bluuebunny/crossref_metadata_2025_split huggingface dataset:
The microRNA144/451 Locus Enhances Nuclear FOXO3a Activity to Protect Erythroid Cells against Oxidant Stress
Authors: dos Santos Camila O. , Yu Duonan , Amigo Julio , Kandros Eugene , Valentine Eric R , Shelat Suresh , Dore Louis C , Yao Yu , Souza Janine D’ , Ghaffari Saghi , Paw Barry H. , Orange Jordan S , Weiss Mitchell J. 
Published: April 2020

Abstract
MicroRNAs (miRNAs) regulate tissue development by destabilizing cellular mRNAs or blocking their translation. We previously described two conserved erythroid miRNAs, miR144 and miR451, which are encoded by a single gene locus that is regulated by the essential hematopoietic transcription factor GATA-1. Morpholino-induced inhibition of miR451 impairs erythropoiesis in zebrafish. To gain further insight into miR144/451, we deleted the locus in mice. Mutant animals are born at the normal Mendelian ratio and exhibit no obvious structural defects. At baseline, loss of miR 144/451 causes mild anemia and reticulocytosis with moderate abnormalities in erythrocyte morphology. However, compared to littermate controls, miR144/451 null animals exhibit 33% greater depletion of circulating erythrocytes after phenylhydrazine-induced oxidant stress. At baseline, the mutant erythrocytes exhibit increased levels of reactive oxidant species, exquisite sensitivity to hydrogen peroxide induced hemolysis, and 50% reduced catalase activity compared to controls. Catalase is a downstream effector in an erythroid anti-oxidant program regulated by the forkhead transcription factor Foxo3a. In the mutant erythroblasts, mRNAs encoding catalase and glutathione peroxidase 1, another Foxo3a-regulated anti-oxidant, were reduced 2 and 1.7 fold, respectively. Messenger RNAs encoding several additional Foxo3a target genes, including Cdkn1b and Btg1 were also significantly reduced. Quantitative confocal fluorescence microscopy demonstrated that although total cellular Foxo3a protein was similar in wt and miR144/451 null erythroblasts, nuclear Foxo3a was reduced by 40% in miR144/451 null erythroblasts with a corresponding relocation of the protein to the cytoplasm. To explain this, we showed that miR451 (but not miR144) directly blocks translation of Ywhaz mRNA encoding 14-3-3 zeta, a cytoplasmic protein that sequesters Foxo3a away from the nucleus. In agreement, 14-3-3 zeta protein is upregulated by approximately 2-fold higher levels in miR144/451 null erythroblasts. Together, our findings suggest a model in which miR451 represses 14-3-3 zeta expression, which releases Foxo3a for translocation to the nucleus, thereby activating an anti-oxidant program of gene expression. This illustrates a new miRNA-regulated pathway through which erythroid cells are protected against oxidant stress.
Bortezomib and Dexamethasone as Maintenance therapy in Relapse/Refractory multiple myeloma Patients
Authors: Benevolo Giulia , Larocca Alessandra , Pregno Patrizia , Gay Francesca , Botto Barbara , Orsucci Lorella , Evangelista Andrea , Palumbo Antonio , Vitolo Umberto , Boccadoro Mario 
Published: April 2020

Abstract
Background: Despite the advances in the treatment for multiple myeloma (MM) in the past decade, it still remains an incurable haematological disease and the majority of patients still experience relapse. Bortezomib is a novel proteasome inhibitor approved for the treatment of MM patients. Several studies have demonstrated its efficacy as front-line therapy and in relapsed, advanced MM but no data are available as maintenance therapy (MT) in refractory/relapsed MM.
Patients and methods: The aim of this study is to evaluate the safety and the efficacy of bortezomib/dexamethasone MT (1.3 mg/mq bortezomib on days 1 and 15; 20 mg dexamethasone on days 1–2 and 15–16 in a 28-day cycle for a total of 6 cycles) in patients with refractory/relapse MM who responded to salvage therapy.
