bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of the rotation number on flow and heat transfer in contra-rotating disk cavity with superposed flow
Authors: Chen Shu-xian , Zhang Jing-zhou
Published: October 2019

The turbulent fluid flow and convective heat transfer in counter-rotating disk cavity with central axial air inflow and radial air outflow are numerically studied based on the finite volume method. Efforts are focused upon the influence of the rotation number Rt on the flow structure, cooling performance, sealing effect, and surface tangential friction characteristics in the cavity. The stagnation point where the radial outward flow along the upstream disk driven by the rotation force meets the radial outward flow along downstream disk driven by the combination of rotation force and inflow inertial force moves from upstream disk wall to the shroud with increasing Rt. At the Rt far smaller than 1, the fluids in the core region between two disks rotate with the upstream disk like a rigid body, and the tangential velocity of the rotating core decreases with the increase of the disk cavity radius, which is different from the Batchelor-type flow. At the Rt larger than 1, the fluids on the upstream disk side rotate like the Batchelor-type flow, while the sandwich rotation disappears in the fluid on the downstream disk side. The temperature on the upstream disk wall increases and then decreases with increasing values of Rt, and the critical value of Rt for the change of temperature variation is assessed to be at about Rt = 0.69. The temperature and radial temperature gradient of the downstream disk wall decrease with increasing Rt. With increasing Rt by increasing the disk rotation rate, the pressures near the downstream disk decrease, while the frictional moments on rotating disks increase. Due to the effect of flow structure, the frictional moment on the upstream disk is smaller than that on the downstream disk.
Evaluation of particle size distribution of granular blasting materials based on the fractal theory
Authors: Ji Jiejie , Yao Qiang , Wu Faming , Li Hongtao
Published: October 2019

The particle size distribution of granular blasting materials has a vital influence on the filling quality of earth-rockfill dams. The engineering experience-based method used to evaluate the particle size distribution has shortcomings at both the theoretical and practical aspects. This article proposes a new evaluation method based on the fractal theory. Grading sieve tests on granular materials, mass fractal analysis of particle size distribution, and a probability distribution model test of the characteristic parameters are used to revise the functional relationships between the fractal dimension D and the uniformity coefficient/curvature coefficient ( Cu/ Cc) of the grading curve. The feasibility of using D to evaluate the particle size distribution and the optimal fine grain content is then analyzed and determined. According to the results, the geometric shapes of the granular blasting materials have fractal characteristics, and their particle size distribution has a fractal distribution. The ranges of D where the rockfill and transition materials have a good particle size distribution are D = 2.254–2.529 and D = 2.358–2.559, corresponding to optimal fine grain content of 1.953%–11.805% and 10.268%–23.123%, respectively. Fractal dimension has a solid theoretical basis and strong practical applicability as an evaluation index for the particle size distribution of granular blasting materials.
Effects of a new drag reduction agent on natural gas pipeline transportation
Authors: Ma Yachao , Huang Zhiqiang , Lian Zhanghua , Chang Weichun , Tan Huan
Published: October 2019

Pipeline transportation is the major way to transport natural gas. How to reduce energy dissipation and retain the gas delivery capacity are the main problems of pipeline transportation. In this article, a new drag reduction agent named CPA is synthesized. An experimental investigation on the roughness-reducing effect of CPA on the inner surface of the pipeline is carried out. The effect of CPA on natural gas flow regime in the near-wall region of the pipeline is researched with Fluent software. Field tests for calculating the drag reduction rate of CPA are performed. The results show that CPA can reduce the roughness of the inner surface effectively, and the maximum roughness-reducing percentage is 38.74%. Meanwhile, CPA can reduce the frictional drag and thereby improve transportation capacity of pipelines. After injecting CPA, the streamline of the natural gas in the near-wall region is more consistent. The velocity fluctuation decreases by 93.2%. The mean turbulence intensity decreases by 53.01%. The pipeline pressure further decreases the roughness of the inner surface of the pipeline. The field test shows that the maximum drag reduction rate of CPA is 25%, and it is suitable for application in gathering and transportation pipelines of high flow velocity and turbulent rough region.
National trends in hospital admission, case fatality, and sex differences in atrial fibrillation-related strokes
Authors: Jewett Gordon AE , Lindsay M Patrice , Goia Cristina , Zagorski Brandon , Kamal Noreen , Kapral Moira K , Demchuk Andrew M , Hill Michael D , Yu Amy YX
Published: October 2019

Background and aim

Atrial fibrillation is associated with increased risk of ischemic stroke and its global prevalence is increasing. We aimed to describe the contemporary temporal trends in hospital admissions, case fatality rate, as well as sex differences in atrial fibrillation-related stroke in Canada.

Methods

We conducted a retrospective cohort study using Canadian national administrative data to identify admissions to hospital for stroke with comorbid atrial fibrillation between 1 April 2007 and 31 March 2016. We determined temporal trends in the crude and the age- and sex-standardized admission and case fatality rates. We also evaluated for any sex differences in these outcomes.

