ESPON Project 2.3.1., Application and effects of the ESDP in Member States. First Interim Report

This First Interim Report includes the initial results of the project “Application and Effects of the ESDP in the Member States” within the ESPON Programme 2000-2006. The focus of the study is the application of the European Spatial Development Perspective (ESDP), which was adopted at the Potsdam European Council meeting in May 1999

Chronostratigraphy and paleomagnetism of Oligo-Miocene deposits of Corsica (France) : geodynamic implications for the liguro-provençal basin spreading

Dans un contexte de convergence entre Afrique et Europe, le domaine méditerranéen occidental est caractérisé par l’ouverture du bassin liguro-provençal au Miocène inférieur et de la mer Tyrrhénienne à partir du Miocène moyen. Ces ouvertures sont précédées par un épisode de rifting oligocène. De nouveaux résultats biostratigraphiques, géochronologiques et paléomagnétiques permettent de proposer une ébauche de stratigraphie intégrée pour la période oligo-miocène en Corse. Les dépôts continentaux syn-rift, dans la région d’Ajaccio, sont datés du Chattien supérieur par la présence d’un mammifère : Pomelomeryx boulangeri. Leur étude paléomagnétique indique une rotation anti-horaire de 44 ± 4 o par rapport à l’Europe stable. Les dépôts du Miocène inférieur se sont mis en place sur une topographie contrastée héritée de la période de glyptogenèse oligocène. Les terrains miocènes les plus anciens contiennent 4 dépôts de coulées pyroclastiques dans le Sud de la Corse. De nouvelles datations par la méthode 40 Ar- 39 Ar leur attribuent des âges compris entre 21,3 et 20,6 Ma (Aquitanien supérieur). Le Miocène marin du bassin de Bonifacio comprend deux formations : à la base, la formation de Cala di Labra, jalonnée de récifs en onlap côtier, est attribuée à la zone à Globigerinoides trilobus ; au-dessus, la formation des calcarénites de Bonifacio est essentiellement constituée par un empilement de dunes hydrauliques. L’absence d’Orbulines montre qu’elle est antérieure au Langhien supérieur (biozone N9). Le Miocène de Saint-Florent comprend 3 formations marines encadrées par deux formations continentales. A la base, la formation continentale de Fium’ Albinu (contemporaine de la partie inférieure de la formation de Cala di Labra) est surmontée par la formation de Torra, rapportée au Burdigalien supérieur en raison de la présence de G. trilobus et G. bisphericus. La formation de Sant’ Angelo (en partie équivalente à la formation de Bonifacio) se développe du Burdigalien terminal au Langhien supérieur. La formation de Farinole est attribuable au Serravallien inférieur (base de la zone à Globoquadrina altispira altispira). La direction paléomagnétique mesurée pour la formation de Fium’ Albinu indique une rotation anti-horaire d’une trentaine de degrés postérieure au début du Burdigalien supérieur. L’ensemble de ces nouveaux résultats paléomagnétiques et des informations antérieures indique que l’ouverture du bassin ligure s’accompagne d’une rotation antihoraire de 45 o de la Corse, qui commence entre 23 et 21 Ma et se termine autour de 15 Ma

Tsunamigenic potential of a Holocene submarine landslide along the North Anatolian Fault (northern Aegean Sea, off Thasos island): insights from numerical modelling

The North Anatolian Fault in the northern Aegean Sea triggers frequent earthquakes of magnitudes up to Mw∼7. This seismicity can be a source of modest tsunamis for the surrounding coastlines with less than 50&thinsp;cm height according to numerical modelling and analysis of tsunami deposits. However, other tsunami sources may be involved, like submarine landslides. We assess the severity of this potential hazard by performing numerical simulations of tsunami generation and propagation from a Holocene landslide (1.85&thinsp;km3 in volume) identified off Thasos. We use a model coupling the simulation of the submarine landslide, assimilated to a granular flow, to the propagation of the tsunami wave. The results of these simulations show that a tsunami wave of water height between 1.10 and 1.65&thinsp;m reaches the coastline at Alexandroupoli (58&thinsp;000 inhabitants) 1&thinsp;h after the triggering of the landslide. In the same way, tsunami waves of water height between 0.80 and 2.00&thinsp;m reach the coastline of the Athos peninsula 9&thinsp;min after the triggering of the landslide. Despite numerous earthquakes of Mw&gt;7 and strong detrital input (on the order of 30&thinsp;cm ka−1), only a few Holocene landslides have been recognized so far, asking for tsunami recurrence in this area.</p

