Preliminary Evidence for Cell Membrane Amelioration in Children with Cystic Fibrosis by 5-MTHF and Vitamin B12 Supplementation: A Single Arm Trial

Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disorders in the Caucasian population caused by mutations of gene for the cystic fibrosis transmembrane conductance regulator (CFTR). New experimental therapeutic strategies for CF propose a diet supplementation to affect the plasma membrane fluidity and to modulate amplified inflammatory response. The objective of this study was to evaluate the efficacy of 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation for ameliorating cell plasma membrane features in pediatric patients with cystic fibrosis.A single arm trial was conducted from April 2004 to March 2006 in an Italian CF care centre. 31 children with CF aged from 3 to 8 years old were enrolled. Exclusion criteria were diabetes, chronic infections of the airways and regular antibiotics intake. Children with CF were supplemented for 24 weeks with 5-methyltetrahydrofolate (5-MTHF, 7.5 mg /day) and vitamin B12 (0.5 mg/day). Red blood cells (RBCs) were used to investigate plasma membrane, since RBCs share lipid, protein composition and organization with other cell types. We evaluated RBCs membrane lipid composition, membrane protein oxidative damage, cation content, cation transport pathways, plasma and RBCs folate levels and plasma homocysteine levels at baseline and after 24 weeks of 5-MTHF and vitamin B12 supplementation. In CF children, 5-MTHF and vitamin B12 supplementation (i) increased plasma and RBC folate levels; (ii) decreased plasma homocysteine levels; (iii) modified RBC membrane phospholipid fatty acid composition; (iv) increased RBC K(+) content; (v) reduced RBC membrane oxidative damage and HSP70 membrane association.5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF.ClinicalTrials.gov NCT00730509