Results

There were 222,100 admissions to hospital for ischemic (n = 182,990) or hemorrhagic (n = 39,110) stroke. Comorbid atrial fibrillation was present in 20.2% of admissions for ischemic strokes and 10.1% for hemorrhagic strokes. Over the study period, the age–sex adjusted proportion of admissions with atrial fibrillation increased from 16.3% to 20.5% (p = 0.02) for ischemic stroke and was stable for hemorrhagic stroke. In-hospital case fatality rate decreased for ischemic stroke with and without comorbid atrial fibrillation. Women aged 65 years and older with ischemic stroke were more likely to have comorbid atrial fibrillation compared to men, while this association was reversed in younger women. There were no sex differences in the case fatality rate for people with atrial fibrillation-related ischemic stroke.

Conclusion

Atrial fibrillation is present in an increasing proportion of people hospitalized in Canada with ischemic stroke and disproportionately affects older women. Renewed focus is needed on atrial fibrillation-related stroke prevention with particular attention to sex disparities.
Clustering stability evaluation method based on SSIM
Authors: Hu Yan Zhu , Hu Yu , Ai Xin Bo , Zhao Hui Yang , Meng Zhen
Published: October 2019

In the clustering validity analysis, three main methods including intra-class cohesion, inter-class separation, and artificial judgment index can be used to evaluate the clustering results. If the clustering result is efficient, it means that the clustering stability is better. However, when those methods are used, it is essential to provide the sample data or clustering algorithms in advance. This paper proposes a clustering stability evaluation method based on the Elliptic Fourier Descriptor structural similarity index (EFD-SSIM), which can evaluate the clustering stability only when the clustering result is available. Its mechanism is that cluster is mapped into 2D graphics, and the degree of intra-class cohesion is measured based on the structural similarity (SSIM) on the graphics. As shown by the experimental results, EFD-SSIM has a good evaluation effect and it is consistent with the existing effectiveness evaluation indices of the clustering algorithm.
Novel therapeutic avenues in triple-negative breast cancer: PI3K/AKT inhibition, androgen receptor blockade, and beyond
Authors: Chan Jack J. , Tan Tira J. Y. , Dent Rebecca A.
Published: October 2019

Multiomic analyses have shed light upon the molecular heterogeneity and complexity of triple-negative breast cancers (TNBCs). With increasing recognition that TNBC is not a single disease entity but encompasses different disease subtypes, a one-size-fits-all treatment paradigm has become obsolete. In this context, the inhibition of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and androgen receptor (AR) signaling pathways have emerged as potential therapeutic strategies against selected tumors. In this paper, we reviewed the preclinical rationale, predictive biomarkers, efficacy, and safety data from early phase trials, and the future directions for these two biomarker-directed treatment approaches in TNBC.
Effects of Inhalation of Geranium Essential Oil on Blood Pressure and Heart Rate in Mice
Authors: Masubuchi Rhuichiro , Watanabe Saori , Satou Tadaaki
Published: October 2019

For a better scientific understanding of the basics of aromatherapy, our research group attempted to clarify the effectiveness of the essential oil from Pelargonium graveolens (EOPG, geranium essential oil), which is used to try and balance the mind-body connection. In order to eliminate any possible placebo effect, we used animal experiments that are considered to be insensitive to the placebo effect. Measurements of blood pressure and heart rate in the mouse tail artery were used as a reflection of the mind-body balance. Thirty minutes after inhalation of EOPG (5 µL/L air) for 90 minutes, blood pressure and heart rate of the mice were measured. EOPG significantly reduced blood pressure and heart rate. To further clarify the factors responsible for these effects, gas chromatography analysis was performed in order to determine the components transferred into the brain after the EOPG inhalation. Linalool, citronellol, and geraniol were detected at concentrations around 0.1 nL/L tissue from the brain after 10 µL/L air inhalation of EOPG. However, these were not detected after a 5 µL/L air inhalation of EOPG, as the levels were below the detection limit. These results suggest EOPG inhalation might lower blood pressure and heart rate, with the expressed effects associated with the transfer of components such as linalool into the brain.
Who Am I? An Ethnographic Study Exploring the Construction of Organizational and Individual Self among Indian IT Employees
Authors: Venkataraman A. , Joshi Chandra Shekhar
Published: October 2019

The self is both a sociological and a psychological construct. It is investigated in this paper from the perspectives of sociology of work, critical management studies and employment relations. Accordingly, drawing upon ethnographic research, this article seeks to unravel how an employee defines herself or himself in two realms—the organizational and the personal—respectively against the background of changing Indian IT industry marked by uncertainty and rising job insecurity. It examines how these two realms converge to bring about an individual’s sense of ‘dasein’ or being. The self is entwined in the value chain of the Indian IT labour process and, within it, soft HRM discursive practices seek to constitute and mould the ‘disciplined confessional self’ who is supposed to be not only autonomous but a proactive and proactive team player. This article identifies the sources from which the self finds definitions and validation in the liquid modern context of the ‘gig economy’. It seeks to reflect upon the ramifications arising out of the interplay between Western and Indian managerial repertoires and, finally, the interplay of caste and class against changing Indian societal norms and expectations. In doing so, it looks at the micro and macro means through which the self seeks to obviate its incoherence and find resonance and fullness. Given the volatile political economy of the Indian IT industry labour process, much of the work is repetitive and fragmented, and individuals feel alienated and burnt out after the initial excitement of experiencing the Sapient or Cisco way of life. They adopt various coping mechanisms reminiscent of Burawoy’s (1985) respondents to fight job insecurity and to secure their peer group’s acceptance. Thus, the onus of negotiating inherent dualities for finding meaning in the organizational realm, and yet leaving room for a transcendental individual coherent self whose larger ‘internal conversation’ transcends the existential concern of the structured antagonism of the wage–employment relationship, lies upon the individual rather than the organization.
Counterfactual Inflation Targeting in Nepal
Authors: Suzuki Tomoya
Published: October 2019