On the equivalence of game and denotational semantics for the probabilistic mu-calculus

The probabilistic (or quantitative) modal mu-calculus is a fixed-point logic de- signed for expressing properties of probabilistic labeled transition systems (PLTS). Two semantics have been studied for this logic, both assigning to every process state a value in the interval [0,1] representing the probability that the property expressed by the formula holds at the state. One semantics is denotational and the other is a game semantics, specified in terms of two-player stochastic games. The two semantics have been proved to coincide on all finite PLTS's, but the equivalence of the two semantics on arbitrary models has been open in literature. In this paper we prove that the equivalence indeed holds for arbitrary infinite models, and thus our result strengthens the fruitful connection between denotational and game semantics. Our proof adapts the unraveling or unfolding method, a general proof technique for proving result of parity games by induction on their complexity

Calculations of the binding affinities of protein-protein complexes with the fast multipole method

In this paper, we used a coarse-grained model at the residue level to calculate the binding free energies of three protein-protein complexes. General formulations to calculate the electrostatic binding free energy and the van der Waals free energy are presented by solving linearized Poisson-Boltzmann equations using the boundary element method in combination with the fast multipole method. The residue level model with the fast multipole method allows us to efficiently investigate how the mutations on the active site of the protein-protein interface affect the changes in binding affinities of protein complexes. Good correlations between the calculated results and the experimental ones indicate that our model can capture the dominant contributions to the protein-protein interactions. At the same time, additional effects on protein binding due to atomic details are also discussed in the context of the limitations of such a coarse-grained model

Theory and simulation of short-range models of globular protein solutions

We report theoretical and simulation studies of phase coexistence in model globular protein solutions, based on short-range, central, pair potential representations of the interaction among macro-particles. After reviewing our previous investigations of hard-core Yukawa and generalised Lennard-Jones potentials, we report more recent results obtained within a DLVO-like description of lysozyme solutions in water and added salt. We show that a one-parameter fit of this model based on Static Light Scattering and Self-Interaction Chromatography data in the dilute protein regime, yields demixing and crystallization curves in good agreement with experimental protein-rich/protein-poor and solubility envelopes. The dependence of cloud and solubility points temperature of the model on the ionic strength is also investigated. Our findings highlight the minimal assumptions on the properties of the microscopic interaction sufficient for a satisfactory reproduction of the phase diagram topology of globular protein solutions.Comment: 17 pages, 8 figures, Proc. of Conference "Structural Arrest Transitions in Colloidal Systems with Short-Range Attractions", Messina (ITALY) 17-20 December 200

Steric constraints in model proteins

A simple lattice model for proteins that allows for distinct sizes of the amino acids is presented. The model is found to lead to a significant number of conformations that are the unique ground state of one or more sequences or encodable. Furthermore, several of the encodable structures are highly designable and are the non-degenerate ground state of several sequences. Even though the native state conformations are typically compact, not all compact conformations are encodable. The incorporation of the hydrophobic and polar nature of amino acids further enhances the attractive features of the model.Comment: RevTex, 5 pages, 3 postscript figure

Role of the Subunits Interactions in the Conformational Transitions in Adult Human Hemoglobin: an Explicit Solvent Molecular Dynamics Study

Hemoglobin exhibits allosteric structural changes upon ligand binding due to the dynamic interactions between the ligand binding sites, the amino acids residues and some other solutes present under physiological conditions. In the present study, the dynamical and quaternary structural changes occurring in two unligated (deoxy-) T structures, and two fully ligated (oxy-) R, R2 structures of adult human hemoglobin were investigated with molecular dynamics. It is shown that, in the sub-microsecond time scale, there is no marked difference in the global dynamics of the amino acids residues in both the oxy- and the deoxy- forms of the individual structures. In addition, the R, R2 are relatively stable and do not present quaternary conformational changes within the time scale of our simulations while the T structure is dynamically more flexible and exhibited the T\rightarrow R quaternary conformational transition, which is propagated by the relative rotation of the residues at the {\alpha}1{\beta}2 and {\alpha}2{\beta}1 interface.Comment: Reprinted (adapted) with permission from J. Phys. Chem. B DOI:10.1021/jp3022908. Copyright (2012) American Chemical Societ

