bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pharicin a, a Novel Natural Compound That Induces Mitotic Arrest and Catastrophe of Cancer Cells by Perturbing Microtubule Dynamics and the Spindle Checkpoint
Authors: Chen Guo-Qiang , Dai Wei , Xu Han-Zhang , Li Dao 
Published: April 2020

Abstract
Microtubule poisons such as taxol represent a potent and effective class of anticancer compounds. In the past decades, great efforts have been directed to identify novel natural products with a mode of action similar to taxol but with a minimal side effect. In this study, we report the functional characterization of a new ent-kaurene diterpenoid termed pharicin A, which was originally isolated from Isodon pharicus leaves, a perennial shrub frequently used in Chinese folk medicine for tumor treatment. Pharicin A induces mitotic arrest in Jurkat, Raji and HeLa based on their morphology, DNA content, histone H3 serine-10 phosphorylation, and mitotic marker protein analyses. Pharicin A stabilizes the formation of mitotic spindles, which is coupled with a rapid accumulation of Cdc20 as well as an increased phosphorylation of Cdc27 and BubR1. Pharicin A treatment also results in an enhanced interaction between Cdc20 and spindle checkpoint components including Mad2 and BubR1. Moreover, pharicin A-induced mitotic arrest in HeLa cells is tightly associated with the presence of lagging/missegregated chromosomes at spindle pole regions, which are highly enriched in BubR1, CENP-E and Sgo1. Although pharicin A stabilizes microtubules both in vitro and in vivo, it induces mitotic arrest in taxol-resistant Jurkat cells. Combined, our study strongly suggests that pharicin A represents a novel class of small molecule compounds capable of perturbing microtubule dynamics and the spindle checkpoint in tumor cells, which merits further preclinical and clinical investigations for cancer drug development.
An in Vitro and in Vivo Evidence That Human Rhabdomyosarcomas (RMS) Express Functional CXCR7 Receptor: Overlapping and Distinct Role of CXCR4-SDF-1 and CXCR7-SDF-1/ITAC Axes in Regulating Metastatic Behavior of RMS Cells
Authors: Grymula Kasia , Tarnowski Maciek , Maksym Radoslaw , Kucia Magdalena , Ratajczak Janina , Ratajczak Mariusz Z 
Published: April 2020

Abstract
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescents and children that frequently infiltrates the bone marrow (BM). We have demonstrated that the a-chemokine stromal-derived factor (SDF)-1-CXCR4 axis (Blood2002;100:2597, Cancer Res. 2003;63:7926, Cancer Res.2007;67:2131) plays an important role in RMS metastases. With the recent description of CXCR7, a new receptor for SDF-1 that also binds the interferon-inducible T cell-alpha chemoattractant (ITAC) chemokine, we become interested in the role of the CXCR7-SDF-1/ITAC axis in RMS metastasis. To address this issue, we evaluated 7 highly metastatic alveolar RMS (ARMS) and 3 less metastatic embryonal RMS (ERMS) cell lines. We found that all RMS cell lines express CXCR7 in contrast to CXCR4, which was expressed by 7 of 10 RMS cell lines only. CXCR4 was expressed at a much higher level by highly metastatic ARMS lines while CXCR7 was expressed at a high level on the less metastatic ERMS lines. More importantly, CXCR7 receptor on RMS cell lines was functional after stimulation with ITAC and SDF-1 as evidenced by MAPKp42/44-, AKT-, and STAT-3 phosphorylation, chemotaxis, cell motility, and adhesion assays. We also noticed that CXCR7 undergoes rapid internalization after stimulation with SDF-1 and ITAC, is a lipid raft-associated receptor, and, surprisingly, has a downregulated expression in response to hypoxia. We also noticed that similarly to CXCR4, signaling from activated CXCR7 was not associated with increased RMS proliferation or cell survival. Thus, we conclude that both CXCR4 and CXCR7 are not affecting proliferation of RMS cells; however they play an important role in both SDF-1- and ITAC-directed metastasis. For the first time, we also identify ITAC as a new regulatory chemokine that is actively involved in this process. In conclusion, since CXCR7 is more important in adhesion and becomes down-regulated during hypoxia, this changes the balance in SDF-1 signaling through both receptors (CXCR7/CXCR4). As a result, SDF-1 activates more robust CXCR4 receptor, which promotes chemotactic responses of RMS cells. As a biological consequence, this switch in SDF-1 receptor signaling increases the metastatic potential of RMS by mobilizing tumor cells to egress from the tumor into the peripheral blood and lymph and migrate to new tissue locations. We also conclude that targeting of the CXCR-SDF-1 axis alone without simultaneous blockage of CXCR7 will be an inefficient strategy to inhibit metastasis of RMS cells. This is currently being tested in our laboratories in immunodeficient mice inoculated with human RMS cells.
In Vivo Modulation of T Cell and Monocyte Function Following Infusion of Mesenchymal Stromal Cells (MSC)
Authors: Castilla-LLorente Cristina , Iwata Mineo , Mielcarek Marco , Abrams V. Kraig , Hwang Billanna , Nash Richard A. , Torok-Storb Beverly 
Published: April 2020