Studio ed analisi del fosfoproteoma della membrana del globulo rosso patologico

Il recente sviluppo di nanotecnologie, di nuovi strumenti per l\u2019analisi e l\u2019elaborazione bioinformatica, la disponibilit\ue0 di enormi database facilmente accessibili, insieme al completo sequenziamento del genoma umano, hanno aperto nuovi orizzonti alla ricerca biomedica. Inoltre, lo sviluppo dell\u2019analisi con gene-micro-array1, che \ue8 in grado di valutare il profilo di espressione di 50.000-80.000 geni contemporaneamente, ha permesso di individuare nuovi geni coinvolti nella patogenesi di diverse malattie e ha senz\u2019altro evidenziato come il prodotto proteico finale spesso non corrisponda al profilo di espressione dei geni corrispondenti, indicando quindi l\u2019esistenza di eventi in grado di intervenire sulle proteine neo-sintetizzate, modificandone la struttura ed influenzandone la funzione come evidenziato dal divario tra genotipo e fenotipo in patologie di interesse medico. In questo contesto, le modificazioni post-traduzionali delle proteine (MPT) costituiscono gli eventi chiave che intervengono sulle funzioni delle proteine e sulle attivit\ue0 cellulari in cui in esse sono coinvolte. Alla luce di queste considerazioni, parte della ricerca biomedica si \ue8 rivolta allo studio delle proteine e delle loro possibili MPT in vari contesti fisiopatologici di biologia cellulare. Ad oggi si conoscono pi\uf9 di duecento tipi differenti di MPT2, tra cui si annoverano la glicosilazione, la solforilazione, la nitrosilazione e la fosforilazione. Tra queste MPT, alcune sono reversibili quali, ad esempio, le fosforilazioni e le nitrosilazioni ed in quanto tali sono importanti nelle diverse attivit\ue0 della cellula, nella regolazione di processi biologici e in risposta a stress cellulari. La fosforilazione delle proteine \ue8 posta sotto il controllo dell\u2019azione combinata di due classi di enzimi: (1) le protein kinasi, che catalizzano il trasferimento di un gruppo fosforile da un composto ad alta energia ad un residuo amminoacidico (fosforilazione); (2) le protein fosfatasi, che catalizzano l\u2019idrolisi del legame fosfoesterico (defosforilazione). Nelle cellule eucariotiche, la forma pi\uf9 diffusa di fosforilazione \ue8 la formazione di un fosfoestere con il gruppo R di tirosine o di serine o treonine3. La tirosina si trova 1000 volte meno fosforilata rispetto alla serina e 100 volte rispetto alle treonina. Esistono anche fosforilazioni che interessano altri tipi di residui amminoacidici come le istidine, le arginine, gli acidi aspartici, le cisteine e gli acidi glutammici, ma la loro funzione non \ue8 stata ancora caratterizzata. L\u2019importanza biologica della fosforilazione/defosforilazione a livello cellulare \ue8 sottolineata anche dall\u2019elevato numero di geni che codificano per kinasi o fosfatasi in differenti modelli cellulari. Si calcola, infatti, che circa il 2% dell\u2019intero genoma umano sia costituito da geni che codificano proteine appartenenti a queste due classi enzimatiche4,5. Le kinasi/fosfatasi e quindi la fosforilazione/defosforilazione delle proteine sono molto importanti nelle diverse attivit\ue0 cellulari quali: il metabolismo cellulare, il ciclo cellulare, il riarrangiamento delle proteine plasma-membrana, il movimento cellulare, la degradazione delle proteine, la formazione di complessi proteici, l\u2019apoptosi e la differenziazione cellulare. Inoltre, nelle cellule eucariotiche i cambiamenti nello stato di fosforilazione delle proteine sono parte integrante delle vie di trasduzione del signaling intracellulare in risposta a stimoli esterni, ed anche del signaling intercellulare. Studi condotti fino ad ora sul profilo di fosforilazione hanno permesso di identificare alcuni gruppi di proteine che pi\uf9 frequentemente, rispetto ad altre, sono interessate da cambiamenti dello stato di fosforilazione, in particolare: le proteine strutturali della membrana, le chaperonine, i fattori di trascrizione e i recettori di membrana. Si stima che il 30% di tutte le proteine cellulari siano fosforilate. Spesso la fosforilazione avviene in siti multipli con effetti distinti sulla funzione della proteina stessa. Ecco perch\ue9 difetti o alterazioni nei geni codificanti per kinasi/fosfatasi o alterazioni a carico di siti fosforilabili/defosforilabili delle proteine possono alterare cos\uec profondamente le funzioni cellulari da contribuire in modo determinante al divario osservato tra genotipo e fenotipo. Solo recentemente, grazie allo sviluppo di nuove tecnologie, supportato dall\u2019aumento delle conoscenze, si \ue8 potuti passare dallo studio di una singola proteina e del suo stato di fosforilazione, allo studio di complessi network di interazioni delle proteine che si esplicano attraverso la fosforilazione dei diversi effettori che vi fanno parte. Si \ue8 passati quindi allo studio del fosfoproteoma, ovvero dell\u2019insieme delle proteine fosforilate in un dato momento cellulare e/o a seguito di un determinato stress a cui la cellula \ue8 stata sottoposta. Nello studio del fosfoproteoma si utilizzano le cosiddette metodiche ad alto livello tecnologico ( o high-throughoutput) tra cui l\u2019elettroforesi bidimensionale su gradiente immobilizzato (2-DE) e la spettrometria di massa (MALDI-TOF). Tali metodiche possono essere associate in un approccio integrato (integrated proteomic) ad analisi in Westernblot con anticorpi specifici, per esempio anticorpi anti-fosfotirosina, che permettono di individuare spot differentemente tirosin-fosforilati in campioni messi a confronto. Successivamente all\u2019analisi di immagine, tali spot/proteine possono essere identificate con precisione utilizzando la spettrometria di massa e la ricerca in banca dati (es NCBI database).Non disponibil

Fraudhunter : a supervised fraud detection tool for Internet banking transactions

LAUREA MAGISTRALENowadays online bank services are used by customers every day. The huge numbers of transactions and accounts are a very rich target for fraudsters, which steal billions of euros every year. For this reason financial institutions must adopt fraud detection systems as countermeasures to react to frauds in time. Our goal is to develop a supervised fraud detection tool that helps the transaction analyst to investigate over suspect transactions. The problem can be defined as a classification task, but particular challenges exist in the domain of Internet banking fraud detection: it is dificult to obtain real large datasets, fraudulent transactions behaviours change over time, frauds are rare, misclassification costs are different. In this work we explore supervised algorithms and techniques in order to test and compare these methods on a real bank transactions dataset that we have obtained from an important Italian bank group. We focus on the optimization of a cost function which gives higher weights to fraudulent transactions. The result is a dynamic system that adapts itself. During training, it aggregates attributes per user for each transaction and builds a Random Forest classifier on the basis of analyst's feedback. We implement FraudHunter, a fully working prototype and a web application that can be effectively used in real contexts. We obtain overall good validation results and our system has very good metrics when compared with other works in literature. In particular it can detect up to 87% of frauds with a very low false positive rate of 0.3%

Problematiche relative alla maturazione e conservazione delle pere invernali. (Ripening and storage of winter pear: problems and solutions)

The pear can be considered one of the most important and worldwide spread fruit crops. Italy plays a primary role in this cultivar

Heat-Shock Protein-70, -27 and Peroxiredoxin-2 Show Chaperone Function in Sickle Red Cells: Evidences from Transgenic Sickle Cell Mouse Model.