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in β-thalassemic mice

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in β-thalassemic mice β-thalassemia (β-thal) is a genetic red cell disorder characterized by chronic hemolytic anemia due to ineffective erythropoiesis and reduced red cell survival.1-3 Chronic transfusion and intensive iron chelation are standard treatments for β-thalassemic syndromes,1 but new therapeutic options are being developed, including gene therapy4 and novel pharmacologic approaches. We have shown that mitapivat, a pyruvate kinase activator, improves anemia and ineffective erythropoiesis in Hbbth3/+ mice, a widely used model for β-thal.5 The effects of mitapivat are not limited to the erythroid compartment: mitapivat also modulates DMT1 expression, controlling iron absorption in the duodenum in Hbbth3/+ mice, with an increase of hepcidin related to the improvement in ineffective β-thalassemic erythropoiesis.5 Results from a phase II trial of mitapivat in non–transfusion-dependent β-thal patients previously demonstrated a sustained long-term increase in hemoglobin (Hb ≥1 g/dL) with improvement of hemolysis and ineffective erythropoiesis.6 Here, we asked whether mitapivat might be a potential therapeutic option also for β-thal patients under chronic transfusion regimen. In order to address this question, we exposed female Hbbth3/+ mice (3-4 months of age) to chronic transfusion with or without mitapivat (50 mg/kg twice daily [BID]). Hbbth3/+ mice were treated by oral gavage with mitapivat (50 mg/kg BID) or vehicle for 10 days, and then transfused with 400 μL washed red blood cells at 40-45% hematocrit (Hct)7 (Figure 1A). We defined Hb values ≤10.5 g/dL as the transfusion threshold, corresponding to the reduction of ~50% of post-transfusion Hb values. Normality of data was assessed with the Shapiro-Wilk test. Two-tailed unpaired Student t-test or two-way analysis of variance with Tukey’s multiple comparisons were used for data analyses. Data show values from individual mice and are presented with mean ± standard error of the mean (differences with P&lt;0.05 were considered significant). As shown in Figure 1B, mitapivat-treated β-thal mice exposed to chronic transfusion displayed a greater sustained rise in Hb from baseline compared to vehicle-treated transfused β-thal mice. This resulted in a longer interval between transfusions (13.8±1.0 days in mitapivat- treated β-thal mice vs. 10.5±1.0 days in vehicletreated β-thal mice; Figure 1C). Chronic transfusion resulted in a significant reduction of splenomegaly in both mitapivat- and vehicle-treated β-thal mice (Online Supplementary Figure 1SA) compared to untreated β-thal mice, but spleen iron accumulation was significantly lower in mitapivat-treated β-thal mice when compared to vehicle-treated β-thal mice (Figure 1D). A significant reduction of both bone marrow and spleen ineffective erythropoiesis was observed in all transfused β-thal mice (Figure 1E; Online Supplementary Figure S1B). Of note, mitapivat- treated transfused β-thal mice showed a slight increase of bone marrow erythropoiesis with a trend towards an improvement of maturation index compared to vehicle-treated transfused β-thal mice evaluated at the end of the study.5 This is most likely related to a protective effect of mitapivat on residual bone marrow and spleen erythropoiesis (Figure 1F). Indeed, plasma erythropoietin was lower in mitapivat-treated transfused β-thal mice than in vehicle-treated transfused β-thal mice (Online Supplementary Figure S1C). Since splenic macrophages contribute to both erythrophagocytosis and iron recycling, we evaluated the functional profile of spleen macrophages in the different mouse groups.8 As shown in Figure 1G, flow cytometric analysis of the surface expression of the M1 marker CD80 and the M2 marker CD206 on spleen macrophages (MΦ) revealed that mitapivat promoted a proresolving profile of splenic macrophages in transfused β-thal mice when compared to vehicle-treated transfused β-thal mice (Online Supplementary Figure S2A). This effect was still observed in non-transfused mitapivat-treated mice compared to vehicle-treated β-thal mice (Figure 1G; Online Supplementary Figure S2A). Collectively, these data support the role of