bluuebunny/crossref_metadata_2025 huggingface dataset:
MDM2 SNP 309 Is Not Associated with Increased Expression Levels of MDM2 Nor Clinical Significance in Childhood Acute Lymphoblastic Leukemia
Authors: He Jing , Gu Lubing , Zhou Muxiang
Published: April 2020

Previously, we reported that MDM2 overexpression by pediatric acute lymphoblastic leukemia (ALL) cells is linked to a poor response to chemotherapy and early relapse of disease. It was recently shown that a single nucleotide polymorphism (SNP) in the MDM2 promoter, SNP309, was related to increased MDM2 expression and poor outcomes for various malignancies. The present retrospective study investigates the association of SNP 309 with MDM2 expression levels and clinical significance in pediatric ALL. Data obtained from 81 children with ALL showed a similar genotype distribution of SNP 309 as seen in other types of cancer and healthy populations: 37 T/T (46%), 37 T/G (46%) and 7 G/G (8%). However, this SNP did not seem to control MDM2 mRNA expression in ALL. We observed that 20 of the 81 ALL cases overexpressed MDM2 mRNA (> 10-fold higher than in normal bone marrow mononuclear cells), of which 8 were SNP 309 T/T, while 12 were T/G (10) or G/G (2) (p>0.5). Furthermore, we found that both MDM2 mRNA and protein were undetectable in 10 of 37 T/G and 3 of the 7 G/G cases. In addition, there were no associations found between SNP 309 and the in vitro response to doxorubicin, nor and early relapse in the 81 ALL patients. Upon review of 23 ALL cell lines, we found there was a relatively higher percentage of T/G (57%) than in fresh ALL samples (46%). However, the expression level of MDM2 was not related to SNP 309, but was rather closely associated with p53 status: All 11 ALL lines with wild-type p53 overexpressed MDM2 and all 12 ALL lines with mutant p53 or with a p53-null phenotype expressed no or low levels of MDM2, regardless of their SNP 309 status. These results suggest that in pediatric ALL, the SNP 309 status affects neither the expression of MDM2 nor the clinical prognosis.
The Role of MLL in DNA Damage Checkpoint and Its Implication in Leukemogenesis
Authors: Liu Han , Westergard Todd D , Chen David Y , Cheng Emily H.-Y. , Hsieh James J.-D.
Published: April 2020

Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which results in human cancers. Hence, it is of great significance to discover and investigate novel key constituents of the mammalian DNA damage response network. Human chromosome band 11q23 translocation disrupting the MLL gene leads to poor prognostic leukemias. MLL is a transcription co-activator that maintains HOX gene expression. The importance of HOX gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models have indicated that incurred HOX gene aberration alone is insufficient to initiate MLL leukemia. Thus, additional signaling pathway must be involved, which remains to be discovered. Our recent studies demonstrated an intimate relationship between MLL and the cell cycle(Takeda et al. 2006, Genes & Development, 20, 2397–2409; Liu et al. 2007, Genes & Development, 21, 2385–2398). More importantly, our studies uncovered a critical role of MLL in executing the S phase checkpoint. We showed:

Over-expression of MLL induces an S phase block. MLL accumulates in the S phase upon DNA damage. MLL deficiency results in radioresistant DNA synthesis (RDS) and chromatid-type chromosomal abnormalities, two signature characteristics of S phase checkpoint defects.

We further determined the underlying mechanisms concerning the DNA damage-induced MLL accumulation. Our data showed that MLL is phosphorylated after DNA damage, which in turn blocks its degradation by SCFSkp2 in the S phase and results in the ultimate accumulation. Our data revealed the link between MLL and the S phase checkpoint, which provides novel insights into the mammalian cell cycle checkpoint network and human leukemia pathogenesis. Future studies utilizing murine leukemia models will be performed to examine whether MLL translocation compromises the S phase checkpoint and if the resulted dysfunction contributes to MLL leukemogenesis.
Loss of Growth Arrest DNA Damage 45b (GADD45b) Enhances Oncogenicity in BCR/ABL-Induced Chronic Myelogenous Leukemia
Authors: Sha Xiaojin , Hoffman Barbara , Liebermann Dan A.
Published: April 2020

Growth arrest DNA damage 45b (GADD45b) is upregulated during myeloid lineage terminal differentiation. It is also involved in G2/M cell cycle arrest and apoptosis in response to exogenous stress stimuli. To investigate the effect of GADD45b in the development of leukemia, lethally irradiated mice were reconstituted with either wildtype or gadd45b null myeloid progenitors which retrovirally expressed 210-kD BCR/ABL fusion oncoprotein. We found that both wildtype and gadd45b null myeloid progenitors expressing BCR/ABL induced chronic myelogenous leukemia (CML)-like disease between 11days to 22days after bone marrow transplantation in recipients. However, gadd45b null recipients had a shorter latency (11days–19days) compared to those of wildtypes (14days–22days). This result implies that GADD45b acts as a supressor of CML. Data will be presented on further analyzing the phenotypes of these mice.
Lack of Drug-Drug Interaction Between ATI-5923, a Novel Vitamin K Epoxide Reductase Inhibitor, and Amiodarone, in Beagle Dog
Authors: Choppin Anges , Irwin Ian , Lach Leang , Davies Adrian , Shao Lei , Becker Cyrus , Palme Monica P. , Paliard Xavier , Bowersox Scott , Dennis Donn , Druzgala Paliard
Published: April 2020

