bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increased Unfolded Protein Response in Hematopoietic Cells from Cyclic Hematopoietic Dogs.
Authors: Meng Ronghua , Upchurch David , Niemeyer Glenn P. , Lothrop Clinton D. 
Published: April 2020

Abstract
Canine cyclic hematopoiesis, a model of human cyclic neutropenia/congenital neutropenia (CN), is caused by a mutation in the AP3b1 gene. Human CN is caused by mutations in the Ela2 gene. The exact mechanism(s) whereby mutations in the Ela2 and AP3b1 cause CN or CH is not well understood. Elastase is directed to the primary granules by AP3 in myeloid progenitor cells, suggesting abnormal trafficking of elastase as a cause for CN/CH. Discontinuous percoll gradients on PMN cells from normal and CH dogs were performed. Fractions corresponding to the cytosol, plasma membrane, nuclei, primary, secondary and tertiary granules were collected and assayed for elastase and myeloperoxidase (MPO) enzyme activity and protein levels by Western blot using canine specific antibodies. Percoll density gradient fractionation results indicated that MPO, a primary granule protein that is not an AP3 cargo protein, is present in the primary granules in approximately equivalent amounts in both normal and CH dog PMN’s. Elastase was also localized in the primary granule fraction from both normal and CH dog PMN, but with a lower amount in CH dogs. Elastase was not present in the plasma membrane fraction in either CH or normal dog PMN’s. Quantitatively the CH dog primary granules had 10–20% of normal dog primary granule elastase. These results suggest PMN’s from CH dogs correctly sort elastase to the primary granules but at a lower level compared to PMNs from dogs without AP3b1 defect. Glucose-regulated protein (GRP78/BiP) is an indicator of ER stress. ER stress leads to activition of the unfolded protein response (UPR) and is cytoprotective. However, prolonged UPR leads to apoptosis. Analysis of GRP78 expression in PMN’s and bone marrow cell cultures from normal and CH dogs stimulated with SCF and G-CSF demonstrated 3–4 fold increase of GRP78 in CH cells compared to normal dog cells. These results indicate mistrafficking or accumulation of misfolded elastase induces the UPR in myeloid precursor cells and disrupts normal PMN production in CH dogs. These results are consistent with in vitro studies in which over-expression of mutant but not normal human elastase induces the UPR in myeloid cells and cell death. These results suggest that induction of the UPR is a common event, in myeloid progenitor cells from patients and animal models with either Ela2 or AP3b1 mutations, which ultimately results in congenital and/or cyclic neutropenia.
Directed Differentiation: Evolution towards Human Application.
Authors: Colvin Gerald A. , Dooner Gerri J. , Dooner Mark S. , Aliotta Jason , Politopoulou Galatia , Abedi Mehrdad , Quesenberry Peter J. 
Published: April 2020

Abstract
Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential. This is in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. We have reproducibly induced directed stem cell differentiation towards megakaryopoiesis by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. Murine experiments have been performed on highly purified quiescent G0–1 lineagenegative rhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor (TFS), they synchronously pass through cell cycle. Megakaryopoiesis is focused at early to mid S-phase, returning to baseline before initial cell division. Population based differentiation cultures after 14-days produced up to 49% megakaryocytes with stem cells sub-cultured during early-mid S-phase with little to no production with colonies cultured from stem cells in G0–1 or G2 phase at time directed differentiation signaling. Gene expression showed over 2 fold increases in FOG, Nfe2 and Fli1. Clonal studies confirm the results. In one experiment, 33% of clonally derived colonies that grew from early-S phase cells and 10% of colonies that grew from mid-S phase cells had megakaryocytes present compared with 0% for G0–1 and G2 cells. We have now worked with human lineagenegative double-effluxed-rhodaminelow double-effluxed-Hoeschtlow G0–1 stem cells. When expose to TFS cytokines, there initial cell cycle lasts more than 80 hours opposed to CD34+ cells and murine LRH cells which have divided by 44–48 hours. This human population of stem cells comprises approximately 0.01% of CD34+ cells and has tremendous promise in replicating our murine work, elucidating opportunities for human translational work targeting patients that have a block of differentiation toward megakaryopoiesis i.e. sub-sets of autologous transplant or myelodysplastic syndrome patients.
Perturbing Hemostasis by Altering the Kinetics of the Interactions between GPIb Apha and the A1 Domain of VWF: A Mouse Tale.
Authors: Diacovo Thomas G. , Tan Kui , Liddington Robert C. , Chen Jianchun 
Published: April 2020

