bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of NPM in the Control of Genetic Stability and Tumorigenesis.
Authors: Grisendi Silvia , Bernardi Rosa , Rossi Marco , Cheng Ke , Khandker Luipa , Manova Katia , Pandolfi Pier Paolo 
Published: April 2020

Abstract
Nucleophosmin (NPM, B23, NO38) is a nucleolar protein directly implicated in cancer pathogenesis as the NPM gene is found mutated and rearranged in a number of haematological disorders. In addition, the region of chromosome 5 where NPM maps to (5q35) is deleted in a proportion of de novo human myelodysplastic syndromes (MDS), and loss of chromosome 5 is extremely frequent in therapy-related MDS (t-MDS). NPM is a multifunctional protein, and its role in oncogenesis is controversial as NPM has been attributed both oncogenic and tumour suppressive functions. To study the function of Npm in vivo, we generated a hypomorphic Npm mutant series [Npm+/− &lt; Npmhy/hy &lt; Npm−/−] in the mouse, and we found that Npm is essential for embryonic development and the maintenance of genomic stability. Npm−/− and Npmhy/hy mutants display aberrant organogenesis and die between E11.5 and E16.5 due to severe anemia resulting from defects in primitive haematopoiesis. Furthermore Npm inactivation leads to unrestricted centrosome duplication and genomic instability. We demonstrate that Npm is haploinsufficient in the control of genetic stability and that Npm heterozygosity accelerates oncogenesis both in vitro and in vivo. Importantly, Npm+/− mice develop a haematological syndrome with features of human MDS. Our findings unravel an essential developmental role for Npm and implicate its functional loss in tumourigenesis and MDS pathogenesis.
The Cyclin-Dependent Kinase Inhibitor p57KIP2 Functions as a Tumor Suppressor Gene in Human Leukemia.
Authors: Kuang Shao-Qing , Ling Xiaoyang , Sanchez-Gonzalez Blanca , Yang Hui , Garcia-Manero Guillermo 
Published: April 2020

Abstract
p57KIP2 is a cyclin-dependent kinase inhibitor with a role in the regulation of cell cycle progression, proliferation, differentiation, and development. Disruption any of these biological processes could result in malignant transformation. We have previously reported that epigenetic inactivation of p57KIP2 by aberrant DNA methylation is associated with a worse prognosis in patients with adult acute lymphocytic leukemia (Blood2003;101:4131–6 and JCO2005;23:3932–9). To further study the role of p57KIP2 in human leukemia we have developed several knock-in and knock-down cellular models of p57KIP2 in human leukemia cell lines. First, we established the methylation/expression patterns of p57KIP2 in 21 leukemia cell lines of both myeloid and lymphoid origin and of different phenotypes. We found that p57KIP2 was methylated and silenced in all except 6 (28%) cell lines. Methylation of the p57KIP2 promoter region correlated with gene silencing, and lack of protein expression, and treatment of methylated/silenced cell lines with a 5-aza-2′-deoxycytidine (DAC), a demethylating agent, resulted in p57KIP2 gene and protein re-expression. In unmethylated p57KIP2 cell lines, p57KIP2 mRNA was rapidly up-regulated by stimulation by growth-inhibitory signals such as TNF alpha, LPS and TGF beta, and when cell-cycle progression was arrested. In contrast, treatment with dexamethasone, DAC or the histone deacetylase inhibitor SAHA did not have such an effect. The reverse was observed in methylated cell lines. Transient transfection of p57KIP2 in silenced cell lines resulted in cell-cycle arrest at the G0/G1 phase. Stable transfection of p57KIP2 using a lentivirus system in Raji cells, in which p57kip2 is methylated and silenced, resulted in the induction of apoptosis and the suppression of cell growth in soft agar. Stable transfection of p57KIP2 in Jurkat cells, in which p57KIP2 is unmethylated and expressed, resulted in partial inhibition of cell growth, and increased susceptibility to the induction of apoptosis by staurosporine. Knock-down of p57KIP2 using an shRNA lentivirus in K562 cells, in which p57kip2 is not methylated and is expressed, promoted cell growth and proliferation in soft agar. These studies provide evidence that p57KIP2 has the properties of a bona fide tumor suppressor gene in human leukemia and provide further weight to the notion that p57KIP2 has an important role in human leukomogenesis.
MLL-AF9 and FLT3 Cooperation during Myeloid Leukemogenesis: Development of a Model for Rapid Testing of New Therapeutics.
Authors: Stubbs Matthew C. , Krivtsov Andrei , Kim Yong-mi , Wright Renee D. , Feng Zhaohui , Agarwal Jyoti , Kung Andrew L. , Armstrong Scott A. 
Published: April 2020

