bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Novel C to A Substitution in the CCAAT Box of beta Globin Gene.
Authors: Refaldi Chiara , Di Pierro Elena , Mocellini Maria C. , Cappellini Maria D. 
Published: October 2019

Abstract
The promoter of the human beta-globin gene contains three positive cis-acting elements required for maximal transcription: the CACCC box located between −86 and −90, the CCAAT box located between −72 and − 76 and the TATA box located between −28 and −31 relative to the start site of transcription. Naturally occurring mutations within the TATA and the CACCC box regions have been recorded in patients with beta+ thalassemia. Mutations within the TATA box disrupt assembly of the basal transcription complex, while mutations at the CACCC box prevent binding of an erythroid-specific transcription factor EKLF. Surprisingly, no mutations have so far been identified in the highly conserved element CCAAT box and the transcription factors responsible for the regulatory activity of the CCAAT site in vivo have been less intensively studied. We report a novel mutation −76 C>A (HBB c. −126) detected by sequencing analysis of beta globin gene in a Italian beta+ thalassemic patient. The transversion C>A hits the first nucleotide in the CCAAT box of the beta globin gene. The carrier, a male 44 years old, shows a mild hypochromic and microcytic anaemia with reduced mean corpuscular volume and mean corpuscular haemoglobin (MCV 75 fl, MCH 25 pg) and Hb A2 level slightly increased (3.9%). Recently, studies in vitro in gel-shift and reporter assays, investigating the transcriptional activity of human beta globin CCAAT box, have identified five factors: NF-Y (CP1) a ubiquitous CCAAT box binding complex, GATA-1 an erythroid-specific transcription factor, C/EBPbeta, C/EBPgamma and C/EBPdelta members of CCAAT/enhancer-binding protein family involved in hemapoietic regulation. This represents the first report of a natural mutation of the human beta-globin CCAAT box and confirms its functional significance for in vivo transcription.
Contribution of Platelets and Erythrocytes to Thrombin Generation in Patients with β-Thalassemia.
Authors: Valles Juana , Piñon Marta , Dasi Maria A. , Moscardo Antonio , Fuset Maria P. , Latorre Ana , Santos Maria-Teresa 
Published: October 2019

Abstract
Patients with β-thalassemia show an abnormally high proportion of red blood cells (RBCs) with exposed phosphatidyl serine (PS) molecules on the RBC membrane, which may contribute to thrombophilia in these patients (1). We have reported previously (2) that thromboxane A2 and free-arachidonate—substances released from activated platelets—induce PS exposure in a subpopulation of normal RBCs. The aims of the present study were first, to evaluate the presence of activated circulating platelets in patients with β-thalassemia, and second, to determine tissue factor (TF)-initiated thrombin generation in plasma and the influence of RBCs, platelets and their cooperation on this process in normal subjects and patients with β-thalassemia. Thrombin generation was assessed according to Hemker et al. (3) in 12 patients with β-thalassemia diagnosed by molecular biology, age 5–33 years and in 18 normal subjects. Two patients had thalassemia major (T-M) requiring blood transfusion every 3 weeks and the analysis were performed just prior transfusion. Two had thalassemia intermedia (T-I) with esplenectomy without transfusion requirements and eight had thalassemia minor (T-m). Thrombin generation was evaluated in platelet-poor plasma (PP), platelet-rich plasma (PRP), RBCs in the plasma (P-RBC) and in the presence of both cell types (PRP+RBC). The presence of activated platelets in circulation was evaluated in whole blood by flow cytometry. We monitored P-selectin (CD-62) and the activated αIIbβ3 receptor, defined as PAC-1 binding. The percentage of platelets exposing activation markers CD-62 (mean± SEM, 0.72± 0.17 vs. 4.03± 1.39) and PAC-1(0.75± 0.14 vs. 12.26± 6.47) were significantly higher in patients than in control subjects (P < 0.05). In normal subjects, the presence of erythrocytes in plasma (P-RBC) triggered a quicker thrombin generation and a significant elevation in the thrombin peak (32 ± 5 nM in PP vs. 86 ± 16 nM in P-RBC; P < 0.05), in the absence of cell lysis. These effects of RBCs were markedly higher in the patients with β-thalassemia, who had 2.5-fold higher thrombin generation at 3 min and higher peak thrombin levels (86 ± 16 nM in controls vs. 130 ± 15 nM in patients; P < 0.05). Thrombin generation in PRP was also higher in the patients with thalassemia at early times (3 min; 19 ± 3 nM in controls vs. 43 ± 8 nM in patients; P < 0.05). Importantly, the rate and peak of thrombin generation were significantly amplified by the presence of both cell types (PRP+RBCs), suggesting a biochemical cooperation between activated platelets and RBCs for thrombin generation. This was enhanced in patients with β-thalassemia, particularly those with T-I. In conclusion, chronic platelet activation in patients with β-thalassemia may influence TF-induced thrombin generation in platelets and erythrocytes. Thrombin generation appears to be a novel mode of platelet-erythrocyte cooperation in thrombogenesis.
Liver Iron Measurement by SQUID Compared to Liver Biopsy.
Authors: Pakbaz Zahra , Fischer Roland , Butz John , Foote Drucilla , Hackney-Stephens Ekua , Fung Ellen , Fagaly Bob , Harmatz Paul , Vichinsky Elliott 
Published: October 2019