The Nepal Rastra Bank (NRB) pegs the Nepalese rupee to the Indian rupee, meaning that the NRB effectively transfers control over the country’s monetary policy to India. It is therefore argued that Indian inflation causes Nepalese inflation. This study investigates whether the NRB should have instead floated the Nepalese rupee and targeted inflation. For this purpose, a dynamic stochastic general equilibrium (DSGE) model under a fixed exchange rate regime is estimated using Nepalese data during the 1993–2016 period. The estimated model is used for simulations under counterfactual scenarios for which the NRB floated the Nepalese rupee and followed a rule to target inflation during the study period. Findings show that inflation targeting would have significantly increased the welfare cost associated with business cycles to risk-averse household, owing to interest-rate volatility. The volatilities of consumption and labour services would have become larger.

JEL Classification: E31, E32, E52, F41, F45
Injury Rate in TackleBar Football
Authors: Toninato Joseph , Healy Tim , Samadani Uzma , Christianson Eric
Published: October 2019

Background:

As football comes under greater scrutiny because of concussions and other injuries, many people are searching for safer alternatives, especially for youth athletes. The injury rate in TackleBar football, one such alternative, is not currently known.

Purpose:

To describe TackleBar football and calculate its injury rate over a single season to compare it with both traditional tackle and flag football.

Study Design:

Descriptive epidemiology study.

Methods:

TackleBar football was described, and injuries of male athletes (age range, 9-14 years) participating in TackleBar football were tracked over the course of a season (August 5 through October 27, 2018) and compared with published injury rates for traditional tackle and flag football.

Results:

The overall injury rate of TackleBar football was found to be 0.31 per 1000 athlete-exposures (AEs), which is lower than youth tackle (2.25-2.60/1000 AEs) and flag (5.77/1000 AEs) football.

Conclusion:

TackleBar football may represent a reasonable alternative to tackle and flag football with regard to injuries.
Involvement of CGRP receptor RAMP1 in cluster headache: A Swedish case-control study
Authors: Michalska Julia M , Ran Caroline , Fourier Carmen , Steinberg Anna , Sjöstrand Christina , Waldenlind Elisabet , Belin Andrea Carmine
Published: October 2019

Background:

Increased levels of the potent vasodilator calcitonin gene-related peptide (CGRP) have been found in ipsilateral jugular vein blood during the active phase of cluster headache (CH) and this is hypothesized to cause distinctive vasodilation. The receptor activity-modifying protein 1 (RAMP1) is part of the CGRP receptor complex responsible for ligand binding and specificity and therefore constitutes a promising candidate gene for CH. The aim of this study was to investigate the possible genetic association of RAMP1 with CH in Sweden, with focus on two RAMP1 single nucleotide polymorphisms, rs3754701 and rs7590387, and quantify RAMP1 mRNA expression levels in biological tissue from CH patients and controls.

Methods:

rs3754701 and rs7590387 were genotyped by quantitative polymerase chain reaction (qPCR) in 542 CH patients and 585 control subjects. RAMP1 mRNA expression was determined by reverse transcription qPCR in tissue from 12 CH patients and 12 controls.

Results:

We identified a significant difference between the CH patient and control groups for rs3754701 ( p = 0.0088). In addition, RAMP1 mRNA expression was enhanced in primary fibroblasts from CH patients compared to controls ( p = 0.0073).

Conclusion:

The association between rs3754701 and CH and the enhanced RAMP1 mRNA expression in CH patients support the hypothesis that CGRP and its receptor component RAMP1 are involved in CH pathophysiology.
ON THE INFLUENCE OF STILBAMIDINE UPON MYELOMA CELLS
Authors: SNAPPER I. , SCHNEID B.
Published: October 2019

Injections of stilbamidine cause morphological changes in myeloma cells. During this treatment large basophilic granules appear in the cytoplasm which show a tendency to become confluent. These granules stain red with pyronine and can be visualized in the supravital stain with neutral red. One of the main constituents of these inclusions consists of ribose nucleic acid.

These morphological changes seem to be limited to myeloma cells, since in none of the other bone marrow elements do comparable granules or inclusions develop.
Distinct c-Myb Regulation by HoxA9, Meis1 and Pbx1 in Haemopoietic and Leukaemic-Like Stem Cells.
Authors: Dassé Emilie , Volpe Giacomo , del Pozzo Walter , Frampton Jonathan , Dumon Stephanie
Published: October 2019

