IN VITRO FERROPORTIN EXPRESSION IN NON-TRANSFUSION DEPENDENT THALASSEMIA DURING ERYTHROID DIFFERENTIATION

INTRODUZIONE Le \u3b2-talassemie sono una delle malattie genetiche pi\uf9 frequenti in tutto il mondo con 270 milioni di portatori e 350.000 nuovi nati affetti all\u2019anno. Questa malattia \ue8 geneticamente caratterizzata dalla perdita di produzione della catena \u3b2 globinica dell'emoglobina adulta, dovuta a diverse mutazioni nel gene della \u3b2-globina. Poich\ue9 il gene beta \ue8 espresso su entrambi i cromosomi 11, possiamo avere due differenti tipi (e con differente gravit\ue0) di beta talassemia a seconda dell\u2019assenza di entrambi o di un solo gene della beta globina: nel primo caso si ha la \u3b2 talassemia MAJOR o trasfusione dipendente, nel secondo caso si ha la \u3b2 talassemia MINOR o INTERMEDIA trasfusione indipendente. I nostri studi si concentrano su quest\u2019ultima. L'assenza della catena \u3b2 globina comporta diverse conseguenze per l'organismo come: - Eritropoiesi inefficace - Il sovraccarico di ferro - Il danno ossidativo Finora sono stati condotti molti studi in diversi campi (genomico, proteomico e nel metabolismo ferro) per garantire una maggiore comprensione di questa malattia. Recentemente si \ue8 scoperta una nuova proteina che potrebbe essere un eventuale regolatore o responsabile del sovraccarico di ferro nella \u3b2 - talassemia; questa molecola \ue8 la ferroportina. La Ferroportina (FPN) \ue8 l'unico esportatore di ferro finora conosciuto. Essa \ue8 espressa in diversi tipi di cellule, tra cui gli enterociti duodenali, gli epatociti, i macrofagi e gli eritroblasti. Pochi anni fa, \ue8 stata segnalata l'esistenza di due trascritti alternativi della FPN con o senza le Iron Responsive Elements (IRE) sul loro promotore (FPN1A e FPN1B rispettivamente). L'espressione delle diverse isoforme della ferroportina nonch\ue9 i meccanismi che la regolano nelle cellule eritroidi della \u3b2 talassemia non-trasfusione dipendente (NTDT) non sono ancora noti. SCOPO Studiare il profilo di espressione delle due isoforme della ferroportina durante il differenziamento eritroide in colture controllo e di NTDT e chiarire i meccanismi che regolano la loro espressione. MATERIALI E METODI Per questi studi \ue8 stato usato un modello di eritropoiesi in vitro derivato da cellule CD34+ provenienti da sangue periferico di volontari sani (controllo) e pazienti NTDT. Il profilo dell'espressione genica delle due isoforme (FPN1A e FPN1B) \ue8 stato valutato allo stadio basale (giorno 0) e al giorno 7 e 14 della cultura (stadio di pro eritroblasti e di eritrociti ortocromatici rispettivamente) mediante la tecnica di real-time PCR (2-dCt). La percentuale relativa di ogni isoforma \ue8 stata calcolata sulla base dell\u2019espressione della ferroportina totale (FPN1A + FPN1B). La concentrazione di ferro intra and extracellulare \ue8 stata analizzata utilizzando un kit di Ferro Assay (Biovision). In esperimenti indipendenti, colture di controllo e NTDT sono state trattate con: ferro (Ferro Ammonio Citrato [FAC] 100\u3bcM), Desferal (DFO, 4\u3bcM), protoporfirina (SNPP IX 50-20\u3bcM), eme (Emina 20-10\u3bcM) o perossido di idrogeno (H2O2 0,1mM) per indagare su un possibile ruolo di questi composti nella regolazione della ferroportina. L\u2019espressione della FPN \ue8 stata valutata al 14esimo giorno in condizioni standard e nei trattati mediante la tecnica di real-time PCR (2-ddCt; cellule non trattate utilizzate come calibratore). RISULTATI L'espressione ferroportina aumenta durante il differenziamento eritroide, raggiungendo il livello massimo di espressione allo stadio di eritroblasti (giorno 14 di coltura) sia nel controllo sia negli NTDT. La FPN1A \ue8 l'isoforma pi\uf9 espressa in entrambe le condizioni. La sua espressione \ue8 pi\uf9 elevata negli stadi iniziali e finali dell\u2019eritropoiesi (giorno 0 e 14), mentre l'espressione della FPN1B \ue8 maggiore nella fase intermedia di differenziamento eritroide (giorno 7). Degno di nota, l'espressione della FPN1B, anche se inferiore rispetto alla 1A, \ue8 significativamente maggiore nelle culture NTDT rispetto ai controlli, in particolare al giorno 14. La concentrazione di ferro intracellulare \ue8 diminuita in modo significativo durante il differenziamento eritroide (dal giorno 7 al giorno 14), sia nei controlli sia negli NTDT, tuttavia, al giorno 7 (stadio di eritroblasti) i livelli di ferro nelle culture NTDT sono notevolmente inferiori rispetto ai controlli. L'aggiunta di FAC, DFO, SnPP IX ed Emina nei controlli e nelle colture di NTDT non ha modificato l'espressione della ferroportina rispetto ai non trattati. L\u2019H2O2 aggiunto ai controlli aumenta l'espressione di entrambe le isoforme della ferroportina (FPN1A: cellule non trattate: 1; H2O2: 1.33 FPN1B: cellule non trattate: 1; H2O2: 2.04). I livelli di ferro intra ed extracellulari riflettono i risultati genetici: c'\ue8 stato un aumento di ferro extracellulare causa di un aumento di espressione FPN. CONCLUSIONI L'espressione della ferroportina aumenta durante il differenziamento eritroide sia nei controlli sia nelle culture NTDT, suggerendo il suo ruolo nell\u2019esportare il ferro intracellulare in eccesso. In entrambe le condizioni, la FPN1A \ue8 l'isoforma pi\uf9 espressa. Tuttavia, l'espressione dell\u2019isoforma 1B non responsiva al ferro, anche se minore rispetto a FPN1A, \ue8 significativamente maggiore nei NTDT rispetto ai CTRL. In