Abstract
Effective hemostasis relies on ability of circulating platelets to be rapidly deposited at sites of vascular injury. Critical to this process is the formation of adhesive bonds between the platelet receptor GPIb alpha and the A1 domain of VWF, an interaction that is rapid and reversible due to the relatively high rate of bond formation and dissociation. The importance of maintaining the kinetic properties of this interaction within a critical range is suggested by the bleeding phenotype that results from gain- or loss-of function points mutations associated with VWD. To demonstrate the an alteration in bond kinetics can have a dramatic effect of hemostasis, we generated a mouse in which a single point mutation was introduced into the vWF-A1 domain that results in a shortening of the time it can maintain contact with GPIb alpha (aka - increase in off-rate). The mutation was chosen based on our crystal structure data of the recombinant mouse A1 domain protein and its effect on mouse platelet adhesion to the immobilized mutant protein under flow conditions. Homozygous animals were viable and exhibited normal Mendelian ratios. Both VWF antigen levels as determined by ELISA and multimer banding pattern were identical to WT littermate controls. Moreover, transcription of the A1-A2-A3 region of VWF was comparable to controls as determined by RT-PCR. Despite the similarities in biochemical and molecular parameters to WT littermates, mutant A1 domain mice had a significant prolongation in bleeding time as measured by severing a small portion of their tail. Moreover, perfusion of whole blood from these animals over surface-immobilized collagen resulted in &gt;50% reduction in platelet accumulation at 1600 s-1. This abnormality in platelet adhesion was also observed in vivo. Although mutant A1 domain mice were able to form thrombi upon laser-induced injury to arterioles located within the cremaster muscle, these thrombi were unstable and rapidly dissolved. In conclusion, we are the first to demonstrate that an alteration in the kinetic properties of a receptor-ligand bond can have a significant effect on biological processes such as hemostasis. Our results also provide insight into mechanisms by which point mutations associated with certain types of VWD may result in bleeding in afflicted individuals.
Mitochondrial Proteome from Human Peripheral Blood Cells.
Authors: Sarry Jean-Emmanuel , Danet-Desnoyers Gwenn-ael , Carroll Martin , Emerson Stephen G. , Daldal Fevzi , Dancis Andrew 
Published: April 2020

Abstract
Mitochondria play a special role in iron metabolism as the site of heme synthesis for hemoglobin. Mitochondria also function in cellular respiration, apoptosis, amino acid synthesis, Fe-S cluster formation and repair, and redox homeostasis; different blood cell lineages depend on some or all of these diverse mitochondrial functions. Mitochondrial abnormalities in hematopoietic stem cells might manifest themselves in proteomes of all the hematopoietic lineages. Therefore, we have begun characterization of mitochondria from different peripheral blood cell populations: platelets, lymphocytes, neutrophils and reticulocytes with the objective of comparing their function and proteomes in normals and in certain disease states. The procedures utilized as starting material a blood draw of approximately 80 ml from normal volunteers. The peripheral blood samples were separated by centrifugation and Hypaque density gradient into platelet, mononuclear cell, neutrophil and red cell populations. The red cells were further sorted by density gradient and magnetic cell sorting with specific CD71 microbeads to obtain enrichment of reticulocytes (reticulocytes retain their mitochondria and lose these upon maturation into mature red cells). The various cell fractions were evaluated by cell counting, flow cytometry and staining for morphology and identification. In accordance with differences in size and surface characteristics of these cell types, different procedures for cell rupture were utilized: shearing with a home-made device using ball bearings (mononuclear cells, neutrophils), nitrogen cavitation (platelets) and hypotonic shock (reticulocytes). Mitochondria were prepared by differential centrifugation and Percoll density gradient separation. The mitochondria were evaluated by fluorescence microscopy, flow cytometry, marker enzyme activity (succinate dehydrogenase) and Western blotting with compartment-specific antibodies. Mitochondrial protein profiles were obtained using 2-dimensional gel electrophoresis coupled to mass spectrometry. From 80 ml blood, 50 million lymphoctes were obtained equivalent to 150 microgram mitochondrial protein and 10 fold enrichment of succinate dehydrogenase activity. In parallel, K562 cell mitochondria were studied. The imaging analysis revealed significant differences in the protein patterns due to hematopoietic cell lineage. This work seeks to establish a proteomic database of shared and distinct erythroid, myeloid and lymphoid mitochondrial proteins that will form the basis of future studies of blood diseases in which perturbations of mitochondrial proteins are expected to occur. We are especially interested in examining the mitochondrial proteome and correlating with mitochondrial function in myelodysplasia and sideroblastic anemia.
Identification of Transcription Factor AP-1 Bound to Untranslated 5′ Regulatory Region DNA of mdr1 Gene with Atomic Force Microscopy.
Authors: Chen Bao-An , Xiong Jie , Ba Long , Gao Feng , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Cheng Jian , Wang Jun , Zhao Gang , Lu Zu-Hong , Chen Ning-Na , Fei Fei 
Published: April 2020

