bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stage-Specific Switches in Alternative Pre-mRNA Splicing during Late Erythropoiesis Are Conserved from Mouse to Human
Authors: Gee Sherry , Villalobos Jonathan , Yamamoto Miki , Clark Tyson A. , Kang Jeong-Ah , Wickrema Amittha , Chasis Joel Anne , Conboy john G. 
Published: April 2020

Abstract
Spatial and temporal regulation of alternative pre-mRNA splicing determines which exons are incorporated into mature mRNA, modulating mRNA coding capacity to ensure synthesis of appropriate protein isoforms throughout normal differentiation and development. During erythropoiesis, a stage-specific switch in pre-mRNA splicing activates incorporation of protein 4.1R exon 16, thereby increasing 4.1R affinity for spectrin and actin and mechanically strengthening red blood cell membranes. We are exploring the hypothesis that stage-specific changes in pre-mRNA splicing regulate expression of other critical genes during terminal erythropoiesis. Last year we described exon microarray and RT-PCR studies that revealed several novel pre-mRNA splicing switches in terminally differentiating human erythroid progenitors. These alternative splicing events involved well-annotated exons with consensus exon-intron boundaries, supporting a model in which these events represent a regulated alternative splicing program rather than a breakdown of splicing integrity in late erythropoiesis. Here we report additional evidence for this model by showing that several erythroid stage-specific switches in alternative pre-mRNA splicing are conserved between human and mouse. Primary mouse splenic erythroblasts from FVA-infected mice were cultured in vitro under differentiation conditions and used as the source of RNA for analysis of murine erythroid splicing events. From a total of seven internal cassette exons whose splicing was activated in late human erythroblasts, five exhibited an analogous splicing switch in murine erythroblasts. Comparative genomic analysis showed that these alternative exons are embedded in regions of unusually high sequence conservation among vertebrate species, suggesting that important regulatory signals are contained within the adjacent introns. Indeed, the flanking introns for several of these exons contain binding motifs for Fox2, an RNA binding protein and known splicing regulator for many tissue-specific splicing events. Further analysis of the conserved erythroid splicing events revealed the following:
three splicing switches occur in transcripts encoding RNA binding proteins (MBNL2, HNRPLL, and SNRP70), suggesting significant changes in the RNA processing machinery of late erythroblasts; and three of these alternative exons encode premature stop codons that could induce nonsense mediated decay (NMD) and contribute to down-regulation of these genes during terminal erythropoiesis.
Consistent with the latter hypothesis, inhibition of NMD in murine erythroblast cultures led to increased accumulation of mRNA isoforms containing the premature stop codons. Together these results suggest the existence of a highly regulated alternative splicing program that is critical for late erythroid differentiation.
Activation of TAO2 Kinase by Arsenic Trioxide in Leukemic Cells
Authors: McNeer Jennifer L. , Dolniak Blazej , Kroczynska Barbara , Sassano Antonella , Platanias Leonidas 
Published: April 2020

Abstract
Arsenic Trioxide (As2O3) has major efficacy in the treatment of acute promyelocytic leukemia (APL), but its use in other malignancies is limited by the need for high intracellular concentrations to induce apoptosis. Prior work in our laboratory has demonstrated that the p38 MAP kinase (MAPK) pathway is activated following treatment of cells with As2O3 and exhibits negative regulatory effects on As2O3-induced apoptosis and growth suppression. In the current study, we sought to identify upstream effector mechanisms by which the p38 pathway is activated by As2O3 in leukemic cells. We found that the MAPK kinase kinase TAO2 (thousand and one amino acid protein kinase 2) is phosphorylated on Ser181 after treatment of NB4, NB4.306, and U937 cells with arsenic. Such phosphorylation was rapid, occurring as early as after 5 minutes of As2O3 treatment. In addition, our data indicate that such phosphorylation occurs downstream of As2O3-induced redox reactions, as demonstrated by increased phosphorylation in cells pretreated with the oxidizing agent buthionine sulfoximine (BSO) and decreased phosphorylation following pretreatment with the reducing agent dithiothreitol (DTT). Arsenic treatment of the cells also resulted in activation of the kinase domain of TAO2, as evidenced in in vitro kinase assay studies using ATF2 as an exogenous substrate. siRNA-mediated TAO2 knockdown resulted in inhibition of As2O3-induced p38 phosphorylation, suggesting that this kinase acts as an upstream effector of the arsenic-activated p38 MAPK pathway. Moreover, in studies to determine the functional relevance of TAO2 in the induction of As2O3-dependent antileukemic responses we found that siRNA-mediated TAO2 knockdown enhanced the suppressive effects of As2O3 on KT1-derived leukemic progenitor (CFU-L) growth in clonogenic assays in methylcellulose. Altogether, our data demonstrate that TAO2 is activated during arsenic treatment of leukemic cells lines and acts as an upstream activator of the p38 MAPK pathway. Such activation appears to occur in a negative feedback regulatory manner to compensate for the suppressive effects of As2O3 on leukemic cell growth. Importantly, these findings raise the possibility that targeting TAO2 may provide a novel approach to enhance the generation of the antileukemic properties of As2O3.
Differential Glucose Transporter Expression during Perinatal and Postnatal Erythropoiesis
Authors: Montel-Hagen Amelie , Blanc Lionel , Boyer-Clavel Myriam , Jacquet Chantal , Vidal Michel , Sitbon Marc , Taylor Naomi 
Published: April 2020