Abstract
Human leukemias harboring chromosomal translocations involving the Mixed lineage leukemia (MLL, HRX, ALL-1) gene possess high-level expression, and frequent activating mutations of the receptor tyrosine kinase FLT3. We used a murine bone marrow transplant model to assess cooperation between MLL translocation and FLT3 activation, and demonstrate that MLL-AF9 expression induces leukemia in approximately 70 days whereas the combination of MLL-AF9 and FLT3-ITD does so in less than 30 days. In both cases, the resulting disease is a highly aggressive, clonal, acute myelogenous leukemia. Detailed immunophenotypic analysis demonstrates expansion of an IL-7Rα− Lin− Sca-1− c-Kit+ CD34int. granulocyte macrophage progenitor (GMP)-like population in both the MLL-AF9 and MLL-AF9/FLT3-ITD induced leukemias. Other progenitor populations such as IL-7Rα− Lin− Sca-1− c-Kit+ CD34+ FcγRII/IIIlo common myeloid progenitors (CMPs) and IL-7Rα− Lin− Sca-1− c-Kit+ CD34− FcγRII/III− megakaryocyte erythroid progenitors (MEPs) are absent in the bone marrow from leukemic mice. Secondary transplantation of splenic cells from diseased mice established that leukemic stem cells are present at a very high frequency of approximately 1:100 in both diseases. Cooperation between MLL-AF9 and FLT3-ITD was further verified by real-time assessment of leukemogenesis using non-invasive bioluminescence imaging. We developed a transgenic mouse model that expresses luciferase under control of the ubiquitin promoter to provide luminescent bone marrow cells that can be used as donor cells in bone marrow transplant assays. In these experiments, the development of leukemia was followed in secondary recipients via bioluminescent imaging. We used this model to confirm cooperation of MLL-AF9 and FLT3-ITD, and to demonstrate that MLL-AF9/FLT3-ITD induced leukemias are sensitive to FLT3 inhibition in a 2–3 week in vivo assay. These data (1) show that MLL-AF9 induces acute myelogenous leukemia with a high frequency of leukemic stem cells and expansion of a GMP-like progenitor population, (2) demonstrate that activated FLT3 cooperates with MLL-AF9 to accelerate leukemogenesis, and (3) provide a new genetically-defined model system that should prove useful for rapid assessment of potential therapeutics in vivo.
Endogenous Thrombin Potential (ETP) for Assessing the Risk of Recurrent Venous Thromboembolism.
Authors: Eichinger Sabine , Hron Gregor , Weltermann Ansgar , Kollars Marietta , Kyrle Paul A. 
Published: April 2020

Abstract
Venous thromboembolism (VTE) is a multifactorial chronic disease. The number and severity of risk factors determine the risk of recurrence. A laboratory method that measures the overall thrombophilia is required.
In a prospective cohort study, we measured ETP in 778 patients with a first unprovoked VTE. Patients were followed after discontinuation of anticoagulants for an average of 49 months. The study endpoint was recurrent symptomatic VTE. ETP was determined by a commercially available (research use only) assay (Dade Behring, Marburg, Germany) in platelet poor plasma by use of a chromogenic substrate and automatic registration as well as computer assisted calculation of thrombin generation over time. Patients with recurrence had higher ETP than those without recurrence (104.2% ± 15.4% vs. 101.4% ±14.2%. Patients with ETP ≥ 100% had an almost two-fold higher relative risk (RR) of recurrence than patients with lower levels (RR 1.6, 95% CI 1.0 – 2.5). At 4 years, the cumulative probability of recurrence was 14.4% in patients with ETP ≥ 100% and 6.1% in those with lower levels (p = 0.05). Patients with ETP ≥ 100% had higher clotting factor levels (Table). ETP was significantly increased in heterozygous carriers of factor II G20210A as compared with patients with wild type factor II (128% ± 18% vs. 100 ± 12%, p &lt; 0.001).
Patients with a first unprovoked VTE and an ETP ≥ 100% have an increased risk of recurrence.
Table Characteristics of 778 Patients with VTE ETP &lt; 100% ETP ≥ 100% p-value Men, no. (%) 160 (42%) 174 (44%) n.s. Age at first VTE (yrs) 46 ± 17 47 ± 14 n.s. Factor V Leiden, no. (%) 124 (32%) 108 (28%) n.s. Factor II G20210A, no. (%) 4 (1%) 49 (13%) &lt; 0.001 Factor VIII (IU/dL) 162 ± 46 168 ± 45 0.09 Factor IX (IU/dL) 113 ± 24 123 ± 27 &lt; 0.001 Factor XI (IU/dL) 102 ± 21 110 ± 24 &lt; 0.001
Deep Vein Thrombosis during Pregnancy in Double Homozygous Carrier of Factor V Leiden and Prothrombin G20210A.
Authors: Martinelli Ida , Battaglioli Tullia , Beretta Angelo L. , Bianchi Marina , Mannucci Pier Mannuccio 
Published: April 2020