Abstract
Multidrug resistance(MDR) phenotype of cancer cells is a major obstacle for cancer chemotherapy, this phenotype is main due to the overexprssion of the mdr1 gene. Transcription factor AP-1, which is one of important regulate proteins in the promoter region of mdr1 gene. To date, no direct data of AP-1-DNA regulating complexes on mdr1 gene. A novel method for identifying DNA-binding proteins from image analysis using atomic force microscopy(AFM) was developed. This study is to image and map the structure of Untranslated 5′ regulatory region DNA of mdr1gene, identificate and analysis of transcription factor AP-1 bound to Untranslated 5′ regulatory region DNA of mdr1 gene complex with atomic force microscopy, to find out the molecule mechanism of multidrug resistance.
Human leukemia adriamycin resistant strain K562/A02 was used as a target cells, transcription factor AP-1 was used as a target protein. Cultivate and PCR technique was used to amplify K562/A02 cells with the 769bp 5′regulatory region DNA of mdr1 gene, which fragment from −755 to +14. The amplified DNA products were purified by the PCR product purification kit, AP-1 of hela nuclear extract were purified by Sephadex spin-column filled with a bed of Sephadex G-100. The target DNA fragments were incubated with the target protein AP-1 at a 1:2 molar ratio in binding buffer and then immobilized the the AP-1-DNA complexes on a freshly cleaved mica surface which treated by MgCl2. AFM was used to idificate image of the structure of target DNA and the AP-1-DNA complexes.
We show here the applicability of AFM in the quantitative analysis of the molecular mechanisms of DNA-protein interaction:
The optimum DNA concentration to yield well-absorbed DNA molecular on the mica surface was found to be 10ng/ul. the contour of target DNA AFM image :the length of the DNA fragment measured by AFM image was 260.13± 2.29nm, the width was 11.88 ± 0.92nm, the mean height was 1.2nm(mean±SD, N = 50). Sephadex spin-column (Ultrafree- MC 0.22) can be used to purificate DNA and protein-DNA complex, the contour data of AP-1 protein AFM image:: the width of proteinlarge was 30±3.2nm, protein small was 19±2.8nm; the height of proteinlarge was 3.8±1.4nm, proteinsmall was 3.2±1.8nm (mean±SD, N = 30). we got the contour data of pro- tein-DNA complexes: the width of protein large was 28±2.7nm, the height was 3.4± 0.94nm (mean±SD, N = 8), the width of proteinsmall was 18±1.7nm, the height was 3.1±2.2nm (mean±SD, N = 22), and deduced the possible AP-1 site on mdr1DNA located between bases −126 and −115bp, this result is in close agreement with the expected −121 and −115 bp values.
the overall connection efficiency of protein was about 10%. The AFM method to visualize individual biomolecules allows us to investigate the conformation of protein-DNA complexes.
Genome-Wide Computational Screen for Blood Stem Cell Enhancers Integrates RUNX1 and the BMP Signaling Pathway into the SCL Transcriptional Network.
Authors: Pimanda John E. , Donaldson Ian J. , Bruijn Marella F. , Kinston Sarah J. , Knezevic Kathy , Huckle Liz , Piltz Sandie , Landry Josette-Renee , Green Anthony R. , Tannahill David , Gottgens Berthold 
Published: April 2020

Abstract
Hematopoietic stem cell (HSC) development is regulated by several signaling pathways and a number of key transcription factors, which include Scl/Tal1, Runx1 and members of the Smad family. However, it remains unclear how these various determinants interact. Using a genome-wide computational screen based on the well-characterized Scl +19 HSC enhancer, we have identified a related Smad6 enhancer that also targets expression to blood and endothelial cells in transgenic mice. We show that at E10.5, Smad6 and Bmp4 transcripts are concentrated along the ventral pole of the dorsal aorta and resemble Runx1 expression in the aorta-gonad-mesonephros (AGM) region where HSCs originate. Moreover, Smad6 binds and inhibits Runx1 activity whereas Smad1, a mediator of Bmp4 signaling, transactivates the Runx1 promoter. Taken together, our results integrate three key determinants of HSC development; the Scl network, Runx1 activity and the Bmp4/Smad signaling pathway.
32D TPO, a TPO-Dependent Subclone of the 32d Cell Line, Expresses CXCR4 and Proliferates in Response to SDF-1.
Authors: Amabile Giovanni , Botta Rosanna , Di Noia Antonella , Alfani Elena , Migliaccio Anna Rita , Migliaccio Giovanni 
Published: April 2020