ATI-5923 is a novel oral anticoagulant that is being developed to prevent thromboembolic events associated with atrial fibrillation, prosthetic heart valves, and deep venous thrombosis. Like warfarin, it is a vitamin K epoxide reductase inhibitor. Unlike warfarin, which is metabolized by CYP2C9 and CYP3A4, ATI-5923 is metabolized by carboxylesterases. Our hypothesis is that ATI 5923 will lead to superior INR (international normalized ratio) control compared to warfarin as it will not be subject to pharmacokinetic interactions observed with many of the drugs that affect warfarin metabolism, leading to either excessive or suboptimal anticoagulation resulting in bleeding or thrombotic complications, respectively. These studies compared the efficacy of ATI-5923 to that of warfarin, when administered either intravenously (IV) or once a day orally, to produce stable anticoagulation in beagle dogs. The drug-drug interaction potential was assessed using co-administration with amiodarone, an inhibitor of CYP3A4 and CYP2C9, with either ATI-5923 or warfarin. Effects on coagulation were assessed by measuring Factor VII and X levels and prothrombin times (PT). Continuous IV infusions (5 to 9 days per dose) and oral administration of ATI 5923 and warfarin (5 to 10 days per dose) caused a dose-dependent decrease in plasma concentrations of Factor VII and Factor X, and an associated increase in PT. On a dose basis, ATI 5923 was a more potent anticoagulant than warfarin. Intravenous fresh frozen canine plasma or subcutaneous vitamin K1 treatment reversed the anticoagulant effects of orally-administered ATI-5923 in adult beagle dogs. Co-administration of amiodarone (40mg/kg for 2 days, 20mg/kg for 6 days) significantly increased the plasma levels and anticoagulation effect of warfarin (0.25mg/kg) in beagle dogs, as measured by increase PT and decrease in factor VII and X. In contrast, amiodarone had no effect on the anticoagulation parameters and plasma levels of ATI-5923 (0.3mg/kg). We conclude that ATI-5923, via a vitamin K-dependent mechanism, causes changes in key parameters of hemostasis in beagle dogs that are consistent with effective anticoagulation but without the important drug-drug interaction commonly observed with amiodarone in warfarin-treated patients.
Ornithine Transcarbamylase Deficiency: A Possible Risk Factor for Thrombosis
Authors: Venkateswaran Lakshmi , Scaglia Fernando , McLin Valerie A , Hertel Paula M , Shchelochkov Oleg A , Mahoney Donald , Karpen Saul J , Yee Donald L
Published: April 2020

We describe four infants with ornithine transcarbamylase (OTC) deficiency, who developed arterial and venous thromboses prior to undergoing liver transplantation. These patients prompted a retrospective chart review of children with inborn errors of metabolism associated with hyperammonemia seen at our institution between 1998 and 2008; no additional cases with thrombosis were detected. Data abstracted from medical records include patient demographics, details about thrombus, treatment, associated risk factors and metabolic abnormalities. OTC deficiency was diagnosed during the first week of life in all patients (median age – 4 days; age range 1–7 days). Thrombotic complications developed prior to liver transplant in all patients (median age - 63 days; age range 6–71 days). One patient had arterial thrombus; the rest developed venous thromboses. Possible risk factors for thrombosis are summarized in the table below.

Risk factors Patient 1 Patient 2 Patient 3 Patient 4 Indwelling catheter at the site of thrombus Yes Yes Yes Yes Hyperammonemia (>94 mmol/L) Yes No Yes Yes Low plasma arginine (<42 mmol/L) Yes Yes Yes Yes Hypercoagulable risk factors None Elevated factor 8, fibrinogen Elevated fibrinogen, low AT III* Dysfibrinogenemia Infection (positive blood culture) None C albicans Coag neg Staph None

Table 1. Patient characteristics that are potential risk factors for thrombosis * AT III - Anti-thrombin III

Multiple pro-thrombotic risk factors were present. Hyperammonemia was seen in 3 patients and low plasma arginine level was present in all patients around the time of thrombosis. As L-arginine is a substrate for nitric oxide synthesis, arginine deficiency leads to low nitric oxide levels. Recent studies have linked nitric oxide insufficiency to platelet hyperaggregability, thrombosis and endothelial damage. Infants with OTC deficiency, indwelling vascular catheters, hyperammonemia and low plasma arginine may be at increased risk for thrombosis. Further studies in patients with OTC deficiency, to evaluate the role of hyperammonemia and low plasma arginine in thrombogenesis are warranted.
A Rapid Multiplexed Genotyping of Hereditary Thrombophilia by SELDI-TOF Mass Spectrometer
Authors: Yang Shangbin , Xu Lihui , Wu Haifeng
Published: April 2020

It is known that approximately 50% of patients presenting with venous thromboembolism are associated with polymorphic SNPs in the genes for coagulation factor V (factor V Leiden), prothrombin (prothrombin G20210A), and MTHFR (C677T). Genotyping these thrombophiliac SNPs helps clinicians to properly manage patients with thrombotic disorders. In this study, we reported a novel method to rapidly genotype all three thrombophiliac SNPs in a multiplexed fashion. First, patient DNA samples were subjected to PCR reactions to amplify and extend the DNA products with masses corresponding to genotypes for each of the three targeted thrombophiliac SNPs. PCR products were then applied to Q10 anionic Protein Chips, undergoing on-chip sample enrichment and clean-up. Finally, genetic variants were genotyped by Surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) mass spectrometry based on their masses. This method offers a rapid turnaround time of less than five hours. The analytical accuracy of each SNP genotyping result has been confirmed by DNA sequencing. Additionally, the genotype results produced by this method were validated by comparing them to the results obtained by the approved method in the clinical reference laboratory. In summary, we have developed a novel approach of multiplex genotyping for known thrombophilic SNPs by SELDI-TOF mass spectrometer. This method is fast, accurate, reproducible, and is ready for immediate application in the clinical laboratory.
Evaluation of the Prothrombotic Risk Factors in Children with Leukemia
Authors: Unuvar Aysegul , Akcay Arzu , Saribeyoglu Ebru T , Tugcu Deniz , Karakas Zeynep , Ozturk Gulyuz , Anak Sema , Agaoglu Leyla , Devecioglu Omer
Published: April 2020