Abstract
Background. Factor V Leiden and prothrombin G20210A are gain-of-function mutations leading to an increased risk of venous thrombosis. They are the most common causes of thrombophilia, being present in their heterozygous form in 15–20% for factor V Leiden and 6–15% for prothrombin G20210A of unselected patients with venous thrombosis. The prevalence of each mutation in the Italian general population is 3%. Homozygous carriers for each mutation are rare, and double homozygous carriers, expected in 1 in 1.200.000 individuals, has never been described in the literature so far.
Patient. On March 3, 2005 a 30-year-old woman was admitted to the Unit of Internal Medicine, Valduce Hospital, Como, for swell and pain in the left leg. She was pregnant at the 25th gestational week. Ultrasound examination diagnosed a deep vein thrombosis of the left external and common iliac veins. She had no symptoms of pulmonary embolism. She was given nadroparin 6000 UI b.i.d. (her weight was 63 kg), elastic stockings, and was discharged on March 9, 2005. Because of worsening of the swell in the left leg, she was re-admitted on March 12, 2005 and the ultrasound examination showed an extension of the deep vein thrombosis to the left femoral-popliteal venous system. The dose of nadroparin was increased to 7000 UI b.i.d. Thrombophilia screening revealed a double homozygosity for factor V Leiden and prothrombin G20210A. Her past personal history was negative for thrombosis, but she was never exposed to high risk situations (surgery, bone fractures, prolonged immobilization, oral contraceptive use, long-haul flights) apart from a previous pregnancy which ended in miscarriage at the 6th gestational week. On June 14, 2004 she had an uneventful vaginal delivery after two hours of labour, with the last nadroparin injection in the evening before, The newborn was a female who weighted 3500 g. Her family history was also negative for thrombosis in first- and second-degree relatives despite both parents were double heterozygotes for factor V Leiden and prothrombin G20210A and brother and sister were homozygotes for factor V Leiden and heterozygotes for prothrombin G20210A.
Conclusions. The relative risk of venous thrombosis in double homozygous for factor V Leiden and prothrombin G20210A is unknown but is predicted to be extremely high. However, the patient described here had her first episode during a trigger situation, i.e., pregnancy, and severe thrombophilia seems to run an uneventful cause in her family in spite of repeated exposures to high risk situations. Hence, we shall advise the patient to stop oral anticoagulant therapy, started soon after delivery, one year after the thrombotic event.
Low Protein Z Levels and the Risk of Venous Thrombosis.
Authors: Martinelli Ida , Battaglioli Tullia , Razzari Cristina , Biguzzi Eugenia , Mannucci Pier Mannuccio 
Published: April 2020

Abstract
Background. Protein Z (PZ) is a vitamin-K dependent protein that serves as cofactor for PZ-dependent protease inhibitor, inactivating factor Xa. Therefore, deficiency of PZ could led to a prothrombotic phenotype. Contrasting data are available on the relationship between PZ and venous thromboembolism (VTE).
Patients and Methods. We carried out a case-control study on 443 patients with deep vein thrombosis of the lower limbs and/or pulmonary embolism who discontinued oral anticoagulant therapy and 394 controls, similar for age and sex. PZ was measured with an enzyme immuno-assay (Diagnostica Stago, France). A thrombophilia screening including DNA testing for factor V Leiden and G20210A prothrombin mutation and measurements of plasma antithrombin, protein C, protein S, and homocysteine (fasting and post-methionine load) was performed. Pregnancy, oral contraceptive use, liver or renal disease were exclusion criteria.
Results. 443 patients and 394 healthy controls who underwent thrombophilia screening were investigated. Median PZ levels (range) were 1.95 μg/mL (0.22–6.26 μg/mL) in patients and 1.79 μg/mL (0.25–7.82 μg/mL) in controls (p=0.07). The odds ratios (adjusted for age, sex and the presence of thrombophilia) for VTE in patients with PZ levels in the lowest quartile was 0.8 (95% CI 0.5–1.1), and remained statistically non significant considering PZ levels below the 10th percentile. However, low PZ levels (lowest quartile) increased the risk of VTE associated with factor V Leiden (odds ratio 14.6, 95% CI 1.9–113), prothrombin G20210A (odds ratio 3.2, 95% CI 1.0–10.3), and hyperhomocysteinemia (odds ratio 4.7, 95% CI 1.7–17.0). These interactions remained, apart for prothrombin G20210A, when PZ levels below the 10th percentile were considered.
Conclusion. Low PZ levels are not an independent risk factor for VTE, but may enhance the relative risk due to already established risk factors, particularly factor V Leiden.
Regulated Shedding of sema4D from the Platelet Surface Produces a Bioactive Second Messenger in Thrombotic Disorders.
Authors: Zhu Li , Bergmeier Wolfgang , Wu Jie , Jiang Hong , Yeboah Nana , Fan Ran , Cieslak Marcin , Milla Marcos E. , Brass Lawrence F. 
Published: April 2020