Abstract Sickle cell disease (SCD) is a worldwide-distributed hereditary red cell (RBCs) disorder characterized by the pathologic hemoglobin-S (HbS). The acute clinical manifestations of SCD are strictly related to HbS polymerization, to generation of dense RBCs, to their interaction with the abnormal activated vascular endothelial cells and to amplified inflammatory response. To identify new biomarkers associated with acute SCD events, we studied the RBCs membrane proteome from human SCD erythrocytes (n=20) fractioned according to density compared to normal RBCs (n=20), we divided RBCs into 2 fractions: fraction 1 (F1) corresponding to the least dense RBCs (density &lt; 1.074) and fraction 2 (F2) corresponding to the densest RBCs (density &gt; 1.095). None of the patients were under hydroxyurea treatment and they did not receive transfusion in the sixmonths before the study. Bi-dimensional electrophoresis (2-DE) followed by MS-analysis of RBCs F1 and F2 from both control and SCD subjects was carried out and 65 proteins differently expressed were identified. We focused on molecular chaperones. In SCD RBCs, we found that the amount of HSP27, HSP70 and Prx-2 recruited to the membrane was higher than in controls, Prx-2 was present as monomers and dimmers that were more abundant in SCD RBCs than in normal controls. Then, we exposed F1 and F2 fractions from normal and SCD patients to in vitro cellular stress (deoxygenation). In sickle RBCs, deoxygenation induced increase membrane recruitment of HSP27 in F1, modulation of HSP70 membrane binding in F2; reduction of Prx-2 monomers with increase Prx-2 dimerization in F1, but slightly changes in Prx-2 monomers in F2. These data suggest that in SCD RBCs HSP-27, -70 and Prx-2 are recruited to the membrane in response to cellular stress, acting as molecular chaperones to assist proteins in regaining their functional conformation. In order to evaluate their role in vivo and because increase serum levels of HSP70 has been recently reported in few SCD patients, we used transgenic sickle cell SAD mice as a model for SCD and exposed to hypoxia (8%-O2) respectively for 2–12–48 hours, mimicking acute human SCD vaso-occlusive-crisis (VOCs). We studies SAD mice and wild-type mice (C57/B6, WT) divided into groups of 6 animals each. We evaluated HSP70, HSP27 and Prx-2 expression on RBCs membrane, hematological parameters, RBCs density, and cation content. We found that HSP70 and HSP27 bound to RBCs membrane respectively after 12 hours and 48 hours (hrs) of hypoxia, while Prx-2 membrane binding was modulated during hypoxia. At the different time schedule, we observed a marked reduction in red cell K+ at 12 and 48 hrs hypoxia, associated with significant increase of the dense RBCs at 48 hrs hypoxia. Our data indicate that in SCD, HSP70, HSP27 and Prx-2 membrane recruitment is modulated in vivo during acute VOCs, supporting their novel role as RBCs membrane protein protectors and as new markers of severity of RBCs membrane damage during acute sickle VOCs.</jats:p

Proteomics of Membrane from Human Sickle and Normal Fractioned Red Cells Identifies Different Expression of Stress-Response Proteins.

Abstract Sickle cell disease (SCD) is characterized by the presence of sickle hemoglobin (HbS), which has the unique property of polymerizing when deoxygenated and thereby triggering erythrocyte sickling and dehydration. Repeated HbS polymerization is associated with generation of dense, distorted red cells characterized by an extensive membrane oxidative damage and abnormal activation of membrane cation transport pathways. The recently developed proteomic technologies represent a useful tool for identification of new functional pathways in sickle red cells. We studied the membrane proteoma of sickle red cells from homozygous patients (SS), not carrying alpha-thalassemia, not undergoing hydroxyurea treatment and not transfused at least three months before the study. The buffy-coat was removed and the red cells were separated into two fractions corresponding to the reticulocytes enriched fraction and to the dense red cell fraction (n= 8). Following osmotic lysis, the red cell ghost proteins were separated by one- dimensional or by two-dimensional gel electrophoresis (1-DE, 2-DE). Using the ImageMaster 2D Platinum (5.0 Amersham-Biosciences) software the 2-DE gels from control and sickle cells were compared, allowing us to identify spots differently expressed in sickle cells compared to normal cells. The selected spots were excised and trypsinized before mass spectrometric analysis and compared with the SWISS-PROT and NCBI non-redundant databases. In sickle reticulocytes enriched fraction compared to the control one, we selectively identified the following cystokeleton proteins: four spots for beta actin, two spots for myosin light chain and a train of four proteins identified as protein 4.1, one of which was phosphorylated on Tyr13, a protein similar to flotillin 2, carbonic anhydrase I and the following stress-response proteins: heat shock protein 70 (HSP70) protein 9B, HSPC108 and peroxiredoxin II (PrxII), which was phosphorylated in Thr142. In dense sickle red cell fraction compared to normal one we selectively identified a train of two spots for 4.1R, band 3 and aldolase and two spots identified as HSP70 protein 5. The identified proteins were then confirmed by western-blotting analysis. These data suggest that in sickle red cells HSP70, HSP108 function may be required by the extensive assistance in the refolding of misfolded proteins most likely related to abnormal sickle red cell oxidative environment as also supported by the presence of phosphorylated peroxiredoxin II, which has been recently proposed as regulator of redox-sensitive signaling in other cell types.</jats:p