mitapivat in reprograming macrophages from proinflammatory to proresolving and repairing the phenotype in β-thal mice with or without chronic transfusion.9 We then evaluated the impact of mitapivat on iron metabolism in transfused β-thal mice. Mitapivat-treated transfused β-thal mice showed lower liver iron accumulation when compared to vehicle-treated transfused β- thal mice (Figure 2A). This might be due in part to the reduction of the transfusion burden but also to the multimodal action of mitapivat, which we previously showed to modulate hepcidin indirectly by the reduction of ineffective erythropoiesis and downregulation of DMT1 expression in the duodenum.5 Indeed, in mitapivat-treated transfused β-thal, we found a significant increase in liver hepcidin/LIC ratio (Figure 2B) and a marked reduction in the percentage of serum transferrin saturation when compared to vehicle-treated transfused β-thal mice (Figure 2C). The reduced transfusion burden observed in mitapivat treated β-thal mice might favorably contribute to the general reduction of iron-overload in β-thal mice exposed to chronic transfusion. Our preclinical results in combination with clinical data from non-transfusion-de- Haematologica | 108 September 2023 2535 LETTER TO THE EDITOR A C E F G D B Continued on following page. Haematologica | 108 September 2023 2536 LETTER TO THE EDITOR pendent β-thal patients treated with mitapivat6 suggest that the increase in the length of time between transfusions with mitapivat treatment may be associated with improvement in the quality of life in patients as well as a decrease in iron-overload-related organ damage. Recent reports in transfusion-dependent β-thal patients have highlighted a correlation between ferritin levels and kidney iron accumulation assessed by magnetic resonance T2* imaging, or in sample analysis from kidney biopsies or autopsy series.10 Kidney iron overload mainly involved the tubular compartment which has been related to chronic anemia and might be reversed by iron chelation.10 In vehicle- treated transfused β-thal mice, we found tubular accumulation of iron, which was significantly reduced in mitapivat-treated transfused animals (Figure 3A). No major difference in creatinine was observed in both β-thal mouse groups exposed to chronic transfusion (Online Supplementary Figure S2B). Previous studies suggest that kidney iron accumulation promotes local oxidative stress, contributing to profibrotic signaling in addition to hypoxia.10,11 MicroRNA (miRNA) let-7b, -c, and -d have been shown to be linked to renal fibrosis throughout the transforming growth factor- β cascade (TGF-β).12 In this study, miRNA let-7b and -d were upregulated in vehicle-treated β-thal mice with or without chronic transfusion (Figure 3B; Online Supplementary Figure S2C), while mitapivat downregulated miRNA let-7b and -d in β-thal mice with or without chronic transfusion (Figure 3B; Online Supplementary Figure S2C). miRNA let-7 have been reported to reduce ATP production by deactivating pyruvate dehydrogenase kinase (PDK).13 Here, we found normalization of the amount of the active form of the TGF-β receptor in β-thal mice treated with mitapivat when compared to vehicle-treated β-thal mice with or without transfusion (Figure 3C). Previously, in β- thal mice, the activation of TGF-β receptor was reduced by chronic transfusion, hypoxia being a trigger of activation of TGF-β receptor.14 Taken together, our data indicate that mitapivat might play a pivotal role in kidney protection by reducing the transfusion burden and iron overload as well as by preserving energy cell metabolism. This might represent an added value of mitapivat as a therapeutic option for patients with β-thal taking iron chelators who develop renal toxicity or chronic kidney disease. Finally, we explored the effects of the co-administration of mitapivat and deferiprone (DFP) on β-thal mice, since iron chelation is part of the gold standard treatment of β- thal patients.1 DFP was administered to Hbbth3/+ mice treated with mitapivat in drinking water at the dosage of either 1.25 or 0.75 mg/mL15 (Online Supplementary Figure S3A). Previously, Casu et al. reported that DFP alone has no effect on hematologic parameters and red cell features in murine β-thal.15 The