Abstract
Proteins that are expressed on the platelet surface can participate in contact-dependent signaling events which modulate thrombus formation or, after being shed from the platelet surface, serve as bioactive messengers that affect the function of nearby cells. Here we show for the first time that platelets express the class IV semaphorin known as sema4D or CD100, and that platelet activation causes the regulated shedding of the sema4D extracellular domain in a biologically-active form. Sema4D is a glycosylated 150 kDa disulfide-linked homodimer that has previously been implicated in interactions between T-cells and B-cells. Platelet activation by collagen, thrombin or PMA causes a transient increase in sema4D surface expression peaking at 15 min, followed by a complete loss of expression over 30–60 minutes. These events are accompanied by the release of the sema4D exodomain as a 130 kDa fragment, leaving a 25–30 kDa transmembrane and cytoplasmic domain fragment that is retained by the platelets. The cleavage event required to produce these fragments is inhibited by metalloprotease inhibitors and abolished in platelets from chimeric mice lacking the metalloprotease known as ADAM17 or TACE (TNF alpha converting enzyme). Incubation of recombinant sema4D with ADAM17 identified a single cleavage site just outside the predicted transmembrane domain. Western blots show that human platelets express ADAM17 in both its immature (zymogen) and mature (active) forms, and indicate that at least some of the ADAM17 is located on the platelet surface. ADAM17-dependent cleavage of sema4D does not require platelet aggregation, but the rate is accelerated when aggregation is allowed to occur and slowed when aggregation is prevented. Under both sets of conditions, cleavage of sema4D occurs to a greater extent and more rapidly than the ADAM17-dependent cleavage of GP Ib alpha, suggesting that there is a hierarchy of proteolytic events when platelets are activated. In terms of biological impact, the shedding of sema4D following platelet activation raises the possibility that the soluble extracellular domain of sema4D serves as a bioactive messenger. Two receptors for sema4D have been identified previously: CD72, which is present on lymphocytes where it regulates the activity of the tyrosine phosphatase, SHP-1, and plexin-B1, which is expressed in endothelial cells. Western blots suggest that both of these receptors are expressed on human platelets and show that SHP-1 is associated with CD72 in resting, but not activated platelets. Taken together, these results demonstrate that sema4D undergoes regulated ADAM17-dependent shedding when platelets are activated, and suggest that this results in the production of a bioactive form of the molecule that can affect responses in nearby platelets, lymphocytes and endothelial cells at sites of thrombosis.
Megakaryocytes Derived from Human Embryonic Stem Cells: A Genetically Tractable System To Study Megakaryocytopoiesis and Integrin Function
Authors: Gaur Meenakshi , Kamata Tamihiro , Wang Shaohui , Moran Barry , Shattil Sanford J. , Leavitt Andrew D. 
Published: April 2020

Abstract
The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits αIIb and β3, is required for platelet aggregation, spreading and hemostasis. Platelet agonists such as thrombin and ADP lead to activation of αIIbβ3, thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to αIIbβ3 triggers cytoskeletal changes and granule release (outside-in signaling). Genetic approaches to characterize the molecular pathways involved in αIIbβ3 signaling are not possible with anucleate blood platelets. Therefore we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs). αIIbβ3 activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, αIIb+/GPIbα+ cells, is readily detectable following stimulation with known platelet agonists. Dose-response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of αIIbβ3 outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of αIIbβ3 activation. Thus, using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and αIIbβ3 signaling in the native cellular environment.
The Mechanism of Cold-Induced Platelet Hyperfunction.
Authors: Han Qiang , Chen Junmei , Peng Yuandong , Bergeron Angela L. , Liu Li , Dong Jing-fei , Lopez Jose A. 
Published: April 2020