colture di controllo, l\u2019espressione della FPN, ed in particolare dell\u2019isoforma 1B, sembra essere regolata dall\u2019aggiunta di H2O2. Questi dati suggeriscono che lo stress ossidativo, particolarmente elevato nelle NTDT, potrebbe essere uno dei principali regolatori dell\u2019espressione dell\u2019isoforma 1B, generando cos\uec un\u2019importante esportazione di ferro dalle cellule NTDT.INTRODUCTION \u3b2-Thalassemias are one of the most frequent genetic disorders worldwide with 270 million of carriers and 350.000 affected new-borns per year. This disease is genetically characterized by the loss of production of the \u3b2 globin chain of the adult haemoglobin, due to several mutation within the beta globin gene. Since the beta gene is expressed on both the chromosomes 11, we can have two different type (and severity) of beta thalassemia depending on the absence of both or just one beta gene: in the first case we have the \u3b2 thalassemia MAJOR transfusion dependent, in the second case we have the \u3b2 thalassemia MINOR or INTERMEDIA, transfusion independent. Our studies are focused on the last one. The absence of the \u3b2 globin chain implies different consequences for the organism like as: - Ineffective erythropoiesis - Iron overload - Oxidative damage Many studies have been conducted so far in different fields (genomic, protein expression and regulation, iron metabolism) in order to guarantee a major comprehension of this disease. Recently a new protein came out as a possible regulator/responsible for the iron overload in \u3b2 thalassemia; this molecule is the FERROPORTIN. Ferroportin (FPN) is the only know iron exporter protein. It is expressed in different cell types including duodenal enterocytes, hepatocytes, macrophages and erythroblast cells. Few years ago it has been reported the existence of two alternative transcripts of FPN with or without an iron \u2013 responsive element (IRE) on their promoter (FPN1A and FPN1B respectively). The expression of the different ferroportin isoforms as well as the mechanisms regulating their expression in erythroid cells in non-transfusion dependent \u3b2 thalassemia syndromes (NTDT) are not known yet. AIM To investigate the expression profile of ferroportin isoforms during erythroid differentiation in control and NTDT cell cultures and to elucidate the mechanisms regulating their expression. MATERIALS AND METHODS An in vitro model of erythropoiesis derived from human peripheral CD34+ cells from healthy volunteers (control) and NTDT patients was used. The expression profiling of FPN isoforms (FPN1A and FPN1B) was evaluated at baseline (day 0) and at day 7 and 14 of culture (pro erythroblasts and orthochromatic erythroblasts stage respectively) by real 12time PCR (2 12dCt). The relative percentage of each isoform was calculated based on total ferroportin expression (FPN1A+FPN1B). The intracellular iron concentration was analyzed by using an Iron Assay Kit (Biovision). In independent experiments, control and NTDT cultures were treated with iron (Ferric Ammonium Citrate [FAC] 100\ub5M), Desferal (DFO, 4\ub5M), protoporfirin (SnPP IX 50-20\ub5M), heme (Hemin 20-10\ub5M) or hydrogen peroxide (H2O2 0.1mM) to investigate a possible role of these compounds in ferroportin regulation; FPN expression was evaluated at day 14 in standard and treated conditions by real 12time PCR (2 12ddCt; untreated cells used as calibrator). RESULTS The ferroportin expression increased during erythroid differentiation; with the highest level at the end of erythroblasts stage (day 14 of cultures) both in control and NTDT cultures. The FPN1A was the more expressed isoform in both conditions. Its expression was higher at the initial and final steps of erythropoiesis (day 0 and 14), while FPN1B expression was higher at the intermediate erythroblast stages (day 7). Noteworthy, the FPN1B expression, although lower compared to FPN1A, was significantly higher in NTDT cultures than in control ones, particularly at day 14. The intracellular iron concentration decreased significantly during erythroid differentiation (from day 7 to day 14) both in control and NTDT cultures, however, at day 7 (early erythroblasts stage) the iron levels in NTDT cultures were notably lower than in controls. The addition of FAC, DFO, SnPP IX and Hemin in control and NTDT cultures did not modify the ferroportin expression compared to untreated. H2O2 added to control cells increased the expression of both ferroportin isoforms (FPN1A: untreated cells: 1; H2O2: 1.33. FPN1B: untreated cells: 1; H2O2: 2.04). The intra and extracellular iron levels reflected the genetic results: there was an increase of extracellular iron due to an increase of FPN expression. CONCLUSIONS The ferroportin expression increases during erythroid differentiation either in control than in NTDT cultures, suggesting its role in exporting the excess intracellular iron. In both conditions, the FPN1A is the more expressed isoform. However, the expression of the non 12iron responsive FPN1B isoform, although lower compared to FPN1A, is significantly higher in NTDT than in control conditions. In control cultures, FPN expression, and particularly the FPN1B isoform, seems to be up regulated by H2O2 addition. These data suggest that the oxidative stress, notably higher in NTDT conditions, could be one of the major regulator of FPN1B expression, with a major iron export from NTDT erythroblast cells