Pediatric patients with malignancy are at high risk of thromboembolic complications due to complex interactions of a variety of factors such as the malignancy, chemotherapy, central venous catheters (CVCs), infections, dehydration, and hereditary thrombophilia. In this study, deficiencies of protein C (PC), protein S (PS), and antithrombin (AT), activated protein C resistance (APCR), the factor V G1691A mutation, the prothrombin G20210A mutation, and increased factor VIII, IX, fasting homocysteine levels were assessed at the diagnosis of leukemia, retrospectively. The aim was to evaluate the role of the thrombophilic risk factors on vascular events during treatment period. Thirty children (11 F, 19 M) with leukemia (24 patients with ALL, 4 AML, one biphenotypic leukemia, one infant leukemia) were enrolled in this study. The median age of the patients was 56 months (range 7–215 months). All patients had CVCs, and thromboprophylaxis was not given to the any patients. We detected main prothrombotic risk factors at the diagnosis of leukemia as following: low PC (8/30), PS (11/30), AT activity (3/25), APCR (5/22), increased FVIII (5/22), FIX (2/22), and homocysteine levels (2/8), and FV Leiden mutation (1/24; heterozygous). The prothrombin G20210A mutation was not detected in any patient. The parents of these patients with abnormal test results were also evaluated to discriminate congenital or acquired deficiencies. In addition; PC, PS, AT levels were followed-up, and MTHFR genotype for the patients with high homocysteine levels was studied, also. In spite of, 24 of these 30 patients had at least one abnormal laboratory test result at the diagnosis of leukemia, venous thrombosis (7 with CVC-related venous thrombosis, one CVC-related jugular vein thrombosis and VOD, one vena saphena parva thrombosis) developed in 9 of 24 patients. In conclusion, leukemic children with at least one prothrombotic risk factor have high venous thrombosis risk. Further trials are needed to clarify the necessarity of screening program for thrombophilia and thromboprophylaxis in children with leukemia and CVCs, and, especially for the patients carrying hereditary prothrombotic risk factors.
The Function of Toll-Like Receptor 4 Expressions on Human Platelet in Platelet Activation
Authors: Ma Liping , Nie Da-Nian , Wang Xiu-Ju , Xie Shuang-Fen , Li Yi-Qing , Wu Yu-Dan , Pei Zhi-Xin , Zhang Cheng , Yin Song-Mei
Published: April 2020

Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) in host. Infection increases risk for hemostasis, thrombosis, DIC, and tissue repair. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (P-selectin) is one of the indexes to determine platelet activation. Experiment was designed to study whether TLR4 is expressed on human platelet, and what is the function of TLR4 in platelet activation induced with LPS. Platelet suspensions from 10 heath people were treated with LPS of different concentrations for 1 hour, which were 0mg/L(control),0.1mg/L,0.5mg/L,1mg/L and 5mg/L, respectively. The expressions of TLR4, P-select on platelets and PAI-1 in platelets were detected through flow cytometry (FCM) and western blot(WB) methods. ADP-induced platelet aggregation was measured by LG-PABER aggregometer. Compared with control, the expressions of TLR4,P-selectin on platelets and PAI-1 in platelets after treated with LPS of 0.5mg/L,1mg/L and 5mg/L were increased (P<0.05). positive correlation was observed between TLR4 and P-selectin on platelets, but between TLR4 and PAI-1 in platelets. LPS of all concentrations did not affected ADP-induced platelet aggregation. Therefore, it is evident that functional TLR4 is expressed on human platelet. TLR4 on platelet might be one of the important intermedia between platelet activation and LPS or bacteria, and contribute to the inflammatory process in host. It is also worthy to study whether LPS affect platelet aggregation induced by other inductors.
Baseline Platelet Reactivity as Determined by TRAP-6 Induced Aggregation in Whole Blood Is Related to the Rate of Non-Responsiveness to Clopidogrel
Authors: Spannagl Michael , Jambor Csilla
Published: April 2020

Background: Platelet inhibition by clopidogrel is often determined using ADP induced aggregometry. TRAP-6 stimulates platelets via the PAR receptors and is influenced to a much lesser extend by aspirin or clopidogrel. The value of the determination of TRAP-6 induced aggregometry in patients treated with clopidogrel is unknown. We evaluated the relation of clopidogrel non-responsiveness as determined by multiple electrode aggregometry (MEA) (1) vs. TRAP-6 induced aggregation. MEA provides a measurement of platelet aggregation in whole blood by monitoring changes in electrical impedance via disposable test cells each containing two pairs of electrodes (1).

Methods: Following IRB approval hirudin-anticoagulated blood (Dynabyte, Munich, Germany) was sampled from 993 patients. Aggregation induced by arachidonic acid (0.5 mM), ADP (6.4 μM) or TRAP-6 (32μM) was determined in whole blood using a new generation impedance aggregometer. The device is called Multiplate analyzer (Dynabyte, Munich, Germany), indicating the multiplicity of channels and sensors per channel of the device. After dilution (1:2 with 0.9% NaCl solution) of hirudin-anticoagulated whole blood and stirring for three minutes in the test cuvettes at 37°C, the activator was added and aggregation was continuously recorded for six minutes. The increase of impedance due to the attachment of platelets to the electrodes is detected for each sensor unit separately and transformed to arbitrary aggregation units. The aggregation is quantified as area under the curve (AUC).