Abstract 1431

Poster Board I-454

The transcription factor c-Myb is an essential regulator of haemopoiesis and its expression is deregulated in several types of leukaemia. Although some c-Myb functions have been defined, the mechanisms involved in the control of its expression have yet to be elucidated. Previous studies have suggested that transcription initiation at the c-myb gene is constitutive, and that the level of mRNA is regulated by an elongation-blocking mechanism operating in its first intron. Here, we define and compare mechanisms influencing c-myb expression in haemopoietic stem cells (HSCs) versus leukaemic stem cell (LSC)-like cells. Using a nuclease sensitivity assay we have defined several potential regulatory elements in both HSC and LSC-like model cell lines. These hypersensitive sites are in the proximal promoter and the first intron, the latter correlating with the position of the putative transcription elongation regulatory region. Moreover, the hypersensitive sites are located in regions of sequence conservation and encompass a number of potential binding sites for homeodomain (HD)-containing proteins. In this study, we were able to demonstrate that the HD-containing transcription factors HoxA9 and Meis1, which are highly expressed in HSCs and whose co-expression in mouse bone marrow leads to rapid development of acute myeloid leukaemia (AML), are necessary but not sufficient for c-myb expression. In addition, we show that the pre-B-cell leukaemia transcription factor-1 (Pbx1), known to be a key binding partner of HD-containing factors, is indispensable in the regulation of c-myb expression. Comparing the effects of altered levels of HoxA9, Meis1 and Pbx1 in HSCs versus LSCs suggests that distinct mechanisms involving dimeric or trimeric complexes operate to regulate c-myb expression in these two stem cell types.

Disclosures

No relevant conflicts of interest to declare.
Lnk Constrains Oncogenic JAK2-Induced Myeloproliferative Disease in Mice.
Authors: Bersenev Alexey , Wu Chao , Balcerek Joanna , Tong Wei
Published: October 2019

Abstract 1437

Poster Board I-460

Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2), which in turn triggers a cascade of downstream signal transduction pathways that regulate key aspects of cell development. Mice that lack the inhibitory adaptor protein Lnk harbor a vastly expanded HSC pool with enhanced self-renewal. We previously demonstrated that Lnk controls HSC self-renewal predominantly through the Mpl/JAK2 pathway. Lnk binds directly to phosphorylated tyrosine 813 in JAK2 upon Tpo stimulation. Moreover, Lnk-deficient HSCs display potentiated JAK2 activation.

Dysregulation of cytokine receptor signaling pathways frequently lead to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML) in human patients. Moreover, the JAK2 V617F mutation has been observed at high frequency in several myeloproliferative diseases (MPDs). The JAK2V617F retains Lnk binding, suggesting that alterations in Lnk could influence MPD development. Indeed, we found that loss of Lnk accelerates and exacerbates oncogenic JAK2-induced MPD in mouse transplant models. Specifically, Lnk deficiency enhanced cytokine signaling, thereby augmenting the ability of oncogenic JAK2 to expand myeloid progenitors. To test whether the interaction between Lnk and JAK2V617F directly constrains MPD development in mice, we transplanted wild-type bone marrow cells expressing the JAK2V617F/Y813F double mutant that does not interact with Lnk (WT;JAK2VF/YF). WT;JAK2VF/YF engrafted mice exhibited increased myeloid expansion when compared to WT;JAK2VF mice, and conferred accelerated polycythemia vera development in secondary transplants. In summary, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may constrain leukemic transformation conferred by oncogenic JAK2.

Disclosures

No relevant conflicts of interest to declare.
Stromal Microenvironment Regulates Progenitor Survival through Nitric Oxide Production.
Authors: Jouni Dima , Zhang Yanyan , Bouamar Hakim , Wittner Monika , Vainchenker William , Louache Fawzia
Published: October 2019

Abstract 1440

Poster Board I-463

Nitric oxide (NO) is a small gaseous molecule with diverse roles including the regulation of cell proliferation, differentiation, apoptosis, adhesion and migration. NO is derived from L-arginine by the nitric oxide synthase (NOS) family of enzymes. At least three distinct NOS isoforms have been identified in mammalian cells including the endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). Recently, we have shown that NO donors induce CXCR4 expression in human CD34 positive cells suggesting that NO production may regulate the migration and adhesion of hematopoietic progenitors. To determine the in vivo relevance of these findings and define the role of NO in the biology of hematopoietic progenitor cells, we first investigated NOS expression in hematopoietic and non-hematopoietic cells. Using quantitative reverse transcription-PCR analysis, we demonstrate that nNOS, and eNOS isoforms are highly expressed in Human Umbilical Vein Endothelial Cells (HUVEC) and osteoblastic cell lines, but there was no or low expression of NOS in hematopoietic cells including cell lines and mature blood cells except macrophages that express high level of iNOS. In agreement with RNA analysis, we found large amounts of nitrite (a stable derivative of NO) in the culture medium of stromal (MS-5) and osteoblasic (MG-63) cell lines. To determine the effects of NO on hematopoietic progenitor cells survival, cord blood CD34+ were cultured on MS-5 cells in the presence/absence of NOS inhibitor (L-NAME) for three days, then hematopoietic progenitor numbers were determined by culture in a semi-solid medium and colonies were quantified 14 days later. Treatment with L-NAME reduced colony number by 33, 42 and 54% with 10, 100 and 500 μM L-NAME concentrations, respectively. This effect is NO specific since the diminution of progenitor number was reverted by adding NO donor in the culture medium.

These results indicate that NO is produced by bone marrow stromal cells. This production regulates the survival of hematopoietic progenitors in vitro. Further experiments are needed to determine if these effects are dependent on the regulation of CXCR4/SDF-1 signaling.