Results: From October 2004 until April 2008, 40 MM patients have been enrolled. The characteristics of the patients were as follows: 20 males and 20 females, median age was 70 years (IQR:66–75). Median haemoglobin value was 10.85 (IQR:10.175–12.225) and 7 (18%) patients had a renal failure. Skeletal disease was present in 27 (68%) patients. The median number of prior therapies was 2 (1–4). The salvage therapy included bortezomib as single agent or in combination with steroids and/or thalidomide in 12 patients (30%). Median time from diagnosis to the first dose of MT was 41 months (IQR:26–59). The median number of bortezomib infusion was 8 (1–18). After a median follow up of 13 months (IQR:6–31), 10 patients died for PD and 4 patients for infections. The MT improved the quality of response and converted in 1 CR and 4 VGPR the PR after salvage therapy in 5 patients; moreover, we observed a remarkable decreased of M component in 11 patients. The median time to progression was 23 months (95%CI: 8-not reached) with a progression free-survival at 1 year of 69% (95%CI: 50–82). The overall survival at 1 years was 63% and the cumulative incidence of death due to PD adjusted for competitive risk event was 25% (95%IC:10–41). In a univariate analysis the response rate to MT was not significantly affected by age, sex, number or type of previous therapy and haemoglobin concentration. Non-dose-limiting toxicities included neuropathy grade 1 (12 pts), herpes zoster reactivation (2 pts) and gastrointestinal affections (1 pts).
Conclusion: The combination bortezomib/dexamethasone can be safely administered as a maintenance therapy in relapse/refractoy MM. These preliminary data suggest that bortezomib/dexametasone MT can improve remission duration and also quality of response with an acceptable toxicity.
The JAK2V617F Mutation Is Present in the Liver Endothelial Cells of Patients with Budd-Chiari Syndrome
Authors: Sozer Selcuk , Fiel Isabel M. , Schiano Thomas , Feller Faye , Mascarenhas John , Hoffman Ronald 
Published: April 2020

Abstract
Post-natal vasculogenesis has been reported to be derived from a hierarchy of circulating endothelial progenitor cells (EPC). Some of these EPC are of myeloid origin while others have a more robust proliferative potential and are solely of endothelial cell (EC) origin. A number of groups have hypothesized that EC dysfunction might contribute to the hypercoagulable state associated with polycythemia vera (PV) by orchestrating the recruitment of blood elements to sites of injury or by regulating vascular tone. The JAK2V617F mutation is present in &gt;95% of patients with PV. In addition, some individuals with normal blood counts who develop splanchnic vein thrombosis, including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT) have been reported to have JAK2V617F positive hematopoiesis (45% and 34%, respectively) indicating that this thrombotic tendency might precede the development of PV. We explored whether this activating mutation is present in EC in the vessels of patients with BCS. We tested this hypothesis by studying EC in venules of liver biopsy specimens of patients with BCS with (n=2) or without PV (n=1) and PVT (n=2) without PV using laser capture microdissection (LCM) followed by nested PCR or RT-PCR in order to determine if EC were JAK2V617F positive and were of hematopoietic or EC origin. EC from the hepatic venules of BCS and PVT patients were captured by LCM from hematoxylin and eosin stained sections of archival formalin-fixed paraffin-embedded liver biopsy tissue specimens. EC were identified by their fusiform nuclei and their location along the lining of the hepatic venules. Hepatocytes were identified by their morphology as having round centrally placed nuclei and abundant cytoplasm with a trabecular arrangement. At least 10 EC and 10 hepatocytes from each biopsy specimen were captured and DNA or RNA was extracted. The JAK2V617F mutation was detected using nested allele-specific PCR. The hepatocytes of each patient contained exclusively wild type JAK2 while the EC of the two BCS patients with PV were homozygous for the JAK2V617F. The EC of the other BCS patient and 2 PVT patients who did not have PV contained exclusively wild type JAK2. The EC identity of these cells in the PV patients was confirmed by the presence of the EC transcripts (VE-Cadherin and VEGF-R2) and the absence of hematopoietic cell transcripts (CD45 and CD14). These findings indicate that hepatic venule EC in BCS patients with PV are JAK2V617F positive. The presence of JAK2V617F in both endothelial cells and hematopoietic cells belonging to such patients suggests that PV originates in an adult hemangioblast like cell in such patients. Further studies are required to understand the consequence of JAK2 mutation in EC as related to their propensity to thrombosis in PV.