Abstract
Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.
Role of New Tyrosine Kinase Inhibitors in Osteoblastogenesis
Authors: Tibullo Daniele , Giallongo Cesarina , La Cava Piera , Guagliardo Provvidenza , Cavalli Maide , Romano Alessandra , Conticello Concetta , Chiarenza Annalisa , Palumbo Giuseppe A. , Di Raimondo Francesco 
Published: April 2020

Abstract
It has been reported that imatinib mesylate (IM) may affect bone tissue remodeling mainly by both an inhibitory activity on osteoclastogenesis and an induction of osteoblastogenesis. Dasatinib (DA) and Nilotinib (NI) are new generation tyrosine kinase inhibitors presently approved for chronic myeloid leukemia patients after imatinib failure. We therefore evaluated possible effects of DA and NI on osteoblatic differentiation of Mesenchymal Stem Cells derived from bone marrow (BM-MSCs). BM-MSCs are multipotent non-haematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, skeletal myocytes and nervous cells. Mesenchymal stem cells (hBM-MSCs) were obtained from bone marrow samples of normal healthy adult bone marrow donors, isolated by density gradient (mononuclear fraction) and cultured either in standard medium (SM) or in osteogenic medium (OM) (0.2 mM ascorbic acid, 0.1 μm dexamethasone and 10 mM β-glycerophosphate) with or without DA 2nM or NI 100nM. Osteogenic differentiation of hBM-MSCs was evaluated by changes in morphology, presence of mineralized nodules (evidenced by Alizarin red) and expression of osteoblast-associated genes such as osteocalcin (OCN), RUNX2 and Bone morphogenetic protein (BMP-2) evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by Scion Image. After 21days of culture, in comparison to control cultures, hBM-MSCs placed in OM, DA, NI and DA+OM, NI+OM exhibited changes in cell morphology from a spindle-shaped fibroblastic appearance to a rounder more cuboidal shape and the cells formed an extensive network of dense multilayered nodules (extracellular mineralization).
Table I indicates mRNA expression of osteogenic markers in different culture conditions and shows that both DA and NI alone or in combination with OM, increase RUNX2, OCN, and BMP-2 expression.
SM DA NI OM DA + OM NI + OM SM= standard medium, OM= osteogenic medium, DA= dasatinib, NI= nilotinib In summary, our data show that both DA and NI, as already reported IM, may induce osteogenic differentiation of mesenchymal cells thus indicating that they potentially favour osteoblastogenesis. RUNX2 1,59 0,20 2,09 0,16 4,2 0,31 2,86 0,25 4,41 0,41 4,18 0,24 OCN 2,57 0,28 3,2 0,14 3,14 0,09 3,59 0,17 3,6 0,28 3,62 0,25 BMP-2 1,55 0,19 2,27 0,17 4,16 0,27 2,84 0,28 4,43 0,30 4,21 0,30
LSD1-Mediated Epigenetic Modification Is Important for TAL1 Function
Authors: Hu Xin , Li Xingguo , Ybarra River , Valverde Kristell , Fu Xueqi , Noguchi Constance , Qiu Yi , Huang Suming 
Published: April 2020