Results: 618 patients were on chronic therapy with 100 mg aspirin/d, 295 patients on clopidogrel 75 mg/d (237 on dual therapy). Aspirin therapy had no influence on TRAP-6- induced aggregation (112+−260 vs. 110+−25; mean +− sd; aspirin vs. no aspirin, p> 0.05 Mann-Whitney U-test). In contrast clopidogrel induced a minor but significant reduction of TRAP6-induced aggregation (107+− 277 vs. 113+−246; p<0.05). Patients on clopidogrel had a significantly lower ADP induced aggregation (41+−30 vs. 86 +−33) compared to controls (p<0.05), and patients on aspirin had a significantly lower arachidonic acid induced aggregation compared to controls (23+−28 vs. 94 +− 35, p<0.05). 89 of 295 patients (30%) were stratified as clopidogrel non-responders based on the cut-off presented in (2). Patients were divided into 4 quartiles (q1-4) depending on their TRAP-6 induced aggregation. The rate of non-responsiveness in Q1 was 8.1%, in Q2 20%, in Q3 38% and 54% in Q4. ADPinduced aggregation was significantly higher in Q4 than in any other quartile (p<0.05).

Conclusion: A high TRAP-6 induced aggregation is associated with an increased rate of clopidogrel non-responsiveness as determined by multiple electrode aggregometry. TRAP-6 induced aggregometry seems to indicate the global platelet potential and might allow a prediction of clopidogrel responsiveness before the induction of therapy.
Agonist and Antagonist Effects of Diadenosine Tetraphosphate, a Platelet Dense Granule Constituent, on Platelet P2Y1, P2Y12 and P2X1 Receptors
Authors: Chang Hung , Yanachkov Ivan B , Michelson Alan D , Li YouFu , Barnard Marc R , Wright George E , Frelinger Andrew L
Published: April 2020

Introduction: Diadenosine 5′,5‴-P1,P4- tetraphosphate (Ap4A) is stored in platelet dense granules, but its effects on platelet function are not well understood. In the current study, we examined the effects of Ap4A on signaling through the human platelet purinergic receptors P2Y1, P2Y12 and P2X1.

Methods: Flow cytometry was used to measure the effects of Ap4A in the presence or absence of ADP on:

P2Y12-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), P2Y1- mediated increase in platelet cytosolic Ca2+ (measured with FLUO-4), and c) P2X1- mediated intraplatelet entry of extracellular Ca2+ (measured with FLUO-4) (Figure). ADP-stimulated platelet shape change (P2Y1-mediated) and aggregation (P2Y1- and P2Y12-mediated) were measured optically.

Results: Ap4A inhibited 3 μM ADP-induced:

platelet aggregation (IC50 9.8 ± 2.8 μM, n = 3), P2Y1-mediated shape change, P2Y1-mediated increase in platelet cytosolic Ca2+ (IC50 40.8 ± 12.3 μM, n = 3), and P2Y12-mediated decrease in VASP phosphorylation (IC50 >250 μM, n = 3).

In addition to inhibiting these ADP-induced effects on P2Y1 and P2Y12, Ap4A in the absence of added ADP decreased VASP phosphorylation, albeit with significantly reduced potency compared to ADP. This Ap4A-induced reduction of VASP phosphorylation was reversed by addition of AR-C69931, a selective P2Y12 antagonist. Ap4A, therefore, has partial P2Y12 agonist properties. Ap4A also had agonist effects on platelet P2X1 receptors, as evidenced by intraplatelet entry of extracellular Ca2+. All these effects of Ap4A were significant at pathophysiologic concentrations. Ap4A had no agonist effects on platelet P2Y1 receptors.

Conclusions: Ap4A, a known constituent of platelet dense granules, is

an antagonist of platelet P2Y1 and P2Y12 receptors, where it inhibits the effects of ADP, an agonist of P2X1 receptors, and a partial agonist of P2Y12 receptors (Figure).

Figure Figure
Hyperlipidemia Could Activate Platelet and Induce the Production of Plasma Inflammatory Factors in Rabbits, Which Lead to Atherosclerosis
Authors: Xie Shuangfeng , Yin Songmei , Nie Danian , Li Yiqing , Wang Xiuju , Ma Liping , Xiao Jie
Published: April 2020

Background: It had been proved by epidemiology and clinical trials that hypercholesterolemia (HC) and atherosclerosis (AS) had close relations. Lipid could take part in many pathological processes of the AS. Platelets played an important role in this process as an inflammatory mediator. We Chose Platelet aggregation maximum (PAG(M)) and plasma P-selectin as the index of platelet activation, try to explore the transfer of platelet activation and lymphocyte inflammatory reaction in atherosclerosis.

Design: We set up the rabbit hyperlipidemia atherosclerosis model through 12 weeks’ hyper lipid feeding. PAG(M), concentrations of plasma P-selectin, Interleukin (IL)- 6, IL-1β, tumor necrosis factor (TNF)-α and mRNA expression of these factors in the lymphocyte were measured at 0 week, 6th week and 12th week time point.

Results: At the 6th week time point of the hyper lipid feeding, the rabbit had elevated PAG(M) (68.12%±4.73% vs. 62.67%±3.13%; P=0.002, n=20). The PAG(M) was maintained in the high level at 12th week time point (68.83%±3.35% vs. 62.67%±3.13%; P=0.001, n=20). At the 6th week time point of the hyper lipid feeding, the rabbit had elevated plasma P-selectin, IL-6, IL-1β and TNF-α (6th week vs. 0 week, n=20, P-selectin P=0.005, IL-6 P=0.000, IL-1β P=0.001, TNF-α P=0.001). The expressions of IL-6/mRNA, IL-1β/mRNA and TNF-α/mRNA in lymphocytes were elevated too (6th week vs. 0 week, n=20, IL-6/mRNA P=0.000, IL-1β/mRNA P=0.002, TNF-α/mRNA P=0.000). At the 12th week time point, all of the parameters were maintained in the high level, while there had no significant difference between 12th week time point and 6th week time point.