Impaired Function of Hematopoietic Stem Cells with Abrogated Hypoxia Induced Expression of Vascular Endothelial Growth Factor
Authors: Rehn Matilda , Reckzeh Kristian , Olsson André , Axelson Håkan , Cammenga Jörg 
Published: April 2020

Abstract
Hypoxia has been suggested to be a characteristic of the bone marrow (BM) microenvironment where hematopoietic stem cells (HSC) reside. The major molecular response to hypoxia is the activation of hypoxia inducible factors (HIF). These transcription factors activate gene expression by binding to hypoxia response elements (HRE) in the promoter region of target genes, such as vascular endothelial growth factor (VEGF). It has been demonstrated that VEGF is indispensable for HSC survival in the adult and that VEGF mediated HSC survival is cell intrinsic. We are therefore investigating the role of hypoxia-regulated VEGF expression in HSC survival. For our studies we use the transgenic mouse model VEGFδ/δ. In this model, the HRE in the VEGF promoter is mutated which prevents HIF binding. Thus, hypoxia-induced VEGF transcriptional activation is abrogated while basal VEGF expression remains unaffected. Steady state hematopoiesis was not affected by the mutation since distribution between the B-cell, T-cell and myeloid lineages in both peripheral blood and BM of mice homozygous for the mutation was comparable to wild type animals. Furthermore, we could not detect any differences in the frequency of lineage-, Sca1+, c-kit+, CD34- cells in the BM between VEGFδ/δ and wild type mice. However, when assessing HSC functional properties in transplantation assays, BM from mice homozygous for the mutation was demonstrated to have dramatically reduced HSC function. Only around 60 % of lethally irradiated recipients that were transplanted with VEGFδ/δ BM could be rescued while recipients receiving wild type BM survived as expected. In addition, during early hematopoietic reconstitution, a skewing towards myeloid differentiation at the expense of B-cell differentiation could be observed in peripheral blood of recipients reconstituted with VEGFδ/δ BM. Moreover, in a competitive repopulation assay, VEGFδ/δ BM was shown to have a severe competitive disadvantage compared to wild type BM. Our findings indicate an important role for hypoxia related VEGF expression in reconstituting HSCs and suggest that low oxygen levels might be an important characteristic of the HSC niche.
The Role of Akt3 in Platelet Activation
Authors: O’Brien Kelly A , Stojanovic Aleksandra , Hay Nissim , Du Xiaoping 
Published: April 2020

Abstract
Akt is a family of serine/threonine kinases that are activated downstream of Phosphoinositide 3 Kinases (PI3K). There are three known Akt isoforms: Akt1, Akt2, and Akt3. Both Akt1 and Akt2 have been shown to be expressed in platelets and play important roles in PI3K-dependent platelet activation signaling. Previous studies have concluded that Akt3 is not expressed in platelets; however, we show that Akt3 is in fact a major Akt isoform present in platelets, and demonstrate that Akt3 plays an important role in platelet activation. In order to determine if Akt3 mRNA is present in platelets, RNA was isolated from platelets and RT-PCR experiments were performed using Akt3 specific primers. Akt3 mRNA was detected in both human and mouse platelets, and the possibility that Akt3 mRNA came from leukocyte contamination was excluded because Akt3 was not detected with RT-PCR using RNA purified from the same number of leukocytes as that in platelet preparation. To assess if Akt3 protein is present in platelets, western blot analysis was performed using an Akt3 specific antibody. Results revealed that Akt3 is highly expressed in human and mouse platelets. In order to assess the relative amount of Akt3 present in platelets, total Akt levels and phosphorylated Akt levels were measured in Akt3 knockout mouse platelets in comparison with wild type mouse platelets. Akt3 knockout mouse platelets showed a significant decrease in total Akt, and also a significant reduction in phosphorylated Akt, as indicated by Western blot analysis of phospho-Thr308-Akt and phospho-Ser473-Akt antibodies. Thus, our data indicates Akt3 is a major Akt isoform expressed in platelets. To determine the role of Akt3 in platelet activation, platelets from wild type mice and Akt3 knockout mice were stimulated with low concentrations of platelet agonists. Akt3 knockout mouse platelets exhibited impaired aggregation and ATP secretion in response to low dose thrombin and collagen compared with wild type controls, indicating that Akt3 plays an important role in promoting platelet activation. In order to assess the signaling molecules downstream from Akt3, Akt3 knockout and wild type mouse platelets were stimulated with low dose thrombin and phosphorylation of GSK-3β, an enzyme recently reported to negatively regulate platelet function downstream from Akt1 or Akt2 in platelets, was assessed. Western blot for phospho-Ser9 of GSK-3β in Akt3 knockout mouse platelets stimulated with thrombin showed less phosphorylation compared to Akt1 or Akt2 knockout mouse platelets, indicating that Akt3 is the major Akt isoform responsible for GSK-3β phosphorylation. Thus, our data shows that Akt3 is a major Akt isoform in platelets and plays an important role in platelet activation. Since phosphorylation of GSK-3β at Ser9 has been reported to reverse the inhibitory effect of GSK-3β on platelet function, our data also suggest that the stimulatory role of Akt3 in platelet activation may be mediated by negative regulation of GSK-3β function.