Abstract
Glucose is a major source of energy for living organisms and its transport in vertebrates is a universally conserved property. Of all cell lineages, human erythrocytes express the highest level of the Glut1 glucose transporter with &gt;200,000 molecules/cell. However, we recently reported that erythrocyte Glut1 expression is a specific trait of vitamin C-deficient mammalian species, comprising only higher primates, guinea pigs and fruit bats (Montel-Hagen et al., Cell, 2008). We now show that in all other tested mammals, including mice, rats, dogs and cows, Glut1 is in fact transiently expressed in erythrocytes during the neonatal period. This is in marked contrast with humans, where Glut1 is present at equivalently high levels on both neonatal and adult RBC. In mice, we found that Glut1 expression was not associated with primitive erythropoiesis but was highly expressed during definitive fetal erythropoiesis. Indeed, this transporter was present at significantly earlier stages of erythropoiesis in fetal spleen and liver than immediately following birth. It was therefore important to determine whether erythrocyte Glut1 expression in mice is specifically associated with fetal erythropoiesis or alternatively, is common to any physiological state where an extensive erythropoiesis is provoked. Induction of a hemolytic anemia in adult mice resulted in a massive erythropoiesis with significant increase in glucose uptake but notably, Glut1 was not detected. Rather, in these conditions as well as following birth, Glut4, an insulin-sensitive transporter previously thought to be responsible for glucose uptake in muscle and adipose tissue, was highly expressed. Following birth, the concomitant repression of Glut1 and induction of Glut4 was associated with a significantly augmented ratio of the Sp3 to Sp1 zinc-finger transcription factors. Thus, in contrast to humans, murine Glut1 is highly expressed during definitive erythropoiesis and is then downregulated at birth. Further erythroid development is characterized by the upregulation of a distinct glucose transporter, Glut4. Expression of distinct glucose transporters in nonhuman erythrocytes, regulated at the transcriptional level, therefore characterizes different states of erythroid development and differentiation.
Characterization of Leukemia-Inducing Genes Using the Retroviral cDNA Library
Authors: Jang Suhwa , Lee Young , Kim Byoung Kook , Chung Hee Yong 
Published: April 2020

Abstract
Acute myeloid leukemia is a disease caused by oncogenic change(s) of hematopoietic stem cells or progenitor cells. The purpose of this research is to characterize the gene involved in the leukemia induction processes by screening the retroviral cDNA libraries of cellular oncogenes, homeobox genes. The cDNA libraries were constructed by cloning the individual genes to a MSCV retroviral vector backbone. For convenience of detecting the transduced cells and their protein products, the MSCV retroviral vector was modified to include HA tag and GFP marker and ninety cellular oncogenes and 30 homeobox genes were individually cloned and the structural integrity was verified. To screen for the leukemia-inducing genes, 5-FU treated mouse bone marrow cells were transduced with retroviral mixtures of oncogenes, and the cells were transplanted into ten lethally irradiated mice. All ten mice developed acute leukemia between the eight and ten weeks post-transplantation. The oncogenes that were responsible for the leukemia induction were characterized by genomic DNA PCR of the leukemic cells of each mouse. Surprisingly, all ten mice had c-myc genes in their leukemic cells. However, except for three mice, all the mice have additional oncogenes within the leukemic cells. The list of the additional genes include; RAB3D, RAB7B, PDGF-beta, CRK, and PIM-2 and Ras. Theses results show that the c-myc is the major leukemia-inducing oncogenes in our system. In addition, since the initial transduction rate of 5-FU-treated bone marrow just prior to in vivo transplantation was 10.56%, it is highly unlikely that all these additional oncogenes were present in the transplantable leukemic cells just by chance. Therefore, the retroviral cDNA library-mediated leukemia induction system we developed may be an useful system in systematically screening the cooperating oncogenes in leukemia induction. We are currently verifying the cooperating potential of the genes co-transduced with c-myc in inducing leukemia in the same animal model.
Risk Determinants of Arterial Thrombosis Increase the Risk of Intrauterine Growth Restriction (IGR)
Authors: Gerhardt Andrea , Scharf Rudiger E. , Zotz Rainer B. 
Published: April 2020