Abstract
TAL1/SCL is critical for normal and abnormal hematopoiesis by regulating hematopoietic stem/progenitor cell growth and differentiation. However, it is still unclear how its transcriptional activities are controlled during hematopoiesis. Here, we undertook the biochemical isolation of TAL1-associated protein complexes in erythroleukemia cells and showed that TAL1 interacts with histone demethylase LSD1 complexes containing LSD1, CoREST, HDAC1 and HDAC2. Interestingly, although TAL1 specifically colocalizes with LSD1 at the target gene promoter p4.2 in undifferentiated MEL cells, the recruitment of LSD1 is decreased at the p4.2 promoter upon induced MEL differentiation indicating that LSD1 may differentially regulate TAL1 target genes during differentiation. The siRNA-mediated knockdown of LSD1 in MEL and ES cells resulted in the derepression of p4.2 by increasing dimeH3K4 at their promoter region, respectively. Finally, we demonstrated that TAL1-associated LSD1 complexes, H3K4 demethylase, and histone deacetylase activities are coordinately regulated during erythroid cell differentiation. Thus, the data suggest that LSD1 mediated epigenetic modification may affect hematopoiesis and leukemogenesis through its association with the lineage-specific transcription factor TAL1.
A New Animal Model for Anemia Induced by Environmental Low-Temperature: Physiology of Erythrocyte Production and Circulation
Authors: Maekawa Shun , Iemura Hitomi , Kuramochi Yuko , Kosaka-Nogawa Nami , Nishikawa Hironori , Aizawa Youichi , Kato Takashi 
Published: April 2020

Abstract
To survive, organisms must adapt to changes in the ambient environment. Here, we describe a new model of anemia based on exposure of African clawed frog, Xenopus laevis to low-temperature. Frogs exposed at low-temperature (5ºC) for five days had decreased numbers of peripheral blood erythrocytes, leukocytes, and thrombocytes as well as low hemoglobin levels. By contrast, spleen erythrocytes increased in number. Cell counts returned to normal in frogs re-warmed at ambient temperature (22ºC) for two days. To confirm these observations in vivo, we labeled peripheral blood cells with fluorescent reagent CFSE. During five days at 5ºC, labeled erythrocytes in peripheral blood decreased in number while those in spleen increased. When the temperature was raised to 22ºC, however, their numbers increased in peripheral blood. The findings suggested that exposure to low-temperature resulted in splenic pooling of peripheral erythrocytes. Accordingly, we looked at recovery from anemia induced by phenylhydrazine (PHZ) in this model. PHZ-treated frogs maintained at 22ºC decreased numbers of peripheral erythrocytes that were minimal on day 8, and increased gradually thereafter. In the liver, we found erythrocyte progenitors expressing erythropoietin receptor and GATA1-A detected by reverse transcription polymerase chain reactions and immunocytochemical staining but no mature forms. In PHZ-treated frogs exposed to 5ºC, peripheral erythrocyte counts remained minimal from day 8, and reversibly recovered when temperature returned to 22ºC. Erythrocyte progenitors were present in liver on day 8 but absent on day 12. Conversely, mature erythrocytes were absent in liver on day 8 but present on day 12. Finally, to learn whether the progenitors proliferate and differentiate without migrating from liver to peripheral blood, we treated frogs with thymidine analog bromodeoxyuridine (BrdU). In frogs kept at 22 ºC, BrdU-labeled erythrocytes were abundant in both liver and peripheral blood. However, frogs cooled at 5ºC had labeled cells in liver but few in peripheral blood. The findings suggest low-temperature exposure cause this anemia by impairing migration of mature/immature erythrocytes from the liver. In summary, this amphibian model offers a new perspective for investigating physiological effects of environmental temperature on vertebrate erythropoiesis.
Excessive Hematopoietic Stem Cell Proliferation Leads to Chromosomal Instability
Authors: Souza Liliana , Hawk Natalyn , Sengupta Sweta , Cabrera Carlos , McLemore Morgan L. 
Published: April 2020