Conclusion: Hyperlipidemia could activate platelet. Increased PAG(M) and plasma P-selectin represented the platelet activation. Hyperlipidemia could induce the production of plasma IL-6, IL-1β and TNF-α. The increased plasma IL-6, IL-1β and TNF-α were partly from the lymphocyte secretion because The IL-1β/mRNA, IL-6/mRNA and TNF-α/mRNA expression of lymphocyte elevated. So in rabbit hyperlipidemia atherosclerosis model, platelet activation and inflammatory factors increasing was the main mechanism, while the lymphocyte took part in the inflammatory process.
Prevalence, Clinical and Instrumental Correlates of Myocardial Fibrosis and Necrosis by Delayed Contrast Enhancement Cardiovascular Magnetic Resonace in Thalassemia Major Patients
Authors: Pepe Alessia , Favilli Brunella , Positano Vincenzo , Capra Marcello , Maggio Aurelio , Pinto Carmen Lo , Spasiano Anna , Forni Gianluca , Filosa Aldo , Borgna-Pignatti Caterina , Cianciulli Paolo , Giuffrida Gaetano , Casini Tommaso , Derchi Giorgio , Lombardi Massimo
Published: April 2020

Cardiovascular Magnetic Resonance (CMR) by delayed enhancement (DE) has proven to visualize myocardial scarring, but no dedicated studies are available in thalassemia major. Aim of our study was to investigate the prevalence, extent, clinical and instrumental correlates of myocardial fibrosis or necrosis by DE CMR in thalassemia major patients. CMR-DE was performed in 115 thalassemia major patients. Myocardial iron overload was determined by multislice multiecho T2*. Cine images were obtained to evaluate biventricular function. All patients gave written informed consent to the protocol. The project was approved by the institutional ethics committee. DE areas were present in 28 patients (24%). Extent of DE was 3.9±2.4 %. In 26 patients the location of fibrosis was not specific and patchy distribution was prevalent. Two patients showed transmural DE following coronary distribution. The DE group was significantly older (P=0.004). DE correlated with cardiac risk factors (P=0.01), history of cardiac complications (P=0.001), anti-HCV antibodies (P = 0.04) and ECG-changes (P=0.002). We did not find significant relation of DE with heart T2* values and biventricular function. Figure shows a thalassemia major patient with no myocardial iron overload (all 16 segments T2* values > 20 ms) (A) and transmural DE (black arrows) following coronary distribution in the apical region (B,C). In conclusion, in thalassemia major patients the significant presence of myocardial fibrosis/necrosis seems to be a time dependent process correlating with cardiovascular risk factors and cardiac complications. HCV infection could be a causal agent in the pathogenesis of myocardial scarring. ECG-changes showed a good accuracy in predicting myocardial scarring.

Figure Figure
Endothelial Differentiation of Human Embryonic Stem Cell and Functional Blood Vessels Formation in Vivo
Authors: Li Zongjin , Smith Bryan , Huang Mei , Xie Xiaoyan , Gambhir Sanjiv Sam , Wu Joseph
Published: April 2020

Background: Human embryonic stem (hES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of hES cell-derived endothelial cells (hESC-ECs), use molecular imaging techniques to examine their survival and function in vivo.

Methods and Results: Here we introduced two-step procedures to increase endothelial differentiation efficiency of hESCs by subcultured embryoid bodies (EBs) in collagen. Single cell suspensions from EBs sprouting in collagen were obtained by treatment with collagenase and Liberase Blendzyme IV and CD31/CD144 positive cells were isolated by FACScan. After isolation, these hESC-ECs express endothelial cell markers similar to HUVEC, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) in vitro. Real time PCR array described increasing endothelial transcription. Using whole genome microarrays, we investigated the hESCs derived endothelial cells (hESC-ECs) transcriptome that occur among sequenced hESCs differentiation processes and human umbilical vein endothelial cells (HUVECs). We found that hESC-ECs expressed endothelial gene at pattern similar to HUVECs. By intravital microscope, we demonstrated that hESC-ECs can form function vessels with blood flow.

Conclusion: Taken together, two-steps procedures increased the endothelial differentiation efficiency hESCs, and hESC-ECs can form functional vessel in vivo.
Differential Effects of TAK-442, a Factor Xa Inhibitor, and Ximelagatran, a Thrombin Inhibitor, on Bleeding: Possible Role of Differences in Effects on Factor V-Mediated Feedback on Blood Coagulation Cascade
Authors: Konishi Noriko , Hiroe Katsuhiko , Imaeda Yasuhiro , Fujimoto Takuya , Kubo Keiji , Kawamura Masaki
Published: April 2020

Thrombin generation serves to amplify the coagulation cascade via positive feedback activation of factor V (FV) and factor VIII. We hypothesized that factor Xa (FXa) inhibitors, unlike thrombin inhibitors, would not block the feedback activation of the coagulation cascade but would have a favorable anticoagulating profile—sufficient to prevent thrombus formation, yet not interfere with hemostatic plug formation. TAK-442 is a newly synthesized, selective FXa inhibitor that strongly inhibits FXa (with a Ki value of 1.8 nM), and displays more than 440x selectivity toward FXa than other serine proteases. In the present study, we compared the effects of TAK-442 versus ximelagatran on FV-mediated positive feedback in vitro, and on their antithrombotic and hemorrhagic effects in a rat model of venous thrombosis.