Negative Regulation of Gq-Mediated Pathways in Platelets by G12/13 Pathways through Fyn Kinase
Authors: Kim Soochong , Kunapuli Satya P. 
Published: April 2020

Abstract
Platelets contain high levels of Src family kinases (SFKs) suggesting an important role for these enzymes in platelet function. In this study, we have investigated the regulation of platelet function by SFKs downstream of G12/13 pathways. Calcium-independent platelet shape change induced by selective G12/13 stimulation with YFLLRNP was potentiated with a small mobilization of intracellular calcium in the presence of SFK inhibitors PP1 or PP2, which was abolished by the chelation of intracellular calcium, suggesting that SFKs downstream of G12/13 negatively regulate calcium mobilization in platelets. In addition, PP1 or PP2 caused a leftward shift of human platelet aggregation, secretion, and calcium response induced by low concentrations of agonists that activate platelets through G12/13 signaling such as PAR1-activating peptide SFLLRN and PAR4-activating peptide AYPGKF. However, 2-MeSADP-induced calcium response and platelet aggregation were not affected by the presence of PP1 or PP2, suggesting that SFKs downstream of G12/13, but not Gq/Gi, pathways are involved in this platelet response. Moreover, platelet aggregation and secretion caused by combined stimulation of G12/13 and Gi/Gz (YFLLRNP + 2-MeSADP with P2Y1 antagonist/epinephrine) were also potentiated in the presence of PP1 or PP2 confirming the contribution of SFKs downstream of G12/13 as a negative regulator to platelet activation. Potentiation of platelet aggregation in the presence of SFK inhibitors was not affected in the presence of GF109203X, while PP2 failed to potentiate platelet aggregation in the presence of 5,5′-dimethyl-BAPTA indicating that potentiation of cytosolic calcium may have an important role in this enhanced platelet responses by SFK inhibition. Moreover, PP1 or PP2 failed to potentiate platelet responses in the presence of Gq selective inhibitor YM-254890 or in Gq-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of Gq signaling pathways. Importantly, AYPGKF-induced platelet aggregation, secretion, and calcium response were potentiated in Fyn-, but not in Lyn-, deficient platelets compared to the wild-type mouse platelets, whereas 2-MeSADP-induced platelet response was not affected in these platelets. We conclude that SFKs activated downstream of G12/13, but not Gq/Gi, pathways negatively regulate platelet responses by inhibiting intracellular calcium mobilization in platelets through Gq signaling pathways. Specifically, we define that Fyn plays a major role in this negatively regulatory pathway.
Role of Sialic Acid for Platelet Lifespan and Function
Authors: Sørensen Anne Louise , Rumjantseva Viktoria , Nayeb-Hashemi Sara , Patel-Hett Sunita , Richardson Jennifer , Italiano Joseph , Clausen Henrik , Hartwig John H. , Wandall Hans H. , Hoffmeister Karin M. 