Disclosures

Jouni: Région IDF: Employment. Bouamar: Association pour la Recherche sur le Cancer: Employment; Cancéropôle IDF: Employment.
CREB Is Involved in the Regulation of C/EBPβ During 'emergency' Granulopoiesis.
Authors: Hirai Hideyo , Kamio Naoka , Matsusue Akiko , Ogino Shinpei , Kimura Nobuhiko , Satake Sakiko , Ashihara Eishi , Maekawa Taira , Tenen Daniel G. , Imanishi Jiro
Published: October 2019

Abstract 1457

Poster Board I-480

Mobilization of sufficient numbers of granulocytes to the front line of infection is prerequisite for host defense. As granulocytes have a short half-life, the production of granulocytes in the bone marrow must be tightly regulated to meet emergency demands. Our previous findings suggested that granulopoiesis at steady state is largely dependent on CCAAT enhancer binding protein α (C/EBPα) transcription factor (Zhang D.E. et al., PNAS, 1996 and Zhang P. et al., Immunity, 2004), whereas the granulopoiesis during emergency such as infections is dependent upon C/EBPβ (Hirai H. et al., Nat Immunology, 2006). Indeed the transcripts of C/EBPβ in granulocyte precursors were upregulated in response to cytokine stimulation or infection. In order to elucidate the molecular switch between C/EBPα- and C/EBPβ-dependent granulopoiesis, we developed a novel lentivirus-based reporter system. The vector carries two independent expression units, a green fluorescent protein (GFP) driven by a promoter of interest and a mouse Thy1.1 gene under the control of a constitutively active phosphoglycerate kinase (PGK) promoter. The activity of a promoter can be monitored by the intensity of GFP in Thy1.1 positive cells. Using this system, the activity of the C/EBPβpromoter was evaluated in primary bone marrow cells. A series of deletion mutants of the promoter revealed the existence of two cyclic AMP responsive elements (CRE) in the positive responsive elements during GM-CSF induced differentiation. The transcripts of CRE binding (CREB) protein were detected at higher level in hematopoietic stem cells and common myeloid progenitors than other mature cells. When a dominant negative mutant of CREB (S133A), in which the serine residue at 133aa was mutated to alanine, was retrovirally transduced into bone marrow cells, the mRNA of C/EBPβ was reduced and the proliferation/differentiation of granulocyte precursors were significantly impaired. In contrast, a constitutively active form of CREB, CREBDIEDML, facilitated the transcription of C/EBPβ. In addition, CREB is phosphorylated and bound to the CRE in response to GM-CSF stimulation. These data suggest that CREB is involved in the regulation of granulopoiesis through upregulation of C/EBPβ.

Disclosures

No relevant conflicts of interest to declare.
Efficient Induction of Hematopoietic Progenitor/Stem Cell Differentiation by Lentiviral Gene Transduction of TAL1/SCL Into Human ES Cells.
Authors: Kurita Ryo , Miura Yoshie , Okada Michiyo , Kageyama Rui , Hiramoto Takafumi , Yokoo Tomoko , Narusawa Megumi , Suehiro Youko , Inoue Hiroyuki , Marumoto Tomotoshi , Takahashi Atsushi , Tani Kenzaburo
Published: October 2019

Abstract 1477

Poster Board I-500

Human embryonic stem (ES) cells differentiate into three lineages in vitro and in vivo as mouse ES cells. They are therefore highly promising source of various cells/tissues in the regenerative medicine. The current protocols, however, remain to be optimized for the induction of the cells/tissues required. We have recently reported that the lentiviral transduction of TAL1/SCL gene to ES cells derived from the common marmoset, a small nonhuman primate, enables efficient differentiation into hematopoietic progenitor cells even in the absence of stromal cells (Kurita et al. Stem Cells.24:2014-22, 2006). Such culture condition without any stromal cells is considered to facilitate clinical application of ES cell-derived cell/tissues therapy in the regenerative medicine. The present study addressed whether the strategy is also effective in human ES cells. First, we determined optimal culture conditions to induce multilineage hematopoietic differentiation in a human ES cell line, khES-1, kindly provided by Dr. Nakatsuji, Kyoto University, Japan, as assessed by the expression of Brachyury, Flk1 and CD34. We found that the addition of BMP4 and VEGF augmented hematopoietic differentiation of embryoid bodies, and determined optimal concentrations of the cytokines. We established four human ES cell lines stably expressing TAL1/SCL gene by lentiviral transduction. The TAL1/SCL transduction further increased the hematopoietic differentiation under the optimal culture condition as assessed by the expression of CD34, CD235a and CD133. We also observed increased number of hematopoietic progenitor cells derived from two of the TAL1/SCL expressing human ES cell lines by colony-forming assays. Hematopoietic differentiation of the TAL1/SCL expressing ES cells in vivo is also being investigated by transplantation into irradiated immune deficient mice. These results suggest that the combination of optimal culture conditions and lentiviral TAL1/SCL gene transduction is a highly effective strategy to obtain hematopoietic stem cells from human ES cells in the absence of any stromal cells.