Abstract
Liver biopsy, which is the gold standard for liver iron assessment in patients with hemosiderosis, is painful, associated with risk of infection or bleeding, and may be affected by uneven iron distribution and variation in analytical methods. Monitoring iron overload by serum ferritin is still a routine practice although its limitations are well known. Therefore, using non-invasive liver iron assessment by quantitative MRI or by liver susceptometry with a SQUID biomagnetometer (BLS) would be the better alternatives. This report explores the accuracy of liver iron concentration (LIC) measured by BLS at CHRCO compared to liver biopsy, in patients with thalassemia and sickle cell disease. A total of 33 patients with chronically transfused thalassemia (n=16) or sickle cell disease (n= 17) were prospectively assessed for LIC measured by BLS and liver biopsy within 2 month of each other. LIC was measured by a low temperature SQUID biosusceptometer system (Ferritometer®) under the standardized Hamburg-Torino-Oakland protocol. Iron in fresh tissue liver biopsy samples (with a dry weight larger than 1 mg) was measured at Mayo clinics by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Median value for the wet to dry weight of the tissue samples was 3.8 (3.1–6.2). Subjects ranged in age from 7 to 40 years (median: 18 y). Median LIC (wet weight) measured by SQUID was 1915 (415–4826) [mg/g wet weight ], median LIC by biopsy (dry weight) was 12681(1854–32864) [mg/g dry weight]. Serum ferritin ranged from 405 to 9843 (median: 1831). The Spearman rank correlation between wet weight LIC assessed by BLS and dry weight LIC from fresh tissue sample measured by ICP-MS was highly significant (RS=0.93, p<0.0001). The conversion factor between the two methods obtained from a weighted linear regression with zero intercept was 6.1 ± 0.3 (R2 = 0.92, cn2 = 0.5), while the median value for wet-to-dry weight ratio of the actual samples is 3.8. This conversion factor agrees with the value reported earlier by Piga et al (Blood2005; 106(11): 755a). The role of the wet-to-dry weight ratio needs to be explored further as the conversion factor generated from the linear regression is significantly larger.
Fig1. LInear regression LIC (BLS)=(0.165 ± 0.008) *LIC (biopsy) weighted by the uncertainties to the two methods (R2 = 0.92, reduced chi-square χ2= 0.5). Fig1. LInear regression LIC (BLS)=(0.165 ± 0.008) *LIC (biopsy) weighted by the uncertainties to the two methods (R2 = 0.92, reduced chi-square χ2= 0.5).
Bcl11a Causes Leukemia/Lymphoma in Cooperation with Nf1 Inactivation.
Authors: Yin Bin , Jeske-Geurts Jennifer L. , Diers Miechaleen D. , Shannon Kevin M. , Largaespada David A. 
Published: October 2019