beneficial effects of mitapivat on murine β-thal anemia was maintained when mitapivat was co-administered with DFP at both dosages, as supported by the stable and sustained increase in Hb and the reduction in circulating erythroblasts compared to baseline values (Online Supplementary Figure S3B, C). In agreement with Matte et al.,5 we found a significant reduction in α-globin membrane precipitates in red blood cells from mitapivat DFP-treated Hbbth3/+ mice compared with vehicle-treated animals (Online Supplementary Figure S3D). Of note, DFP iron chelation efficacy represented by a change in LIC was preserved in β-thal mice treated with both DFP and mitapivat (Online Supplementary Figure S3E). In conclusion, our study shows for the first time that mitapivat improves the transfusion burden and reduces organ iron overload in β-thal mice exposed to a chronic transfusion regimen. We also observed that mitapivat might protect the kidney against profibrotic stimuli related to local iron accumulation by two different mechanisms: the reduction in transfusion requirement and the local modulation of miRNA involved in profibrotic signal- Figure 1. Mitapivat reduces transfusion burden in β-thalassemia mice exposed to chronic transfusion with associated reprogramming of splenic macrophage phenotype. (A) Experimental study design to assess the effects of mitapivat on hematologic phenotype of β-thalassemia (β-thal) mice exposed to chronic transfusion. (B) Hemoglobin (Hb) changes over time in transfused (Tr.) β-thal (Hbbth3/+) mice treated with either vehicle or mitapivat (50 mg/kg twice daily [BID]) shown as single animals (n=3 vehicle- treated mice; n=4 mitapivat-treated mice). Grey dotted line shows the transfusion threshold (10.5 g/dL). (C) Transfusion time intervals in β-thal (Hbbth3/+) mice treated with either vehicle or mitapivat (50 mg/kg BID). Data are presented as means ± standard error of the mean (SEM) (n=3 vehicle-treated mice; n=4 mitapivat-treated mice); #P&lt;0.05 compared to vehicle-treated transfused β-thal mice. (D) Iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in spleen from Hbbth3/+ mice treated with either vehicle or transfusion plus vehicle or transfusion plus mitapivat. One representative image from 3 with similar results. Left panel: quantification of iron staining in spleen. Data are mean ± SEM (n=3). *P&lt;0.05 compared with vehicle Hbbth3/+ mice and #P&lt;0.05 compared with vehicle-treated transfused Hbbth3/+ mice. (E) Flow cytometric analysis (CD44+Ter119+ and cell size markers, see also the Online Supplementary Figure S2) of bone marrow and spleen from Hbbth3/+ mice exposed to either vehicle or to chronic transfusion with and without mitapivat treatment (see also Matte et al.5). Data are mean ± SEM (n=3-4). *P&lt;0.05 compared with vehicle Hbbth3/+ mice and #P&lt;0.05 compared with vehicle-treated transfused Hbbth3/+ mice. (F) Maturation index as ratio between pop II (Baso E.) and pop IV (Ortho E.) in bone marrow and spleen from Hbbth3/+ mice treated with either vehicle or exposed to chronic transfusion with or without mitapivat, analyzed by flow cytometry. Data are mean ± SEM (n=3-4). (G) Flow cytometric quantification of M1 (CD80) and M2 (CD206) expression on spleen macrophage cell surface from wild-type (WT) or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment. Spleen macrophages (MΦ) were isolated with the GentleMACS cell dissociator (Miltenyi Biotech, Germany). MΦ were identified and gated as CD45+/F4/80+ cells. Anti-CD45 PE-Cy5.5, F4/80 PE, CD206 PerCP-Cy5.5 and CD80 were from BioLegend, USA. Data are mean ± SEM (n=3-4). MFI: mean fluorescence intensity; RBC: red blood cells. Haematologica | 108 September 2023 2537 LETTER TO THE EDITOR A B C Figure 2. Mitapivat-treated transfused β-thalassemia mice show reduced liver iron accumulation and improved iron homeostasis. (A) Left and central panels: iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in liver from wild-type (WT) and Hbbth3/+ mice treated with either vehicle or transfusion (Tr.) or transfusion plus mitapivat. One representative image from 5 with similar results. Right panel: quantification of iron staining in liver. Data are mean ± standard error of the mean (SEM) (n=5). °P&lt;0.05 compared to WT, *P&lt;0.05 compared with