Abstract
Truncation mutations in the granulocyte colony stimulating factor receptor (G-CSFR), common in severe congenital neutropenia (SCN), lead to excessive stem cell proliferation in response to G-CSF. These G-CSFR mutants are (at least indirectly) implicated in the progression of these patients to acute leukemia. Since SCN patients require continuous G-CSF treatment throughout their lifespan, we hypothesize that excessive stem cell proliferation can lead to DNA damage. Stem cells are relatively quiescent and rarely enter the cell cycle under normal conditions. During the cell cycle cells generate approximately 5000 single strand DNA lesions per nucleus (Vilenchik and Knudson, 2003). Approximately 1% of these lesions are ultimately converted to double strand DNA breaks (DSBs). Hematopoietic stem cells are found within the Sca+ ckit+ Lin- (KLS) population. Wild type and mice bearing a mutant G-CSFR similar to that found in patients with SCN were treated with G-CSF. After 21 days of treatment with G-CSF (10 ug/kg/day), the KLS population in the bone marrow increased four-fold in wild type mice and eight-fold in mutant mice. We isolated Lin-Sca+ bone marrow cells from these G-CSF treated mice and evaluated for the presence of double stranded DNA breaks by staining with anti-phospho-H2AX by immunofluorescence. H2AX is a histone whose phosphorylated form localizes to the site of double stranded DNA breaks. The results showed that there is an 8-fold increase in the DSB in wild type Lin-Sca+ and 10-fold in mutant Lin-Sca+ when compared to cells from untreated mice. This data suggests that excessive proliferation can contribute to an increase in DSBs in hematopoietic stem cells. Investigation of potential mechanisms contributing to DSB formation are ongoing. Understanding the causes and trends of chromosomal instability would improve our understanding of leukemogenesis and potentially reveal novel treatment strategies.
Quantification of Mouse Lymphohematopoietic Stem CellsUsing IR-Spectroscopy
Authors: Kapelushnik Joseph , Dror Ziv , Iskovich Sevtlana , Mordechai Shaul , Zelig Udi 
Published: April 2020

Abstract
Stem cells and in particular, Hematopoietic stem cells (HSCs) therapy is valued for its potential to restore damaged or degenerating tissues. Transplantation of HSCs is already a routine procedure for treatment of blood and immune system disorders. Till now complicated and expensive procedures were needed for identification and isolation of HSC. In the present research we propose a simple and reagent-free procedure for identifying and quantifying lymphohematopoietic stem cells (LHSC) using Fourier transform infrared (FTIR) spectroscopy, which is able to monitor content and conformation of all bio-molecules in cells simultaneously. B6D2F1 mice, 6 to 12 weeks of age were used for this study. Bone marrow (BM) from male B6D2F1 mice was drawn. Putative LHSC were isolated from the BM by common methods according to cell size, markers and high levels of aldehyde dehydrogenase. After isolation of LHSC, various dilutions of LHSC among BM cells were performed and measured under FTIR-microscope. Using spectral and statistical analysis, we identified several spectral markers which characterize and distinct LHSC from pure BM. These markers refer to nucleic acids which increase in LHSC due to their extensive proliferation and differentiation. Furthermore, we achieved high sensitivity quantification of LHSC from BM. (about one LHSC per thousand BM cells) Thus, we conclude that the proposed method of FTIR spectroscopy identification and quantification of LHSC may significantly contribute to stem cells research and hearten the use of stem cells in medicine.
Circulating Endothelial Cells: A Potential Parameter of Organ Damage in Sickle Cell Anemia?
Authors: Strijbos Michiel , Landburg Precious , Nur Erfan , Biemond Bart J. , Leebeek Frank , Gratama Jan-Willem , Duits Ashley , Schnog John-John 
Published: April 2020