Abstract
Background: The HPA1-b allele of the beta3-subunit of the essential platelet integrin AlphaIIb-beta3 is a risk factor for increased platelet thrombogenicity, leading to arterial vascular occlusion also triggered by inflammatory endothelial alterations. We performed a case-control study to assess hereditary risk factor for arterial thrombosis in addition to the known risk determinants for venous thrombosis as risk determinants for fetal IGR.
Materials and Methods: 116 women with severe fetal IGR (defined by birth weight below the 5th-percentile for gestational age and sex) and 300 normal women were evaluated. Women with other reasons of IGR (history of venous thrombosis, fetal loss, and preeclampsia, neonatale immunthrombocytopenia) were excluded. The fetuses were born alive after the 24th week of gestation.
Results: A significant risk association could be shown for the HPA-1b/1b genotype OR 3.1 (95%CI 1.2–8.8), increased levels of lipoprotein (a) (&gt;0.2g/l) OR 1.7 (95%CI 1.0–2.9), increased levels of fibrinogen (&gt;median 284mg/dl) OR 2.8 (95%CI 1.8–4.4) increased levels of FVIII:C (&gt;median 147%) OR 1.7 (95%CI 1.1–2.7), and increased levels of von-Willebrand-factor antigen (vWF-Ag) (&gt;median) OR 2.7 (95%CI 1.1–2.7). The combination of risk determinants led to a further increase in risk (e.g. HPA-1b allele, increased lipoprotein (a) and vWF-Ag OR 14.0) There was no significant risk association for factor V Leiden G1691A OR 1.3 (95% CI 0.7–2.81), and prothrombin G20210A mutation. OR 2.0 (95% CI 0.6–6.5)
Conclusions: Increased platelet thrombogenicity via interaction with an inflammatory vascular process and/or via plasma levels of components of hemostasis/receptor ligands appear to be more important in women with IGR than the established risk determinants for venous thrombosis.
Irinotecan and Increased Triglyceride Level in Paraneoplastic Deep Venous Thrombosis (DVT): A Strange Link in An Old Problem
Authors: Giordano Giulio , Farina Giuliana , Tambaro Rosa , Piano Silvia , Papini Stefano , Zappacosta Bruno , Storti Sergio 
Published: April 2020