BAND 3CEINGE (Gly796Arg) Mutation Causes Dehydrated Hereditary Stomatocytosis (DHS) with Dyserythropoietic Phenotype

Abstract Stomatocytosis is an inherited autosomal dominant hemolytic anemia and includes overhydrated hereditary stomatocytosis (OHS), dehydrated hereditary stomatocytosis (DHS), hereditary cryohydrocytosis (CHC) and familial pseudohyperkalemia (FP). Here, we report a novel variant of hereditary stomatocytosis due to a de-novo band 3 mutation due to G&amp;gt;A transition at nucleotide 2500 in exon 17 (p. G796R, band3CEINGE) associated with dyserythropoietic phenotype. This 43-years-old Caucasian female (II-2) with unrelated parents was admitted to our hospital for mild anemia evaluation. The patient was in good health until 7 years when she frequently experienced asthenia. Anemia was first recognized at the age of eighth years with presence of jaundice and hyperchromic urine, but she had never received blood transfusions. We observed a mild hypochromic macrocytic anemia with a hemoglobin level of 11.5 g/dL, a mean cell volume (MCV) of 110 fL, and a mean hemoglobin concentration (MCH) of 36.1 pg, the reticulocyte count was 64 × 103/μL. There was a typical hemolytic features: high levels of indirect bilirubin (3.48 mg/dL) and lactate dehydrogenase ( 567 U/l, v.n. 240– 480 U/l ) with negativity at direct and indirect Coomb’s test. Spleen was enlarged and ultrasonography detected 15 cm of longitudinal size. She was cholecystectomized at the age of 14 years because of numerous symptomatic small stones. Serum iron, soluble transferrin receptor, serum ferritin and transferrin saturation levels were all increased, while the transferrin was in the normal range.Other blood tests including osmotic fragility with incubated and fresh erythrocytes, serum electrolytes, B12 and folate levels, erythrocyte enzyme levels, EMA test and Pink test were normal. Peripheral blood smear showed anisopoikilocytosis with rare stomatocytes and no spherocytes. Bone marrow aspirate showed remarkable dyserythropoiesis with increased number of erythroblasts and binucleate erythroblasts, basophilic erythroblasts with alterations, irregular nuclei maturation, intererythroblastic bridges and erythroblasts with basophilic stippling. She received since the age of 14 yrs a diagnosis for congenital dyserythropoietic anemia type I. Patients red cells showed increase Na+ content and decrease K+ content; reduced Na-K pump activity and increased Na-H exchange, NKCC cotransport and KCC cotransport activities. We then functionally characterized band 3 CEINGE in Xenopus oocytes, showing that the mutated band 3 is converted from anion exchanger (Cl−, HCO3 −) function to unregulated cation pathway for Na+ and K+. The mutated band 3 was also associated with increased tyrosine phosphorylation pattern of some red cell membrane proteins. During erythropoiesis band 3 protein is the last cytoskeletal protein to appear, thus the dyserythropoietic phenotype may be related to a possible role of the mutated band 3 in perturbation of cytoskeleton assembly in the late stage of erythropoiesis, allowing us to conclude for a new variant of stomatocytosis with dyserythropoietic phenotype.</jats:p

A novel erythroid anion exchange variant (Gly796Arg) of hereditary stomatocytosis associated with dyserythropoiesis

Stomatocytoses are a group of inherited autosomal dominant hemolytic anemias and include overhydrated hereditary stomatocytosis, dehydrated hereditary stomatocytosis, hereditary cryohydrocytosis and familial pseudohyperkalemia. This article describes a novel variant of hereditary stomatocytosis due to a de novo band 3 mutation associated with signs of dyserythropoiesis. See related perspective article on page 1039