Abstract
Loss of the NF1 (Neurofibromatosis Type 1) tumor suppressor gene is associated with chronic and acute forms of myeloid diseases including juvenile myelomonocytic leukemia (JMML). In the BXH-2 strain of mice, acute myeloid leukemia (AML) is caused by chronic infection by a murine leukemia virus (MuLV). We have reported the identification of 37 common proviral insertion sites (CIS), loci containing candidate genes whose mutation could cooperate with inactivation of Nf1 to result in BXH-2 AML. Among these candidate genes, Bcl11a, was shown to be expressed in JMML and CMML leukemia samples, and to confer a growth advantage when expressed in Nf1-deficient myeloblast cells. In this report, we have first tried to determine whether dysregulation of Bcl11a affects cellular behavior other than promoting cell growth. Using a granulocyte-colony stimulating factor induced myeloid terminal differentiation of 32D cell culture model, we found no difference in the percentage of differentiated cells between Bcl11a-transduced cells and empty vector-transduced cells, indicating overexpression of Bcl11a may have no influence on myeloid differentiation. Moreover, no effect of Bcl11a on 32D survival after IL-3 withdrawal was detected. To investigate the potential role of Bcl11a in the development of leukemia with Nf1 loss, a bone marrow transplantation assay was then performed. Bone marrow cells were collected from the Nf1(+/+) or Nf1(Flox/Fcr) donor mice carrying pIpC-induced expression of Cre, and transduced with Bcl11a-expressing retroviral construct or empty vector, before transplantation via the tail vein into the lethally irradiated recipient BoyJ mice. Successful bone marrow engraftment, pIpC-induced deletion of Nf1 and delivery of Bcl11a were confirmed by PCR on peripheral blood cells. Interestingly, 5 months after pIpC induction, 10 out of 11 Nf1(Flox/Fcr);Cre+;Bcl11a-transduced bone marrow recipient mice became moribund, whereas no or only 1 out of 10 or 11 recipient mice in each control groups died for unknown reason. The Nf1(Flox/Fcr);Cre+;Bcl11a-transduced mice display myeloid blast-like cells in their peripheral blood, enlarged lymph node and liver, and splenomegaly. Spleen touchpreps also showed excessively infiltration of blast-like cells in their spleens. Unexpectedly, two out of ten moribund recipient mice also developed markedly enlarged thymi. These tumors were demonstrated to be derived from transplanted donor cells by immunophenotyping for the presence of the differential cell surface marker CD45.2, expressed on the donor cells. The nature and cell lineage origin of these tumors are under study. These data show that BXH-2 mouse is a powerful genetic system to dissect Nf1-cooperating genetic events in leukemogenesis, and suggest that overexpression of Bcl11a can induce leukemia/lymphoma with Nf1 gene inactivation.
Interleukin 21-Based Therapy Can Induce Human B Cells To Produce Granzyme B and Express Other Features of Cytotoxic Lymphocytes.
Authors: Jahrsdoerfer Bernd , Blackwell Sue M. , Weiner George J. 
Published: October 2019

Abstract
B cells are not currently known to be capable of producing granzyme B or being cytotoxic. We recently found that human B cells activated with Interleukin 21 (IL-21) and antibodies to the B cell receptor (BCR) or immunostimulatory oligonucleotides (CpG ODN), can produce granzyme B. Further studies were done to assess the biological and potential therapeutic significance of this finding. Granzyme B ELISpot, intracellular staining for granzyme-B, quantitative real time RT-PCR for granzyme B messenger RNA and gene expression array confirmed B cells obtained from the peripheral blood of normal individuals (Fig. 1) and many B cell lines including Namalwa, Daudi, Ramos and EBV-transformed lymphoblasts, can be induced to produce granzyme B. This granzyme B is functional as demonstrated by cleavage of a granzyme B-sensitive colorimetric substrate. IL-21 based treatment also increased the transcription of the gene for perforin and the production of Interferon-γ in select B cell populations. We conclude that IL-21 based therapy can induce B cells to produce functional granzyme B and other components known to be present in the cytotoxic granules of CTL and NK cells. These unexpected findings could have significant implications on our understanding of the role of B cells in immune regulation and for a variety of immune phenomena including auto-, cancer and infectious immunity.
Figure 1. Interleukin 21 (IL-21) induces de-novo synthesis of Granzyme B by activated B cells. Peripheral blood mononuclear cells (PBMC) were stimulated with the above depicted agents for 18 hours. After further incubtion with Brefeldin A for 4 hours cells were harvested, fixed, permeabilized, stained with PE- and PE-Cy5-labeled antibodies against Granzyme B and CD19, and analyzed flowcytometrically. Figure 1. Interleukin 21 (IL-21) induces de-novo synthesis of Granzyme B by activated B cells. Peripheral blood mononuclear cells (PBMC) were stimulated with the above depicted agents for 18 hours. After further incubtion with Brefeldin A for 4 hours cells were harvested, fixed, permeabilized, stained with PE- and PE-Cy5-labeled antibodies against Granzyme B and CD19, and analyzed flowcytometrically.
Effects of Niflumic Acid on Thrombocytic Cytoplasmic Free Calcium and Platelet Aggregation in Patients with Diabetes Mellitus.
Authors: NIE Danian , Yin Songmei , Xie Shuangfeng , Wang Xiaogang , Li Yiqing , Ma Liping , Wang Xiuju , Wu Yudan , Feng Jianhong 
Published: October 2019