Abstract
DVT is present in about 3–15% of cancer patients. Paraneoplastic thrombosis pathogenesis is multifactorial. Chemotherapy is frequently involved in DVT genesis as pro-inflammatory/pro-thrombotic factor. There are few data about incidence of DVT in patients treated with Irinotecan. Aim of our study is to define if thrombotic risk in patients treated with Irinotecan is increased and then which mechanism is probably involved. For this purpose we considered in our patients the complement fractions C3 and C4 and the immune circulating complex (ICC), because they activate macrophages and platelets and increase tissue factor level. Moreover we evaluated also total cholesterol and triglyceride level, cause linked with factor VII activation. We considered C3, C4, ICC, cholesterol and triglyceride level, dose density and chemotherapeutic agents used in 116 patients with solid neoplasms (62colon, 26lung, 18gastric,) and not receiving anticoagulant prophylaxis. Median age was 68.5 years (R 57–83). M/F ratio was 66/40. The threshold value of third quartile was chosen as risk cut-off (C3: 130mg/dl; C4: 32mg/dl; ICC: 2.9 mcg/ml; total cholesterol: 205mg/dl; triglycerides 120mg/dl). The statistical analysis was conducted with Yates corrected chi square test, Fisher’s exact test, Odds Ratio (OR), relative risk (RR). Twenty-four patients (20%) developed DVT. The patients treated with Irinotecan were 42. DVT occurred in 14 of these patients (32%) and in 10 of the 74 patients treated without Irinotecan (14%), with a Yates corrected chi square test of 4.6 (p=0.03), a Fisher’s exact test with p=0.03, an OR of 2.98 (95%CI 1.2–7.3) and a RR of 2.35 (95%CI 1.1– 4.8). C-Reactive Protein and Erythocyte Sedimentation Rate at Pearson’s test were not related with C3, C4, ICC, total cholesterol and triglyceride levels. All patients treated with Irinotecan had advanced stage colon cancer. High levels of triglycerides (&gt;120mg/dl) were present in 17 out of 42 patients treated with Irinotecan (40%) and in 17 out of 74 patients treated without Irinotecan (23%) with a Yates corrected chi square test of 3.1 (p=0.07), a Fisher’s exact test with p=0.05, an OR of 2.2 (95%CI 1–5) and a RR of 1.7 (95%CI 1–3). Although most patients developing DVT have an advanced stage colon cancer and so receiving Irinotecan, the influence of chemotherapy in DVT pathogenesis in our study cannot be excluded. Probably Irinotecan could contribute to DVT increasing triglyceride levels that are linked with in vivo factor VII activation. This relief suggests also that risk factors, other than classical, should be considered in the thrombosis onset; then other drugs (i.e. hypolipemizing drugs) could be useful in paraneoplastic DVT prevention. Nevertheless these data should be confirmed in a larger cohort of patients.
Platelet Reactivity Is Associated with VAMP8 Expression and a VAMP8 3′UTR Polymorphism
Authors: Kondkar Altaf A , Dajiang Liu , Leal Suzanne M , Nagalla Srikanth , Jin Ying , Khan Farhan , Bray Paul F 
Published: April 2020

Abstract
Variations in platelet reactivity can promote either a hemorrhagic or thrombotic phenotype, and platelet granule secretion is essential for platelet activation and normal hemostasis. We have earlier rigorously phenotyped platelet reactivity in 337 healthy subjects and found excellent reproducibility for secretion-dependent platelet functions. Vesicle associated membrane protein (VAMP)-8, a v-SNARE, is the primary regulator of platelet secretion. Our goals were to determine whether the expression of 9 key molecules involved in the platelet secretory pathway – SNAP23, SNAP25, VAMP3, VAMP8, STX2, STX4, STX7, STXBP3, and RAB27B are associated with the well-known variability in platelet reactivity and investigate potential mechanisms. Leukocyte-depleted platelets were prepared from 10 subjects with hyperreactive platelets and 6 subjects with hyporeactive platelets. No significant variations were observed for SNAP25, STX2, STX4 and RAB27B mRNA levels. Although small variations were observed for other genes examined VAMP8 mRNA levels showed the highest variation. VAMP8 mRNA levels were 4.8-fold higher in hyperreactive platelets than hyporeactive platelets (P=0.0023). Immunoblotting showed that VAMP8 proteins were also significantly elevated by 2.5-fold in the hyperresponsive subjects (P = 0.04). An A-to-G transition in the 3′UTR region of the VAMP8 gene (rs1010) has been consistently shown to be associated with a high risk of myocardial infarction (MI) in European Americans [EA]. We genotyped all 337 subjects (182 EA and 155 African Americans [AA]) in our cohort for this rs1010 SNP. The ‘G’ allele frequency was 0.44 and 0.62 in EA and AA, respectively. The ‘G’ allele was associated with enhanced aggregation to epinephrine (P=0.001) and ADP (P=0.001) in an age dependent manner. The 3′ UTR of VAMP8 has at least 10 predicted microRNAs (miRNAs), 8 of which we have identified in platelets and 5 of which encompass the rs1010 SNP. In summary, variable VAMP8 expression levels appear to be a major regulator of platelet reactivity, and a common VAMP8 SNP known to be associated with MI is associated with the platelet aggregation response to epinephrine and ADP. Studies are currently on-going to assess whether this SNP modifies miRNA binding and whether this miRNA regulates VAMP8 expression.
Caffeic Acid Phenyl Ester, a Component of the Chinese Herb Propolis, Inhibits Platelet Aggregation Via Competition with Fibrinogen for Binding to aIIbb3
Authors: Wu Ya Ping , Huisman Albert , Weeterings Cees , de Groot Philip G. , Zhang Yun Xiang , PAN Shao Dong , Wei Ran , Wang Jia Fu 
Published: April 2020