Disclosures:

No relevant conflicts of interest to declare.
Deletion of Bcl-X in the Megakaryocyte Compartment Results in Profound Thrombocytopenia Caused by Impaired Platelet Production and Survival.
Authors: James Chloé , Josefsson Emma C. , White Michael J. , Henley Katya J. , Kile Benjamin T.
Published: October 2019

Abstract 1495

Poster Board I-518

Recent studies have suggested a role for the intrinsic apoptosis pathway in megakaryocytic differentiation and platelet shedding. The intrinsic pathway is regulated by the Bcl-2 family of pro- and anti- cell death proteins. We recently demonstrated that platelet life span is controlled by an intrinsic cell death pathway, whereby the anti-apoptotic protein Bcl-xL constrains the pro-apoptotic activity of Bak to maintain platelet survival. As Bcl-xL is expressed in megakaryocytes, we investigated whether this protein is required for megakaryocyte survival, differentiation and/or platelet shedding. We specifically deleted the Bcl-x gene in the megakaryocyte lineage by crossing mice carrying a floxed allele of Bcl-x with mice carrying a platelet factor 4-regulated Cre transgene. Bcl-xfl/flCre+ mice were profoundly thrombocytopenic (26 ± 5 × 103/μl, n=14) compared with Bcl-xfl/flCre− animals (1157 ± 202 × 103/μl, n=13). Platelet life span in these mice was reduced to only 5 hours, as compared to 5 days in wild type littermates. This result confirmed that Bcl-xL is absolutely required for platelet survival. To determine whether Bcl-x deletion has an impact on platelet production, we analyzed the megakaryocyte compartment in Bcl-xfl/flCre+ and Bcl-xfl/flCre− mice. We observed that the number of megakaryocyte progenitors, and number of megakaryocytes in the bone marrow were increased in Bcl-xfl/flCre+ mice (23 ± 9 megakaryocyte progenitors vs 11 ± 5, and 51 ± 9 megakaryocytes vs 12 ± 1). This result suggested that Bcl-xL is not required for the survival of megakaryocytes or their progenitors. To determine whether Bcl-xL is required for the last step of megakaryocyte differentiation, i.e. platelet shedding, we cultured fetal liver cells with thrombopoietin. Large megakaryocytes were isolated after 3 days of differentiation on discontinuous bovine serum albumin gradient. They were cultured for 3 more days in the same media and the percentage of megakaryocytes displaying proplatelets was determined each day. Interestingly, Bcl-xfl/flCre+ megakaryocytes died much more quickly than Bcl-xfl/flCre− megakaryocytes, and almost none of those that survived were able to form proplatelets. Our study indicates that Bcl-xL is not only essential for platelet survival, but it is also required for the survival of mature megakaryocytes at the stage of platelet shedding.

Disclosures:

No relevant conflicts of interest to declare.
Generation of Functional Lymphoid (Natural Killer) Cells From Human ESC-Derived Hemangioblasts.
Authors: Kimbrel Erin A. , Lu Shi-Jiang , Lanza Robert
Published: October 2019

Abstract 1502

Poster Board I-525

Studies with human and mouse embryonic stem cells (ESCs) have shown that a common precursor to both vascular (endothelial and smooth muscle cells) and hematopoietic cell lineages called the hemangioblast can be produced from ESC-derived embryoid bodies in culture. We have developed a simple strategy to efficiently and reproducibly generate hemangioblasts from multiple hESC lines under serum- and stromal-free conditions, which will be important for their productive use in regenerative medicine. Previous work has shown that hESC-derived hemangioblasts can effectively differentiate into erythroid and myeloid lineages, but their ability to produce lymphoid lineage cells, including those with immunotherapeutic potential, is relatively unknown. Natural killer (NK) cells, which are part of the innate immune system, provide rapid, non-specific responses against viral infection and are involved in tumor cell detection and elimination. Interplay between various activating and inhibitory signals control the three main functions of NK cells, which are cytokine release, natural cytotoxicity, and antibody-dependent cellular cytotoxicity. Using hemangioblasts generated from both H7 and HuES-3 hESC lines, we have been able to produce mature CD56low/−CD16+ NK cells and found that their production does not require the use of stromal feeder layers. The differentiation procedure involves an initial 4 day culture to generate embryoid bodies, followed by a 12-14 day culture in methylcellulose supplemented with a set of cytokines and growth factors for the production and expansion of a hemangioblastic population. An additional 14-17 days in liquid culture plus human serum and a cocktail of cytokines allows for the differentiation of NK cells as assessed by flow cytometry. A non-radioactive cytotoxicity assay similar to the 51Cr release assay shows that these hemangioblast-derived NK cells harbor natural cytotoxicity function as they are able to effectively induce apoptosis in target K562 erythroblastic leukemia cells after a standard 4 hr co-culture. Using hemangioblasts as an intermediary cell source may enhance the capability and/or efficiency of hESCs to differentiate in vitro and importantly, allow for the development of feeder-free systems for the production of cells with immunotherapeutic potential.

Disclosures:

Kimbrel: Stem Cell & Regenerative Medicine International: Employment. Lu: Stem Cell & Regenerative Medicine International: Employment. Lanza: Stem Cell & Regenerative Medicine International/Advanced Cell Technology: Employment.
Increasing C/Ebpα Expression Regulates the Transition of Hematopoietic Stem Cells During Development.
Authors: Ye Min , Zhang Hong , Zhang Pu , Tenen Daniel G.
Published: October 2019