Published: April 2020

Abstract
Although sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet half-life is not fully clarified. We and others have previously provided evidence that thrombocytopenia in mice deficient in the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice) is caused by rapid clearance of the platelets due to recognition of surface galactose by asialoglycoprotein receptor-expressing scavenger cells. Here we report new insight into clearance mechanisms, activation and production of sialic acid deficient platelets. Immunofluorescence staining of organ specimens harvested shortly after platelet transfusion demonstrated the predominant clearance of ST3Gal-IV−/− platelets by liver macrophages and, as previously reported, hepatocytes. The differential cellular clearance pathways were further explored following macrophage depletion in mice using clodronate encapsulated liposomes, a procedure that restored ST3Gal-IV−/− platelet circulation to approximately 40% of normal values, confirming that macrophages play a major role in the clearance of sialic acid deficient platelets and that other clearance pathways are equally important. Ingestion of ST3Gal-IV−/− platelets by hepatocytes was confirmed by in vitro HepG2 phagocytosis assays. We next investigated the function of desialylated platelets. Platelet binding of von Willebrand factor (vWf) upon botrocetin stimulation was 3 fold higher for ST3Gal-IV−/− platelet rich plasma compared to ST3Gal- IV+/+ platelet rich plasma. The circulation time of wild-type platelets transfused into ST3Gal-IV−/− mice was 20% reduced compared to control platelets, indicating that platelet removal could be accelerated due to binding of desialylated vWf to platelets. Loss of sialic acid did not affect platelet production in vitro and in vivo as cultivated megakaryocytes was found to produce proplatelets normally and measurements of reticulated platelets by flow cytometry showed a 3-fold increased thrombocytopoietic activity in the ST3Gal- IV−/− mice compared to wild-type mice. Interestingly, thiazole orange staining revealed a significant negative correlation between platelet size and platelet age for both genotypes, In conclusion, depletion of the ST3Gal-IV gene promotes binding of vWf to platelets, exposes platelet surface galactose residues to the lectin domain of asialoglycoprotein receptors on both hepatocytes and liver macrophages, resulting in rapid platelet clearance from the circulation and supporting previous findings that platelets decrease in size while aging in circulation.
CIB1 Is Required for FAK Activation during Outside-in Signaling through Platelet Integrin αIIbβ3
Authors: Naik Ulhas Pandurang , Naik Meghna Ulhas 
Published: April 2020

Abstract
Platelets play an important role in the processes of hemostasis and thrombosis. Platelet integrin αIIbβ3 mediates bi-directional signaling during these processes. Agonist-dependent activation of integrin αIIbβ3 through inside-out signaling results in high-affinity binding of soluble ligands, such as fibrinogen. Fibrinogen binding induces a cascade of signaling through the integrin, termed outside-in signaling that results in platelet aggregation and clot retraction. Previously, we have characterized CIB1, a calcium- and integrin-binding protein that specifically interacts with the cytoplasmic domain of αIIb. Previous reports using in vitro and ex vivo studies implicated that CIB1 is involved in
maintaining αIIbβ3 in its resting state, agonist-induced activation of the integrin, and outside-in signaling resulting in platelet spreading.
Here, we show that platelet filopodia formation induced by fibrinogen binding to integrin αIIbβ3 needs Ca2+, but is independent of the Ca2+-dependent interaction of CIB1 with αIIb. Additionally, dynamic rearrangement of the cytoskeleton is required for the recruitment of FAK to the CIB1-αIIb complex at the filopodia and FAK activation. Moreover, disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or CIB1 antibody inhibited FAK activation. Furthermore, Cib1 null platelets acquired a spiky morphology and failed to fully spread on immobilized fibrinogen. Interestingly, FAK activation was significantly reduced in Cib1 null platelets exposed to immobilized fibrinogen. Our results suggest that during outside-in signaling, a rise in the intracellular Ca2+ level and filopodia formation occurs prior to the interaction of CIB1 with αIIb. Additionally, Ca2+ bound CIB1 recruits FAK to the αIIbβ3 complex at the filopodia, where FAK is activated, resulting in platelet spreading. Thus, our results have provided a mechanism through which CIB1 regulates outside-in signaling through integrin αIIbβ3.