vehicle Hbbth3/+ mice and #P&lt;0.05 compared with vehicle-treated transfused (Tr.) Hbbth3/+ mice. (B) Liver mRNA expression normalized over liver iron concentration (LIC) as determined using the bathophenanthroline method. Data are presented as means ± SEM (n=3). #P&lt;0.05 compared with vehicletreated transfused Hbbth3/+ mice. (C) Transferrin saturation in Hbbth3/+ mice treated with either vehicle or transfusion or transfusion plus mitapivat. Transferrin saturation was calculated as the ratio between serum iron and total iron binding capacity, using the Total Iron Binding Capacity Kit (Randox Laboratories, UK) and 50 mL of serum, according to the manufacturer’s instructions. Data are presented as means ± SEM (n=3). *P&lt;0.05 compared with vehicle Hbbth3/+ mice and #P&lt;0.05 compared with vehicle-treated transfused Hbbth3/+ mice. RBC: red blood cells. Haematologica | 108 September 2023 2538 LETTER TO THE EDITOR A B C Figure 3. In transfused β-thalassemia mice, mitapivat reduces kidney iron accumulation and downregulates profibrotic kidney miRNA let-7 expression. (A) Upper panels: iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in kidney from wild-type (WT) and Hbbth3/+ mice treated with either vehicle or transfusion (Tr.) or transfusion plus mitapivat. One representative image from 3-6 with similar results. Lower panels: quantification of iron staining in kidney. Data Continued on following page. Haematologica | 108 September 2023 2539 LETTER TO THE EDITOR ing. Finally, the observed reprograming of spleen macrophages toward a proresolving phenotype might represent an added value to the known improvement of ineffective erythropoiesis induced by mitapivat in β-thal mice.5 Thus, the beneficial effects of mitapivat in β-thal mice exposed to chronic transfusion support its use as a potential new therapeutic tool in clinical management of thalassemic patients under chronic transfusion regimen. Authors Alessandro Mattè,1 Penelope A. Kosinski,2 Enrica Federti,1 Lenny Dang,2 Antonio Recchiuti,3 Roberta Russo,4 Angela Siciliano,1 Veronica Riccardi,1 Anne Janin,5 Matteo Mucci,3 Christophe Leboeuf,5 Achille Iolascon,6 Carlo Brugnara7 and Lucia De Franceschi1 1University of Verona and AOUI Verona, Verona, Italy; 2Agios Pharmaceuticals, Inc., Cambridge, MA, USA; 3Deptartment of Medical, Oral and Biotechnology Science, “G. d’Annunzio” University of Chieti, Chieti, Italy; 4Dipartmento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Naples, Italy; 5University Diderot of Paris, Paris, France; 6CEINGE - Biotecnologie Avanzate Franco Salvatore, Naples, Italy and 7Department of Laboratory Medicine, Harvard Medical School, Boston Children's Hospital, Boston, MA, USA Correspondence: L. DE FRANCESCHI - [email protected] https://doi.org/10.3324/haematol.2022.282614 Received: December 20, 2022. Accepted: February 7, 2023. Early view: February 16, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license Disclosures LDF received research funding from Agios during 2015-2022. LD and PAK are Agios employees and stockholders. All other authors have no conflicts of interest to disclose. Contributions LDF, CB, AM and AI designed and carried out research and wrote the paper. PAK, LD and CB critically revised data and wrote the paper. AM, EF, AS and VR carried out cytokine FACS analysis, immunoprecipitation assays and ELISA analysis. RB carried out molecular analysis. EF revised the paper. MM and AR carried out miRNA analysis, analyzed the data and wrote the paper. CL and AJ performed pathology analysis and analyzed data. Funding This study was supported by an Agios Pharmaceuticals, Inc. research collaborative grant to LDF. Editorial assistance was provided by Avant Healthcare, LLC and Excel Medical Affairs, Horsham, UK, supported by Agios. Data-sharing statement All the data and protocols are stored in the Nas Synology DS216se Hard Disk, located at the University of Verona, Verona, Italy and will be made available on request. Please direct requests for original data to the corresponding author. are mean ± standard error of the mean (SEM) (n=3-6). *P&lt;0.05 compared with vehicle Hbbth3/+ mice and #P&lt;0.05 compared with vehicle-treated transfused (Tr.) Hbbth3/+ mice. (B) Relative expression of miRNA let-7b and -7d in kidneys