Abstract
Traditional laboratory markers of disease severity (Hb concentrations, HbF%, LDH concentrations and leukocyte counts) are not powerful enough to identify individuals with sickle cell disease (SCD) at high risk of complications. In several disease states, the number of circulating endothelial cells (CEC) is indicative of the extent of vascular disease and several small studies employing cumbersome techniques have shown increased CEC to occur in SCD as well. In this pilot study we determined if, by employing the CellSearch system, elevated CEC can be detected in SCD and whether the CEC count is related to the presence of organ damage, painful crisis frequency, traditional laboratory markers of disease severity as well as markers of endothelial activation. Peripheral blood was collected from 15 sickle cell patients and 15 healthy sex, age and race matched HbAA controls. Cells positive for CD146 (present on endothelial cells) are immuno-magnetically isolated and stained with nuclear DAPI and anti-CD105 (present on endothelial cells) and anti-CD45 (pan-leukocyte marker). Within the CD146 enriched cell suspension, cells meeting the DAPI+/CD146+/CD105+/CD45− criteria after image cytometry are designated CEC. CEC counts were significantly higher in sickle cell patients (12 cells/mL, IQR 8–29) as compared to controls (4 cells/mL, 3–10) (p=0.007). CEC counts were significantly higher in patients with pulmonary hypertension (PHT) (p=0.02), and increased with the presence of number of affected organs as well (0–4 involved organs, p=0.03), with the number of affected organs being the most important determinant of increased CEC numbers in SCD (R2=0.72, P=0.001). No statistically significant correlation was detected between CEC counts and markers of endothelial activation (serum sVCAM-1 and plasma VWF:Ag levels). CEC counts did not differ between patients on hydroxyurea and those who did not use hydroxyurea. No statistically significant correlations were detected between CEC counts and painful crisis frequency, Hb, HbF%, leukocyte counts, LDH concentrations. In conclusion, elevated CEC’s can be demonstrated in the clinically asymptomatic state in patients with sickle cell anemia with a validated and automated technique and, for the first time, a relation of CEC counts to SCD related organ damage is demonstrated. Furthermore, the extent of EC activation (as assessed by sVCAM-1 and VWF:Ag levels) is not necessarily related to the extent of EC damage (as assessed by CEC counts).
The Plasma Concentration of the Antithrombotic Protein Annexin A5 Is Elevated in Sickle Cell Disease and Rises Further during Painful Crisis
Authors: van Tits Berry , van Heerde Waander , Landburg Precious , Muskiet Frits , Duits Ashley , Schnog John-John 
Published: April 2020

Abstract
Expression of phosphatidylserine (PS) on the membrane surface of red blood cells and circulating microparticles plays an important role in the etiology of the hypercoagulable state of sickle cell disease (SCD), as well as in the reduced red cell life span and adhesive interactions between red cells and endothelium. Annexin A5, an intracellular protein abundantly present in endothelial cells, exhibits high affinity for PS and has been shown to inhibit microparticle-induced PS-mediated thrombin generation. We determined plasma annexin A5 levels and microparticles in 17 sickle cell patients (12 HbSS and 5 HbSC) in steady state and at presentation with a painful crisis. Twenty five HbAA blood donors served as controls. Both annexin A5 levels and microparticle numbers were highest in HbSS patients (5.7 ng/mL IQR 3.7–7.6 and 37.9 nM, IQR 31.9–69.8) as compared to HbSC patients (1.8 ng/mL, IQR 1.7–7.6 and 20.9 nM IQR10.9–29.6) and healthy controls (2.5 ng/mL, IQR 1.4–4.4 and 13.1 nM, IQR 9.5–18.5) (p=0.01 and p&lt;0.001, respectively). At presentation with a painful crisis, annexin V levels and microparticle numbers increased significantly in 16 of 17 patients (p=0.001) and 16 of 17 patients (p&lt;0.001), respectively. Annexin V/microparticle ratio’s were lower at presentation with a painful crisis in 9 patients. In conclusion, sickle cell patients have elevated plasma concentrations of annexin V. The potential role of annexin V in modulating PS related pathophysiological processes in SCD is subject of further investigation.