Abstract
Background: Platelet activation plays an important role in cardiovascular complications which are frequently seen in patients with diabetes mellitus. It has been demonstrated that platelets are hyperactive in diabetes patients and this is supposed to be due to high platelet cytoplasmic free calcium ion concentration ([Ca2+]i). There are chloride channels on the membranes of platelets and the activation of chloride channels may participate in the transportation of calcium. Our previous study also showed that niflumic acid (NFA), a chloride channel blocker, can inhibit the elevation of platelet [Ca2+]i in healthy volunteers. The goal of this study was to investigate effects of NFA on platelet [Ca2+]i and platelet aggregation rate (PAG) in diabetic patients.
Methods: We prospectively enrolled 17 consecutive patients with diabetes mellitus at diagnosis. The control group was formed from 17 healthy volunteers. Platelet [Ca2+]i was detected by Fura-2 fluorescent technique and PAG was detected by platelet aggregometer. We studied the effects of NFA, nifedipine, a calcium channel blocker, and their combining effects on platelet [Ca2+]i and PAG in diabetic patients.
Results: The PAG, platelet resting [Ca2+]i, Ca2+ release and Ca2+ influx in diabetic patients were significantly higher than control group(P<0.05). NFA and nifedipine reduced the platelet Ca2+ influx which induced by thrombin in a dose-dependent manner in diabetic patients. 50% inhibition concentration (IC50) of NFA was 50μmol/L. At this concentration, NFA reduced platelet [Ca2+]i by (54.7±14.5)% and decreased PAG by (32.3±21.4)%. The IC50 of Nifedipine was 7.5μmol/L. At this concentration, nifedipine reduced platelet [Ca2+]i by (17.9±11.9)% and decreased PAG by (32.3±20.4)%. NFA(50μmol/L) combining with nifedipine (7.5μmol/L) inhibited platelet Ca2+ influx induced by thrombin in diabetes patients and their effects were independent.
Conclusions: Platelets in patients with diabetes mellitus are hyperactive. NFA reduces the PAG and platelet [Ca2+]i influx possibly by blocking the chloride channels on platelet membrane in diabetic patients. There are no interactions between NFA and nifedipine on the movement of platelet [Ca2+]i.
CIB1 Positively Regulates Outside-In Signaling through αIIbβ3 by Enhancing FAK and Src Activity.
Authors: Naik Meghna U. , Naik Ulhas P. 
Published: October 2019

Abstract
Integrin αIIbβ3, the platelet fibrinogen receptor, mediates outside-in signaling upon fibrinogen binding. Calcium- and integrin-binding protein 1 (CIB1) specifically interacts with the cytoplasmic domain of αIIb and is involved in platelet spreading. Here we show that CIB1 binding to αIIb is an essential step in the propagation of outside-in signaling through integrin αIIbβ3. Incorporation of BAPTA-AM completely blocked outside-in signaling and CIB-1 binding to aIIb, suggesting that an intracellular Ca2+ rise induced by fibrinogen binding is required for CIB1 interaction with αIIb. When CIB1-binding to αIIb was inhibited by introduction of a function blocking antibody or a synthetic peptide corresponding to αIIb tail, CIB1 localization at the tip of the filopodia, but filopodia formation was not blocked. This result suggests that interaction of CIB1 with αIIb is not required for filopodia formation, but is needed for CIB1 accumulation at the filopodia, as well as platelet spreading. Immunoprecipitation experiments showed that during outside-in signaling, CIB1 associates with FAK. Although association of FAK and CIB1 does not require dynamic rearrangement of the cytoskeleton, their accumulation at the filopodia and the activation of FAK is dependent on cytoskeletal rearrangement, as treatment with cytochalasin D after the platelets form filopodia affects CIB1 localization. Interestingly, the interaction of CIB1 with αIIb upon fibrinogen binding is also necessary for FAK and Src activation. However, Src activity is not required for CIB1 accumulation at the filopodia since this accumulation was not stopped by the Src inhibitor PP2, despite blocking platelet spreading. Our results suggest that during outside-in signaling an intracellular Ca2+ rise occurs that facilitates CIB1 interaction with αIIb. CIB1 recruits FAK to this complex and is localized to the filopodia dependent upon dynamic cytoskeletal rearrangement. Furthermore, activation of Src and FAK requires interaction of CIB1 with αIIb. Although Src activity is not required for the accumulation of CIB1 at the filopodia, it is required for platelet spreading on immobilized fibrinogen. Thus we provide evidence for initial sequence of outside-in signaling in platelets.
Analysis of Immunoglobulin Gene Mutation Status in Lymphoplasmacytic and Splenic Marginal Zone Lymphomas with Bone Marrow Infiltration.
Authors: Nathalie Gachard , Marie Parrens , Barbara Petit , Isabelle Soubeyran , Valerie Costes , Pierre Brousset , Michel Cogne , Dominique Bordessoule , Jean Feuillard 
Published: October 2019