Improving Fission-product Decay Data for Reactor Applications: Part I -- Decay Heat

Effort has been expended to assess the relative merits of undertaking further decay-data measurements of the main fission-product contributors to the decay heat of neutron-irradiated fissile fuel and related actinides by means of Total Absorption Gamma-ray Spectroscopy (TAGS/TAS) and Discrete Gamma-ray Spectroscopy (DGS). This review has been carried out following similar work performed under the auspices of OECD/WPEC-Subgroup 25 (2005-2007) and the International Atomic Energy Agency (2010, 2014), and various highly relevant TAGS measurements completed as a consequence of such assessments. We present our recommendations for new decay-data evaluations, along with possible requirements for total absorption and discrete high-resolution gamma-ray spectroscopy studies that cover approximately 120 fission products and various isomeric states.Comment: Submitted to European Physical Journal

Shell model in the complex energy plane and two-particle resonances

An implementation of the shell-model to the complex energy plane is presented. The representation used in the method consists of bound single-particle states, Gamow resonances and scattering waves on the complex energy plane. Two-particle resonances are evaluated and their structure in terms of the single-particle degreees of freedom are analysed. It is found that two-particle resonances are mainly built upon bound states and Gamow resonances, but the contribution of the scattering states is also important.Comment: 20 pages, 9 figures, submitted to Phys.Rev.

North Atlantic mid-latitude surface-circulation changes through the Plio-Pleistocene intensification of northern hemisphere glaciation

This is the final version. Available from American Geophysical Union (AGU) / Wiley via the DOI in this record.All new data presented are available at https://doi.pangaea.de/10.1594/PANGAEA.892805The North Atlantic Current (NAC) transports warm salty water to high northern latitudes, with important repercussions for ocean circulation and global climate. A southward displacement of the NAC and Subarctic Front, which separate subpolar and subtropical water masses, is widely suggested for the last glacial maximum (LGM) and may have acted as a positive feedback in glacial expansion at this time. However, the role of the NAC during the intensification of northern hemisphere glaciation (iNHG) ~3.5 to 2.5 Ma, is less clear. Here, we present new records from IODP Site U1313 (41°N) spanning ~2.8-2.4 Ma to trace the influence of Subarctic Front waters above this mid-latitude site. We reconstruct surface and permanent pycnocline temperatures and seawater δ18O using paired Mg/Ca-δ18O measurements on the planktic foraminifers Globigerinoides ruber and Globorotalia crassaformis, and determine abundances of the subpolar foraminifer Neogloboquadrina atlantica. We find that the first significant glacial incursions of Subarctic Front surface waters above Site U1313 did not occur until ~2.6 Ma. At no time during our study interval was (sub)surface reorganisation in the mid-latitude North Atlantic analogous to the LGM. Our findings suggest that LGM-like processes sensu stricto cannot be invoked to explain interglacial-glacial cycle amplification during iNHG. They also imply that increased glacial productivity at Site U1313 during iNHG was not only driven by southward deflections of the Subarctic Front. We suggest nutrient injection from cold-core eddies and enhanced glacial dust delivery may have played additional roles in increasing export productivity in the mid-latitude North Atlantic from 2.7 Ma.Funding for this research was provided by IODP France (CTB) and the German Research Foundation (DFG) (grant OF 2544/2 to OF). IB is grateful to the UK IODP for financial support for shipboard and post-cruise participation in IODP Exp. 306. CTB, KT, TDG, LV, CS, and ME acknowledge OSU Pythéas. MMR acknowledges support by the USGS Land Change Science Program. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. PAW acknowledges NERC UK IODP NE/F00141X/1 and a Royal Society Wolfson Merit Award