from WT or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment. Small RNA was isolated from frozen kidneys using a silica spin column-based Quick-RNA kit (Zymo Research), quantified with a UV NanoPhotometer (Implen), and reverse transcribed with the qScript microRNA cDNA Synthesis for RT-PCR (QuantaBio). For real time polymerase chain reaction (RT-PCR) analysis of let-7b and let-7d miRNA, 3 ng of cDNA were used as a template in reaction mixtures (10 mL final volume) including a PowerUp SYBR Green Master Mix (5 mL, Applied Biosystems), miRNA-specific forward and universal reverse primers (1 mL each, miRCURY assays, Qiagen), and PCR-grade water. The expression of the indicated mRNA was quantitated by the comparative ΔCt method. RNU6-1 was used as control for normalization. Data are mean ± SEM (n=3-4). *P&lt;0.05. **P&lt;0.01. ***P&lt;0.001. (C ) Phospho-tyrosine immunoprecipitation of kidneys from WT or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment, using anti-phospho-tyrosine specific antibodies (IP: PY, clone PY99 from SCBT, Santa Cruz, CA and clone 4G10 from Merck KGaA, Darmstadt, Germany), revealed with anti-TGF-β receptor (Rec) specific antibody. GAPDH in whole-cell lysate (WCL) is used as loading control. One representative gel from 4 others with similar results is presented. Blots were developed using the Luminata Forte Chemiluminescent HRP Substrate from Merck Millipore (Armstadt, Germany), and images were acquired with the Alliance Q9 Advanced imaging system (Uvitec, UK). Densitometric analysis of immunoblots is shown on the right. Data are mean ± SEM (n=4). °P&lt;0.05 compared to WT; *P&lt;0.05 compared with vehicle Hbbth3/+ mice, #P&lt;0.05 compared with vehicle-treated transfused Hbbth3/+ mice. RBC: red blood cells. References 1. Taher AT, Musallam KM, Cappellini MD. Beta-Thalassemias. N Engl J Med. 2021;384(8):727-743. 2. De Franceschi L, Bertoldi M, Matte A, et al. Oxidative stress and β-thalassemic erythroid cells behind the molecular defect. Oxid Med Cell Longev. 2013;2013:985210. 3. Rivella S. β-thalassemias: paradigmatic diseases for scientific Haematologica | 108 September 2023 2540 LETTER TO THE EDITOR discoveries and development of innovative therapies. Haematologica. 2015;100(4):418-430. 4. Locatelli F, Thompson AA, Kwiatkowski JL, et al. Betibeglogene autotemcel gene therapy for non-beta(0)/beta(0) genotype beta-Thalassemia. N Engl J Med. 2022;386(5):415-427. 5.Matte A, Federti E, Kung C, et al. The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a betathalassemia mouse model. J Clin Invest. 2021;131(10):e144206. 6. Kuo KHM, Layton DM, Lal A, et al. Safety and efficacy of mitapivat, an oral pyruvate kinase activator, in adults with nontransfusion dependent alpha-thalassaemia or beta-thalassaemia: an open-label, multicentre, phase 2 study. Lancet. 2022;400(10351):493-501. 7. Park SY, Matte A, Jung Y, et al. Pathologic angiogenesis in the bone marrow of humanized sickle cell mice is reversed by blood transfusion. Blood. 2020;135(23):2071-2084. 8. Ramos P, Casu C, Gardenghi S, et al. Macrophages support pathological erythropoiesis in polycythemia vera and betathalassemia. Nat Med. 2013;19(4):437-445. 9. Galvan-Pena S, O'Neill LA. Metabolic reprograming in macrophage polarization. Front Immunol. 2014;5:420. 10. Demosthenous C, Vlachaki E, Apostolou C, et al. Betathalassemia: renal complications and mechanisms: a narrative review. Hematology. 2019;24(1):426-438. 11. Musallam KM, Taher AT. Mechanisms of renal disease in betathalassemia. J Am Soc Nephrol. 2012;23(8):1299-1302. 12. Hong S, Lu Y. Omega-3 fatty acid-derived resolvins and protectins in inflammation resolution and leukocyte functions: targeting novel lipid mediator pathways in mitigation of acute kidney injury. Front Immunol. 2013;4:13. 13.Ma X, Li C, Sun L, et al. Lin28/let-7 axis regulates aerobic glycolysis and cancer progression via PDK1. Nat Commun. 2014;5:5212. 14. Chou YH, Pan SY, Shao YH, et al. Methylation in pericytes after acute injury promotes chronic kidney disease. J Clin Invest. 2020;130(9):4845-4857. 15. Casu C, Aghajan M, Oikonomidou PR, et al. Combination of Tmprss6- ASO and the iron chelator deferiprone improves erythropoiesis and reduces iron overload in a mouse model of beta-thalassemia intermedia