In vitro, TAK-442 gradually inhibited thrombin generation and prolonged prothrombin time (PT) in a dose-dependent manner, while melagatran, an active form of ximelagatran, exhibited a steeper effect at higher doses tested. The PT prolonging potency was increased in FV–deficient human plasma, with CT2 values (the concentration that causes 2 times prolongation of clotting times) of 120 nM for TAK-442 and 32 nM for melagatran, compared with 500 nM and 360 nM for TAK-442 and melagatran, respectively in normal plasma. In the rat model of venous thrombosis, TAK-442 (10 mg/kg, po) prevented thrombus formation by 55% and prolonged PT by 1.3 times of control values; a similar effect was observed in ximelagatran-treated (3 mg/kg, po) animals, with 59% inhibition of thrombus formation and 1.2 times prolongation of PT. TAK-442 at 100 mg/kg, prolonged PT by 2.1 times, with no significant change in bleeding time (BT); in contrast, increasing the dose of ximelagatran to 10 mg/kg, po prolonged PT by 3.9 times and significantly (P<0.025) increased BT.

Our data suggest that the differential effects of the two agents on FV-mediated amplification of thrombin generation may underlie the observation of a wider therapeutic window for TAK-442 than for ximelagatran.
Osteoclast Function Is Essential in the Hematopoietic Stem Cell Niche
Authors: Lymperi Stefania , Horwood Nikki , Dazzi Francesco
Published: April 2020

Hematopoietic stem cell (HSC) function and number are tightly regulated in a specific microenvironment - the HSC niche - constituted of several cell types. Amongst them, osteoblasts have a prominent role since their increment is associated with an expansion of HSC. However, we demonstrated that an overall increase in osteoblasts does not necessarily promote hematopoiesis (Lymperi et al, Blood 2008 111:1173-81). We hypothesised that osteoclast mediated bone resorption, a component of the bone remodelling process which regulates osteoblast formation, takes active part in the HSC niche. To address this question we inhibited osteoclast function with the bone catabolic agents bisphosphonates. In vivo administration of bisphosphonates to C57BL/6 mice altered the architecture of the trabecular bone as defined by micro-CT and histomorphometric analysis. Treated mice exhibited increased bone volume and trabecular number and reduced trabecular separation and trabecular pattern factor compared to the untreated controls. This was accompanied to a decrease in the proportion and absolute number of HSC in the bone marrow (BM) as assessed by both the immunophenotypic enumeration of Lin-Sca1+c-kit+Flk2-HSC and the long-term culture- initiating cell (LTC-IC) assay. The number of the colony forming unit-cells (CFU-C) in the BM of bisphosphonate treated mice was increased, while more Lin-Sca1+c-kit+ enriched cells were found in the S/M phase of the cell cycle. These results indicate that, in osteoclast-impaired mice a proportion of HSC enters the cell cycle changing the balance in favour of hematopoietic progenitors. The ability of BM cells from bisphosphonate treated mice to engraft and reconstitute the hematopoietic system was tested in a competitive transplantation assay, whereby BM cells from treated and untreated mice were transplanted into syngeneic irradiated recipients. The long-term engraftment of treated BM cells was inferior to the one of controls, indicating a decrease in HSC numbers. Furthermore, recipient mice treated with bisphosphonates prior to conditioning and BM transplantation experienced a delayed hematopoietic recovery as compared to untreated recipients. Since it has been shown that parathyroid hormone (PTH) administration increases the number of HSC by stimulating osteoblast numbers, mice receiving micro-pump delivered PTH were also injected with bisphosphonates. We observed that the administration of bisphosphonates abolished the ability of PTH to increase the primitive HSC pool size as measured by FACS analysis of the Lin-Sca1+c-kit+Flk2- BM cells and their capacity to engraft and reconstitute irradiated recipients. Our findings demonstrate that the osteoclast function is fundamental for the HSC niche, possibly by promoting the recruitment of osteoblasts with HSC supporting capacity.
Structural Positioning of the SH2 Domain Is Critical for Bcr-Abl Kinase Activity, Signal Transduction and Oncogenic Transformation
Authors: Hantschel Oliver D. , Grebien Florian , Kaupe Ines , Superti-Furga Giulio
Published: April 2020

We have recently shown that the SH2 domain stimulates c-Abl catalytic activity and substrate phosphorylation. This effect is exerted directly through the establishment of a tight SH2-kinase domain interface in the active conformation of c-Abl (Filippakopoulos et al. (2008) Cell, in press, scheduled to be published on September 5, 2008). Mutations in the SH2 domain that presumably disrupt this SH2-kinase domain interface, such as the Ile164Glu mutation, result in severe impairment of Abl catalytic activity. Thus, correct positioning of the SH2 and kinase domain modules appears to be critical for efficient activation of cytoplasmic tyrosine kinases. Here, we present data showing that the same structural coupling of the SH2 and kinase domain is also a critical factor for full activation of the oncogenic fusion kinase Bcr-Abl. A single point mutation in the SH2 domain (Ile164Glu) led to a dramatic reduction in Bcr-Abl in vitro tyrosine kinase activity and Bcr-Abl autophosphorylation, both on the activation loop (pTyr-412) and the SH2-kinase domain linker (pTyr-245). This resulted in a strong decrease in global cellular tyrosine phosphorylation, as well as decreased phosphorylation of critical downstream mediators of Bcr-Abl signaling. Both wildtype Bcr-Abl, as well as the Bcr-Abl Ile164Glu mutant were able to confer factor independent growth to Ba/F3- and UT-7 cell lines, although to a different extent. Detailed data on the properties of the Ile164Glu mutation in vitro, in imatinib inhibition assays, transformation assays and mouse bone marrow transplant models will be presented. We propose that the structural positioning of the SH2 domain is a crucial factor for constitutive activity, signal transduction and transforming capacity of Bcr-Abl. Besides oligomerization via the N-terminal coiled-coiled domain and loss of the auto-inhibitory N-terminal myristoyl group, the proper positioning of the SH2 domain appears to be another critical factor that is required for constitutive activation of Bcr- Abl, which is the prerequisite for its ability to induce chronic myeloid leukemia (CML). Inhibitors of the SH2-kinase domain interface of Bcr-Abl may comprise alternative or additional points of pharmacological intervention for the treatment of imatinib-sensitive or -resistant CML or Ph+ acute lymphocytic leukemia.
Switch Pocket Inhibitors of the ABL Tyrosine Kinase: Distinct Kinome Inhibition Profiles and in Vivo Efficacy in Mouse Models of CML and B-Lymphoblastic Leukemia Induced by BCR-ABL T315I
Authors: Van Etten Richard A. , Chan Wayne W. , Zaleskas Virginia M. , Walz Christoph , Evangelista Peter , Lazarides Katherine , Betancur Monica , Wise Scott , Petillo Peter A. , Flynn Daniel L.
Published: April 2020