Abstract 1506

Poster Board I-529

During ontogeny, hematopoietic stem cells (HSCs) undergo a change from rapid dividing cells with high reconstitution ability to mainly quiescent cells with lower repopulation capacity. However, little is known about how this switch is regulated. Here we report that levels of C/EBPα, a transcription factor that is frequently disrupted in human acute myeloid leukemia, regulate the proliferation and self-renewal transition of HSCs during development. Loss of C/EBPα in adult mice resulted in a profound expansion of phenotypic HSCs and elevated proliferation rates. Limiting dilution transplantation to measure the frequency of competitive repopulation units (CRU) revealed an increase in the number of functional HSC in C/EBPα-/- mice. Serial transplantation of C/EBPα-/- bone marrow showed advanced reconstitution ability, indicating enhanced self-renewal ability. Interestingly, levels of C/EBPα in HSCs were significantly up-regulated 3 weeks after birth during which HSCs change from an actively cycling state to quiescence in bone marrow. When we conditionally inactivated C/EBPα in mice of different age, we observed a tight correlation between the age-specific levels of C/EBPα expression and the expansion of HSCs. Gene profiling analysis of C/EBPα-/- adult HSCs showed the up-regulation of oncogenes c-myc and n-myc, whose expression can regulate pluripotency and self-renewal of stem cells, as shown by recent induced pluripotent stem cell studies. Knocking down n-myc and c-myc expression in C/EBPα-/- HSCs using shRNA, we observed reduced proliferation and decreased colony formation in serial replating assay, which assesses the preservation of “self-renewal” in the progenitor cell compartment. Consistently, we observed down-regulation of n-myc in HSCs during the transition time, which is reciprocal to C/EBPα expression. Together, our data indicate C/EBPα as a key regulator of HSC self-renewal and proliferation during development, whose levels of expression might control the fetal to adult switch of HSC properties through regulating myc genes.

Disclosures

No relevant conflicts of interest to declare.
Heme Oxygenase-1 Gene Therapy in a Murine Model of Sickle Cell Disease.
Authors: Belcher Belcher D. , Vineyard Julie V. , Bruzzone Carol M , Chen Chunsheng , Beckman Joan D , Ngyuen Julia , Steer Clifford J , Vercellotti Gregory M.
Published: October 2019

Abstract 1527

Poster Board I-550

We previously demonstrated that increases in heme oxygenase-1 (HO-1) and its products, carbon monoxide and biliverdin, inhibit inflammation and vasoocclusion in mouse models of sickle cell disease (SCD). In this study, an albumin promoter driven Sleeping Beauty transposase plasmid with a wild type rat hmox-1 gene (wt-HO-1) transposable element (289 amino acids) was delivered by hydrodynamic tail vein injections to SCD mice. Eight weeks after injection, SCD mice had 3-fold increases in HO-1 activity in their livers, similar to hemin-treated mice. mRNA transcription of the transposable element was confirmed by qRT-PCR RFLP analysis. HO-1 overexpressing mice had marked activation of the phospho-p38 MAPK and phospho-Akt proteins and decreased NF-kB p65 in liver nuclear extracts. Hypoxia-induced stasis, a characteristic of SCD but not normal mice, was inhibited in dorsal skin fold chambers in wt-HO-1 SCD mice. None of these effects were seen in SCD mice injected with Sleeping Beauty containing a nonsense rat hmox-1 gene (ns-HO-1) that encodes for amino acids 1-241 with little or no enzymatic activity. Immunohistochemistry of HO-1 in livers demonstrated perinuclear HO-1 staining in both hepatocytes and sinusoidal Kupffer cells in wt- and ns-HO-1-treated mice. We conclude HO-1 gene therapy targeted to the liver is beneficial in SCD by activating cytoprotective signaling pathways and inhibiting vascular stasis at sites distal to transgene expression.

Disclosures

No relevant conflicts of interest to declare.
Chemo- and Radiotherapy Induce Common Apoptotic Mediators in Hodgkin's Lymphoma.
Authors: Frazzi Raffaele , Nicoli Davide , Iori Mauro , Merli Francesco
Published: October 2019

Abstract 1555

Poster Board I-578

Current therapeutic regimen for classical Hodgkin lymphoma (HL) includes the combined ABVD chemotherapy often followed by radiotherapy. Albeit HL is, in many cases, a curable malignancy, several issues still pose serious threats to the patients. For instance late toxicities, chemoresistance insurgence and relapses represent open and unresolved challenges. We are currently investigating the molecular response of lymphoid cells to doxorubicin treatment and ionizing radiation (IR) in an experimental model represented by HL-derived cell lines. We show the expression and modulation of the stress-inducible protein clusterin/XIP8 (clusterin) in lymphoid cells. The nuclear isoform (having a MW of about 55kDa) is up-regulated upon doxorubicin treatment starting at a concentration of 0.5 micromolar. Interestingly enough, also interferon-gamma determines clusterin and p53 up-regulation starting at 50 nanograms/ml even though just a modest cytostatic effect is observed with this cytokine. High doses of IR increase intracellular clusterin levels, extending to lymphomas a phenomenon first described in the breast carcinoma cell line MCF-7. p53 and Bax are also up-regulated by 12Gy IR at the different time points studied, consistently with the activation of the intrinsic apoptotic pathway. A long-lasting, intracellular clusterin up-regulation observed in surviving cells following irradiation (up to 72h) suggests that this protein is part of the IR-triggered adaptive response. The three cell lines tested here (L-428, KM-H2 and L-540) feature different caspase-3 activity levels. By means of the caspase-3 inhibitor Ac-DEVD-CHO we show here that, in the caspase-3 deficient KM-H2, clusterin is up-regulated by doxorubicin independently of the inhibitor. On the contrary, L-540 shows a different isoforms' expression profile when treated with doxorubicin with or without Ac-DEVD-CHO. This suggests that a linkage exists between caspase-3 activity and clusterin expression.