Abstract
Splenic Marginal-Zone lymphomas (SMZL) and Lymphoplasmacytic lymphomas (LPL) correspond to entities that shared clinical, morphological and immunohistochemical features and are not always easy to discriminate. Immunoglobulin variable heavy chain gene (VH) usage and somatic mutation pattern were analyzed to clarify the relationship and the pathogenesis of these 2 entities.
Bone marrow frozen samples from 27 patients, 16 LPL and 11 SMZL, were studied. B-cell clonality was first analysed using the standard PCR method with the Biomed 2 primers. Informative PCR products were then purified, sequenced and analyzed on IMGT and Ig-Blast websites.
Overall, median rate of somatic mutations was 9% in LPL and 4% in SMZL (p=0.013).
All cases of LPL were mutated. Mutation rate was always higher than 5%. A preferential usage of VH3 gene was observed in 13/16 cases (80%), with a predominance of VH3–23 and VH3–74, both representing 60% of cases. VH4 gene was used only in 2/16 cases (13%). Regarding JH usage, a predominance of JH4 gene rearrangement was observed in 11/16 cases (69%). JH5 and JH6 were used in 20% and 13% of cases respectively.
In the group of SMZL, mutation rates and usage of VH and JH genes were more heterogeneous. Thirty six percent of SMZL were unmutated and 19% had a mutation rate lower than 5%. VH3 was used in 4/11 cases (36%), VH4 in 3/11 cases (27%) and VH1 in 4/11 cases (36%). JH4 and JH6 were used in 4/11 cases (36%) and the JH5 in 1/11 cases (9%).
Therefore, the profile of somatic mutations and the VH and JH segment preferentially used are different in SMZL and LPL, suggesting a different process of antigen-driven selection and of transformation of the initial B-cell clone. These findings consolidate the existence of LPL as a genuine entity, different of SMZL despite their histopathological overlaps.
Expression and Mutation Analysis of the Myc Antagonist Gene Mad1 in Acute Leukemia.
Authors: Pan Ling , Zhang Xuejun , Suo Xiaohui , Wang Fuxu , Niu Zhiyun , Dong Zuoren 
Published: October 2019

Abstract
Objectives: Overexpression of Myc genes has been observed in many solid tumors and hematological malignancies, and Myc proteins are believed to act as transcription factors. In the past decade, several Myc-related genes have been identified, and the interaction mechanisms between these genes have been clarified. Members of Myc superfamily proteins belong to four different families: the Max family of common dimerization partners (including Max and MLx), the Myc family of proto-oncoproteins (including c-, N-, and L-Myc), the Myc antagonists including Mad family (including Mad1 and Mxi1) and Rox (Mnt). Mad1 gene maps to human chromosome 2p13 and encodes a 221-amino acid protein. Mad1 protein was shown to counteract the effects of Myc proteins and to inhibit the proliferation and tumorigenicity of glioblastoma and astrocytoma cells, and therefore was considered to be a kind of tumor suppressor. All Myc super-family members share a highly conserved structural domain, basic helix-loop-helix leucine zipper domain (bHLHZip). This bHLHzip domain is essential for these proteins to function during molecular switching from proliferation to differentiation. To function in vivo, Myc and Mad1 must heterodimerize with Max through the HLHZip motif. Then through the b (basic) motif, the heterodimers bind with CACGTG (E-box) containing DNA to activate or inactivate transcription. The present study is thus designed to examine the expression of Mad1 gene and to search for mutations in bHLHzip encoding domain.
Methods: we screened bone marrow mononuclear cells (BMMNC) from 26 patients with AL and peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, using reverse transcription- polymerase chain reaction, single strand conformation polymorphism analysis and sequencing.
Results: Mad1 gene was expressed in all samples. Two polymorphisms of Mad1 were found in BMMNC from AL patients and PBMNC from health volunteers. A point mutation was detected at a very important position, a substitution of A397G inducing Lys78Glu in two patients. This might have significant implication for the function of Mad1, as Mad1 will heterodimerize with Max at this very point. Mutation at this point could thus disrupt proper intermolecular alignment between Mad1 and Max. The two patients with Mad1 gene mutation had poor clinical outcomes, patient No.9 never achieved CR and died 2 months after diagnosis, while patient No.11 got CR, after two courses chemotherapy, but relapsed quickly and died 3 months after diagnosis.
Conclusion: Mad1 gene expressed and displayed mutations in acute leukemia patients. It would be interesting to know whether these mutations influence either the heterodimerizing or DNA binding potentials of Mad1 protein, by performing functional analysis of the mutant protein.