Acquired resistance to the ABL tyrosine kinase inhibitor (TKI) imatinib is a significant clinical problem for patients with chronic myeloid leukemia (CML), where a major resistance mechanism is mutations in the ABL kinase domain that render the BCR-ABL enzyme insensitive to the drug. The gatekeeper mutation Thr 315 Ile (T315I) arises at high frequency in patients relapsing after sequential therapy with second-line TKIs such as dasatinib or nilotinib (Shah et al., J Clin Invest2007;117:2562), and confers pan-resistance to all ATP-competitive TKIs. Using structure-based rational drug design, we have developed a novel chemical class of compounds that bind to five distinct structural pockets that the ABL kinase uses to switch between the inactive and active conformations. Diversity in these “switch pockets” between kinases can be exploited to fine-tune the selectivity of an inhibitor against the rest of the kinome. Two development candidate ABL inhibitors have emerged from this effort: DCC-2036 and DCC-2157. Both compounds potently (IC50 = 0.8–4.0 nM) inhibit purified ABL in the switch-off (unphosphorylated) and switch-on (phosphorylated) states via a non-ATP-competitive mechanism, and avoid steric clash with Ile 315. Both drugs also impair proliferation and induce apoptosis of Ba/F3 cells expressing a wide variety of BCR-ABL TKI-resistant mutants, including T315I, many P-loop mutants, and the dasatinib-resistant mutant F317L. DCC-2036 is highly selective for ABL, FLT3, TIE2, and Src family kinases, and when dosed at 100 mg/kg/day by oral gavage, significantly prolonged the survival of mice with CML-like myeloproliferative disease induced by retroviral expression of BCR-ABL WT and T315I in bone marrow. Genetic studies in mice (Hu et al., Nat Genet2004;36:453) suggest that the Src inhibitory activity of DCC-2036 would be beneficial against Ph+ B-cell acute lymphoblastic leukemia (B-ALL), and indeed DCC-2036 was superior to dasatinib (Hu et al., PNAS2006;103:16870) in prolonging survival of mice with B-ALL induced by BCR-ABL T315I. By contrast, DCC-2157 is selective for ABL, KIT, and PDGFRA/B kinases, and pharmacodynamic studies demonstrated prolonged (>18 hour) inhibition of phospho-Stat5 in BCR-ABL+ leukemic cells following a single oral 100 mg/kg dose. Treatment of mice with CML-like disease induced by BCR-ABL T315I with DCC-2157 at 50 mg/kg every other day induced virtually complete suppression of leukemia and extensive prolongation of survival. These results demonstrate that switch pocket drug design technology yields ABL TKIs with distinct pharmacologic profiles that are promising new weapons for the treatment of de novo and drug-resistant Ph+ leukemias.
Gene Expression Profiling of Post-Follicular B-Cell Lymphoma
Authors: Chng Wee-Joo , Huang Gaofeng , Kurtin Paul J. , Dogan Ahmet , Remstein Ellen
Published: April 2020

Although classified as marginal zone lymphomas under the WHO classification, the molecular relationship between splenic marginal zone lymphoma (SMZL), extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), and nodal marginal zone lymphoma (NMZL) has not been clarified. Furthermore, lymphoplasmacytic lymphoma (LPL) can show clinical morphologic and immunophenotypic overlap with these entities and may present a diagnostic challenge. In this analysis of gene expression data generated using the Affymetrix U133plus 2.0 chip from 32 SMZL, 25 MALTs (GI, salivary gland and lung), 23 NMZL and 25 LPL, we aim to identify the molecular relationship between these pathological entities using unsupervised methods, identify disease specific signatures and markers using supervised methods and also the functional implications of these signatures using a modified gene-set enrichment analysis. Using hierarchical clustering, MALT lymphoma forms a tight cluster regardless of tumor site. In addition, SMZL and NMZL form another large cluster. LPLs are divided into 2 main clusters with occasional samples interspersed amongst the SMZL and NMZL. There is no correlation between the percentage of CD20-positive B-cells, CD3-positive T-cells or CD138- positive plasma cells or tissue origin of the tumor and the way the samples are clustered. However, the separation of the LPLs into 2 major cluster correspond to the presence and absence of underlying Waldenstrom Macroglobulinaemia (WM), suggesting that the genes distinguishing the 2 clusters are potential markers for differentiating WM LPL from non-WM LPL. Next, using supervised analysis we identified a cluster of genes including MMP7, LTF and SFRP2 with high signals specific to MALTs, while other genes including PRDM1 (BLIMP1), XBP1 and TNFRSF17 (BCMA) were specifically over-expressed in LPL. These may therefore represent novel diagnostic marker differentiating these entities. Several of these are further validated at the protein level using immunohistochemistry on a tissue microarray. Consistent with the unsupervised analysis, SMZL and NMZL have little difference and share the over-expression of CD22 and WNT3 among other genes. Clustering of these samples based on the pathways and genesets that are enriched in the individual tumors as compared to their normal tissue counterpart showed a mutually exclusive pattern with significant enrichment of NFKB-related genesets and genes in LPL and MALT, and significant enrichment of B-cell receptor signaling genesets and genes in SMZL and NMZL. Our analysis, for the first time, describes the molecular relationship between these closely related lymphomas. In the process, we identified novel diagnostic markers that may differentiate these conditions and also new insights into molecular pathways that are differentially activated in the different conditions. These may represent potential therapeutic targets
Aneurysmal subarachnoid haemorrhage: effect of CRHR1 genotype on fatigue and depression
Authors: Vetkas Artur , Prans Ele , Kõks Sulev , Rätsep Tõnu , Asser Toomas
Published: April 2020