Disclosures

No relevant conflicts of interest to declare.
AML1/RUNX1 Mutations in 470 Adult Patients with De Novo Acute Myeloid Leukemia: Prognostic Implication and Interaction with Other Gene Alterations.
Authors: Hou Hsin An , Tang Jih-Luh , Lin Liang-In , Chen Chien-Yuan , Chou Wen-Chien , Tseng Mei-Hsuan , Huang Chi-Fei , Lee Fen-Yu , Liu Ming-Chih , Yao Ming , Huang Shang-Yi , Ko Bo-Sheng , Hsu Szu-Chun , Wu Shang-Ju , Tsai Woei , Chen Yao-Chang , Tien Hwei-Fang
Published: October 2019

Abstract 1564

Poster Board I-587

Somatic mutation of AML1/RUNX1 (RUNX1) gene is seen in acute myeloid leukemia (AML) M0 subtype and AML transformed from myelodysplastic syndrome, but the impact of this gene mutation on survival in AML patients remains unclear. In this study, we sought to determine the clinical implications of RUNX1 mutations in 470 adult patients with de novo non-M3 AML. Sixty-three distinct RUNX1 mutations were identified in 62 individuals (13.2%); 32 were in N-terminal and 31, C-terminal. The RUNX1 mutation was closely associated with male gender, older age, lower LDH value, FAB M0/M1 subtypes and expression of HLA-DR and CD34, but inversely correlated with CD33, CD15, CD19 and CD56 expression. Furthermore, the mutation was positively associated with MLL/PTD (P=0.0061), but negatively associated with CEBPA (P=0.0057) and NPM1 mutations (P=0.0001). AML patients with RUNX1 mutations had a significantly lower complete remission rate, shorter disease-free and overall survival than those without the mutation (P=0.0087, P<0.0001 and P=0.012, respectively). Subgroup analysis of patients with normal karyotype showed that RUNX1-mutation was also closely associated with worse OS and DFS (P= 0.001 and P=0.001, respectively). Multivariate analysis demonstrated that RUNX1 mutation was an independent poor prognostic factor for overall survival (hazard ratio 1.874, 95% CI, 1.101- 3.189, P=0.021). Sequential analysis in 133 patients revealed that none acquired novel RUNX1 mutations during clinical courses. Our findings provide evidences that RUNX1 mutations are associated with distinct biological and clinical characteristics and poor prognosis in patients with de novo AML.

Disclosures

No relevant conflicts of interest to declare.
Hypoxia-Inducible Factor-1 in Multiple Myeloma Progression.
Authors: Ria Roberto , Reale Antonia , Berardi Simona , Piccoli Claudia , Pietro Giulia Di , Basile Antonio , Capitanio Nazareno , Vacca Angelo
Published: October 2019

Abstract 1812

Poster Board I-838

Multiple Myeloma (MM) is a malignancy of immunoglobulin (Ig)-synthesizing plasma cells, that home to and expand in the bone marrow. Similarly to other tumours its development is correlated to the formation of regions of hypoxia, which may be a prognostic indicator and determinant of malignant progression. It is known how in solid tumours the degree of intra-tumoral hypoxia is positively correlated with the expression of the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 is composed of HIF-1á and HIF-1β subunits and its production has been identified as a key element in allowing cells to adapt and survive in a hostile hypoxic environment via a variety of pathways. In hypoxia conditions, the HIF-1á subunit becomes stable and regulates the expression of target genes. When activated HIF-1á also targets those genes which are required for angiogenesis, the development of new blood vessels from an existing vascular network. Angiogenesis represents a constant hallmark of MM progression. In response to hypoxia plasma cells and stromal cells (endothelial cells [ECs], macrophages, mast cells) within the tumour express Vascular Endothelial Growth Factor (VEGF), a mitogen and survival factor specific for endothelial cells. VEGF is the major regulator of tumor-associated angiogenesis. HIF-1á directly activates transcription of the VEGF gene and this leads to autocrine signal transduction that is critical for angiogenesis.

In this study we demonstrate the role of HIF-1á in MM angiogenesis. The constitutive stabilization of HIF-1á contributes to increase angiogenesis in MM. Our data show that HIF-1á is stabilized in the nucleus of MM endothelial cells (MMECs) but not in ECs of Monoclonal Gammopathies of Undetermined Significance (MGECs) and in Human Umbilical Vein ECs (HUVECs) used as controls. Western Blot and Enzyme-Linked Immunosorbent Assay (ELISA) analyses show the overexpression of HIF-1á and the proteic products of its target genes VEGF and VEGF Receptor (VEGFR)-1, in patients with relapsed disease and in MM progression but not in patients with nonactive MM (avascular phase). Moreover, immunofluorescent staining confirm the nuclear stabilization of HIF-1á in MMECs.

At mRNA level all ECs express same quantity of HIF-1á mRNA, as confirmed by RT-PCR and Real-time RT-PCR, indicating that in MMECs the post-trascriptional control is affected. Finally, we show that the inhibition of HIF-1á by siRNA suppresses vessel formation in vitro and promote ECs apoptosis. Our findings indicate that HIF-1á plays an important role in MM progression and that it is correlated to the angiogenic switch from nonactive MM to active MM. Furthermore these data suggest that HIF-1á may represent a target for the MM antiangiogenic treatment.

Disclosures

No relevant conflicts of interest to declare.