Abstract
Background: Propolis is a resinous substance collected by honeybees from various plant sources. Honeybees collect resin from cracks in the bark of trees and leaf buds, masticate it and the partially digested material is mixed with beeswax. The precise composition of propolis varies with the source. There is a long history of the use of propolis, at least to 300BC and its use continues till today. Propolis has a reputation to have a large number of healthy properties among others anti-thrombotic properties. Over 180 different compounds have been identified in propolis, among others caffeic acid phenyl esters (CAPE). We have investigated the effects of CAPE on platelet aggregation.
Results: Platelet aggregation was induced with ADP, collagen or the thrombin analogue TRAP. Different flavoid components present in propolis (CAPE, galangin, acacetin, pinostrobin) were able to inhibit platelet aggregation, CAPE was the most potent one, especially for the collagen induced platelet aggregation. We examined the effect of 100 mM CAPE on the expression of a number of platelet membrane activation markers after addition of collagen and we observed that CAPE inhibited fibrinogen binding and the binding of LIBS antibody against aIIbb3 for 93% and 82%, respectively, while the expression of P-selectin was only inhibited for 37%. To explain this discrepancy, we studied the binding of CAPE to purified aIIbb3 with a Biacore. CAPE binds to aIIbb3 with an affinity constant of about 400 μM. In an ELISA set-up CAPE was able to inhibit the binding of fibrinogen to purified aIIbb3 (figure).
Figure Figure
Conclusion: Propolis contains components such as CAPE that inhibit platelet aggregation via competition with fibrinogen for the binding to aIIbb3.
Interaction Between Genotypes for HFE and TFR2 Genes Mutations and Iron Status in Brazilian Blood Donors
Authors: Cancado Rodolfo D , Santos Paulo CJL , Rostelato Samuel , Terada Cristiane T , Gonzales Iris , Hirata Rosário DC , Hirata Mario H , Chiattone Carlos , Guerra-Shinohara Elvira M 
Published: April 2020

Abstract
Hereditary hemochromatosis (HH) is a disorder characterized by increased intestinal iron absorption, which leads to a progressive accumulation of iron in the body. This iron overload has been associated with mutations in HFE gene (C282Y, H63D and S65C) and other genes. The objectives of this study were to assess the frequencies of functional mutations in HFE and TFR2 genes and to investigate their relationship with the iron status in a sample of blood donors. Blood donors (n=542) were recruited at the Hemocenter of the Santa Casa Hospital, Sao Paulo, Brazil. The genotypes for HFE (C282Y, H63D and S65C) TFR2 (Y250X and Q690P) gene mutations were evaluated by PCR-RFLP. The concentrations of serum iron and total iron-binding capacity (TIBC) were measured by automation system Advia®(Bayer Diagnostics) and serum ferritin by Axsym System®(Abbott Laboratories). The frequencies of HFE 282Y, HFE 63D and HFE 65C alleles were 2.1, 13.6 and 0.6%, respectively. The frequency C282Y allele (2.1%) in Brazilian blood donors is lower than that observed in blood donors from Northern Europe (5.1 to 8.2%, P&lt;0.05). The TFR2 250X and TFR2 690P alleles were not found in these subjects. The iron status was similar between HFE genotypes in women. However, men carrying HFE 282CY genotype had higher serum ferritin and lower TIBC concentrations when compared to the HFE 282CC genotype carriers. HFE 282CY genotype was also associated with higher transferrin saturation in men who donated blood at the first time. Moreover, male donors with HFE 63DD plus 63HD genotypes had higher serum iron and transferrin saturation when compared to those with HFE 63HH genotype. A relationship between HFE CY/HH/SS haplotype and lower TIBC concentrations was also found in men. The HFE 282Y and HFE 65C alleles were rare while the HFE 63D was frequent in blood donors. The mutations in TFR2 gene were not found in this study. The HFE 282Y and HFE 63D alleles were associated with alterations on iron status only in male blood donors.