Abstract
Refrigerated platelets function better when transfused than do platelets stored at room temperature, although chilled and rewarmed platelets do not survive long in the circulation. Here, we describe the one possible mechanism of this increase in the hemostatic function of platelets upon cold treatment. Cold exposure has been demonstrated to cluster GP Ib-IX complexes on platelets, enabling platelet recognition and phagocytosis by macrophages. We find that, in addition to increasing the binding to the leukocyte integrin Mac-1 (aMb2), clustering of GP Ib-IX complexes also augments von Willebrand factor (VWF)-related functions. Before the functional studies, platelets were chilled to 4°C for 2 hr and rewarmed to 37°C for 30 min. Chilled, rewarmed platelets, whether in platelet-rich plasma (PRP), whole blood, or reconstituted whole blood (all anticoagulated with Cirtate Sodium) perfused through a parallel-plate flow chamber (36 dynes/cm2) attached in greater numbers to VWF-coated surfaces than did platelets from the same donor kept at 37°C or 22°C. The increased function was also reflected in increased thrombus number and size on a surface of type III collagen. The surface coverage, an indicator of thrombus formation, increased from 10% to 25% upon cold. Chilled, rewarmed platelets also responded to lower doses of a VWF-specific agonist, ristocetin Chilled, rewarmed platelets showed 80% aggregation while control platelet only had 10% aggregation at low concentration of ristocetin (0.5mg/ml) treatment, and displayed a decreased threshold for shear-induced aggregation, aggregating at lower shear rates than control platelets at both 22°C and 37°C. Finally, when tested in a platelet function analyzer (PFA-100), chilled, rewarmed platelets demonstrated significantly shorter closure times than control platelets using both epinephrine/collagen and ADP/collagen cartridges. The epinephrine/collagen cartridges clotting time of chilled platelets decreased from normal 180 s to 162 s and ADP/collagen cartridges clotting time decreased from 118s to 99s. To exclude that the observed functional increases were not due to changes in GP Iba expression or prior platelet activation, we evaluated by flow cytometry the surface levels of GP Iba, P-selectin, and activated αIIbβ3.(antibody PAC-1 binding). We found no differences in any of these parameters between chilled, rewarmed platelets and control platelets. These results suggest that, if their rapid clearance can be prevented, cold-stored platelets may have enhanced hemostatic function of potential benefit in the bleeding patient. The results also caution, however, that trials will be necessary to exclude potential thrombotic consequences of infusing hyperfunctional, refrigerated platelets.
The Marginal Microtubule Coil in the Resting Blood Platelet Is a Dynamic Bipolar Array.
Authors: Italiano Joseph E. , Richardson Jennifer L. , Schulze Harald , Drabek Ksenija , Bulinski Chloe , Galjart Niels , Shivdasani Ramesh A. , Hartwig John H. , Patel Sunita R. 
Published: April 2020

Abstract
The discoid shape of the resting blood platelet is maintained by its marginal microtubule band. Structural studies have concluded that this band is composed of a single microtubule coiled 8-12 times around the cell periphery. To understand the dynamics of the microtubule coil, we took advantage of EB1 and EB3, proteins that highlight the ends of growing microtubules. Immunofluorescence microscopy with anti-EB1 revealed clear staining of numerous (8.7 +/− 2.0, range 4–12) comet-like dashes in the microtubule coil, suggesting the presence of several microtubule plus ends. Consistent with this observation, rhodamine-tubulin added to permeabilized platelets incorporates at multiple (7.9 +/−1.9) points throughout the microtubule coil. To visualize microtubule dynamics in platelets, we retrovirally directed megakaryocytes to express the microtubule plus-end marker EB3-GFP and isolated platelets released in these cultures. Fluorescence time-lapse microscopy of EB3-GFP-expressing resting platelets revealed multiple microtubule plus ends that grew in both clockwise and counterclockwise directions. Antibodies that recognize tyrosinated tubulin, which preferentially label newly assembled microtubules and not stable microtubules, stain the microtubule coil. These results indicate that resting platelets contain a bipolar array of microtubules that undergoes continuous assembly. When EB3-GFP-expressing platelets are activated with thrombin, the number of polymerizing microtubules increases dramatically and the microtubules grow into filopodia. Collectively, these results suggest that the marginal band of the resting blood platelet is highly dynamic, bipolar, and contains multiple microtubule plus ends. These ends are amplified in platelet activation and point towards the active edges of the cells and the tips of filopodia.