Background

Emotional health disturbances are common after aneurysmal subarachnoid hemorrhage (aSAH) and their causes are largely unexplored. Corticotropin-releasing hormone receptor 1 (CRHR1) is a key factor in stress reactivity and development of mental health disturbances after adverse life-events.

Methods

We explore the effect of CRHR1 genotype on mental health after aSAH in a retrospective cohort study. One hundred twenty-five patients have been assessed using EST-Q mental health questionnaire. Genotyping of CRHR1 single nucleotide polymorphisms (SNP-s) was performed (Rs7209436, Rs110402, Rs242924).

Results

Fatigue was present in almost half of aSAH patients, depression and anxiety in one-third. There was a high prevalence of insomnia and panic complaints. Rs110402 minor allele decreased the risk of depression (OR = 0.25, p = 0.027 for homozygotes). Depression was present in 14% vs 41% in minor and major allele homozygotes, respectively. Rs110402, Rs242924 and Rs7209436 minor alleles and TAT-haplotype, formed by them, were protective against fatigue. After Bonferroni correction only the association of Rs110402 with fatigue remained statistically significant (OR = 0.21, p = 0.006 for minor allele homozygotes). Results remained statistically significant when adjusted for gender, admission state, age and time from aSAH. In multiple regression analysis occurrence of fatigue was dependent on anxiety, modified Rankin score and Rs110402 genotype (R2 = 0.34, p <  0.001).

Conclusions

CRHR1 minor genotype was associated with a lower risk of fatigue and depression after aSAH. Genetic predisposition to mental health disturbances associated with negative life-events could be a risk factor for fatigue and depression after aSAH and selected patients might benefit from advanced counselling in the recovery phase.
Correction to: Efficacy of the interpersonal and social rhythm therapy (IPSRT) in patients with bipolar disorder: results from a real-world, controlled trial
Authors: Steardo Luca , Luciano Mario , Sampogna Gaia , Zinno Francesca , Saviano Pasquale , Staltari Filippo , Segura Garcia Cristina , De Fazio Pasquale , Fiorillo Andrea
Published: April 2020

Following publication of the original article [1], the authors reported an error in the legend of Table 2.
Characteristics of ammonia gas emissions from soybean cultivation soils treated with mixed microorganisms
Authors: Park Jong-Hwan , Lee Su-Lim , Hwang Se-Wook , Eom Ju-Hyun , Kim Seong-Heon , Kang Se-Won , Cho Ju-Sik , Seo Dong-Cheol
Published: April 2020

This study was conducted to evaluate (i) the characteristics of ammonia gas emissions from soybean cultivation soils amended with varying levels of urea and soil water, and (ii) the rate of reduction in ammonia emissions that could be obtained by applying mixed microorganisms (MM) to the urea-treated soils. The ammonia gas emissions from all treatments except the control were highest on day 2 of a laboratory-scale experiment and decreased gradually thereafter. The ammonia gas emissions from the soils increased with increasing urea and soil water contents. However, there were less emissions from soils treated with MM than those from the urea only treatment, and emissions also decreased significantly as the concentration of MM increased. In a field-scale experiment, the total cumulative emissions of ammonia from soil treated with a combination of chemical fertilizers and MM was reduced to 85.8% of that from the soil treated with chemical fertilizers only. Although we infer that MM can be used as an agent to reduce ammonia gas emissions from actual soils used for soybean cultivation, our knowledge of the processes involved in reducing ammonia emissions using microbial treatment is still limited. Consequently, further studies are required to investigate the efficient control of ammonia gas emissions from agricultural soils through the application of microorganisms.
Experimental and numerical study on the bearing capacity of encased stone columns
Authors: Hataf N. , Nabipour N. , Sadr A.
Published: April 2020

Stone columns have attracted considerable attention as a ground improvement techniques recently. Performance of stone columns, however, depends on confining pressure offered by the surrounding soils. In the case of very soft soils, the lateral confining pressure may be insufficient and this may lead to column bulging failure. Encapsulating the individual stone column enhances the lateral resistance against bulging by additional confining pressure. In this study, the influences of the length of encasement and type of aggregate materials on the bearing capacity of a single stone column in both dry sand and clay bed were investigated. The results indicated a clear improvement by encasing the half-length of the column. The numerical study also showed the effect of reinforcement stiffness reduces with increase in the cohesion of the clay. Parametric study showed that the effect of encasement stiffness is insignificant for clay bed with high values of cohesion.