved in asthma and COPD pathogenesis, are relevant in finding new possible diagnostic/prognostic biomarkers and for the validation of new drug targets. In this context, current advances in proteomic approaches, especially those based on MS, provide new tools to facilitate the discovery‐driven studies of new biomarkers in respiratory diseases and improve the clinical reliability of the next generation of biomarkers for these diseases consisting of multiple phenotypes. This review will report an overview of the current proteomic methods applied to the discovery of candidate biomarkers for asthma and COPD, giving a special emphasis to emerging MS‐based techniques.

Orthoformimycin, a selective inhibitor of bacterial translation elongation from Streptomyces containing an unusual orthoformate.

Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-G-dependent mRNA translocation is inhibited by orthoformimycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected.

Chrolactomycins from the actinomycete actinospica.

Examination of the metabolites produced by an Actinospica strain led to the identification of 6-hydroxychrolactomycin (compound 1), which is produced along with minor amounts of chrolactomycin (compound 2). The structure of 1 was established on the basis of extensive spectroscopic analysis, including one- and two-dimensional NMR. Compound 1 showed antimicrobial activity against Gram-positive bacteria, although it was generally less active than 2.

Poster 1002: Galectin-3 as predictive biomarker of airways remodeling modulation in omalizumab treated severe asthma patients

Background A significant effect of Omalizumab treatment on bronchial remodeling modulation(reduction of Reticular Basal Membrane-RBMthickening) by means of histological evaluation of bronchial biopsies was documented after therapy (Riccio et al. 2012). But we surprisingly found two groups , Responders-R (reduction of tickening of RBM) and Non Responders-NR (increasing or stable RBM). We then applied the proteomic analysis to the bronchial specimens. We used MudPIT (Multidimensional Protein Identification Technology) proteomic approach, a highthroughput methodology that allows the identification of hundreds/thousands of proteins for a single complex sample, evaluation of differential abundance, characterization of involved molecular pathways and sub-typing diseases (Brambilla et al., 2012).

Analytical Methods for Characterizing Bioactive Terpene Lactones in Ginkgo Biloba Extracts and Performing Pharmacokinetic Studies in Animal and Human

Ginkgo biloba is an ancient Chinese tree, appeared more than 250 million years ago, and the only surviving member of Ginkgoacea family [Schmid, 1997]. Ginkgo biloba was used as herbal remedy for many centuries in China, and now its extracts are one of the most widely used herbal products in the world, especially in the United States and in Europe [Blumenthal, 2000; Mahadevan & Park, 2008]. Ginkgo biloba extract is considered an alternative medicine for the treatment and/or the prevention of different pathologies and in some cases it could be suggested to be used as complementary of the mainstream medicine [Ernst, 2000]. In fact, over the past decades, there was a steady growth trend in the use of these alternative treatments. In particular, concentrated and partially purified products, containing Ginkgo biloba active constituents, have been marketed widely in the world for the treatment of cognitive deficits and other age-associated impairments [Kanowski et al., 1996; Le Bars et al., 1997]. Furthermore, it has been used as therapeutic compound for many other chronic and acute forms of diseases such as cardiovascular and bronchial pathologies [Diamond et al., 2000]. In view of the large market as well as the keen interest in the use and rediscovery of these herbal products throughout the world, the quality control of Ginkgo biloba extracts becomes necessary, in order to guarantee their clinical efficacy and safety. Therefore, it is important to monitor simultaneously the bioactive constituents present in Ginkgo biloba extracts, optimizing the analysis time and reducing costs. In fact, in the recent years, numerous groups reported in literature different analytical methods, using various chromatographic conditions and spectophotometric technologies, to create quick, accurate and applicable analytical approaches for the identification and the chemical structure characterization of Ginkgo biloba constituents. Ginkgo biloba extracts contain a large number of representative constituents such as terpenoids, polyphenols, allyl phenol, organic acids, carbohydrates, fatty acids and lipids, inorganic salts and amino acids. However, the pharmacological activity of Ginkgo biloba