Abstract
Both Thrombopoietin (TPO) and Stromal Derived Factor 1 (SDF-1) have been reported to regulate megakaryocyte differentiation in vivo and in vitro. Using growth factors [G-CSF, M-CSF, GM-CSF, erythropoietin (EPO) or thrombopoietin (TPO)], as selective pressure, factor-dependent clones were consistently isolated from the murine multipotential IL-3-dependent cell line 32D. The different clones acquired a phenotype consistent with the growth factor for which they were dependent. In particular, the EPO-dependent clones showed erythroblast morphology and expressed high levels of CD71 and TER119, on the other hand, the TPO-dependent clones were megakaryocytes in morphology and expressed CD41 and CD61 (see Figure). The TPO-dependent subclones retain the capacity to grow in IL-3, but respond to neither EPO, G-CSF or GM-CSF. Here it is shown that the TPO-dependent clone, but not its EPO-dependent sister (see figure) or parental 32D cell lines, express robust level of CXCR4 on their surface. 32D TPO cells are also capable to respond to SDF-1 (the CXCR4 ligand). In fact, these cells die in 3 days in the absence of growth factors but proliferate equally well (from 5x104 to 6–9x105 cells/mL) in the presence of TPO (100 ng/mL) and/or SDF-1 (100 ng/mL), retaining a robust megakaryocytic phenotype in both culture conditions. By expression profile 32D TPO cells growing in TPO express the same level of Gata1 (both ~2−ΔCt = 3.6 ± 0.5x10−2 ) and Platelet Factor-4 (both 2−ΔCt = 3.5±1x10−1) more Gata2 (2−ΔCt = 2.3±0.1x10−1 vs 1.3±0.2x10−1, respectively), and Fog1 (2−ΔCt = 4.5±1x10−2 vs 3.1±0.5x10−2, respectively), and less Acetylcholinesterase (2−ΔCt = 4.96±1x10−5 vs 6.68±2x10−5, respectively), and Von Willebrand Factor (2−ΔCt = 2.7±0.3x10−2 vs 3.4±0.5x10−2, respectively), than those growing in SDF-1. These data indicate that a TPO-dependent cell line acquires the ability to respond to SDF-1, supporting the notion that megakaryocytic differentiation can be sustained equally well by TPO or SDF-1. To our knowledge, the 32D TPO cell line is the first megakaryocytic cell line that is dependent for growth on the presence of SDF-1. As such, it might represent a suitable model to compare TPO and SDF-1 signalling in megakaryocytes.
Figure Figure
Differential Expression of Hematopoietic Genes in Murine Embryonic E14 Stem Cells upon G-CSF and IL-3 Stimulation.
Authors: Stoelting Stephanie , Fiedler Anja , Wagner Thomas , Peters Stefan O. 
Published: April 2020

Abstract
Introduction:
G-CSF is used widely in humans as in stem cell mobilization protocols for clinical transplantation. Even though it’s mechanisms of action on differentiated cells are well described, it’s effects on stem cells are widely unknown. IL-3 acts as a survival factor without differentiational effects. The presence of G-CSF and IL-3 receptors on embryonic stem cells have been described. We here use murine embryonic E14 stem cells (ESC) to study cellular and molecular events induced by G-CSF and IL-3 during early hematopoietic development.
Methods:
Cells were precultured in standard medium (DMEM, 20% FCS) for 6 days and then kept as embryoid bodies (EBs) for 0, 5, 10, 15, 20, 25, 30 days either in standard or medium continuously supplemented with G-CSF (50 ng/ml) and/or IL-3 (10 ng/ml). Suspended cells were analyzed for CD 90 (thy1) positivity by flow cytometry. CD13 stained cells of embryoid bodies were examined by fluorescent microscopy. cDNA was analyzed for PU.1, GATA-2, Flk-1, SCL, Epo, HoxB4, LMO2, LMO4 and cyclin D1 activation by Taqman PCR.
Results:
First hematopoietic specific CD90 positivity is found on embryoid bodies at day 6+5, either with or without cytokine stimulation. There is a marked increase in hematopoietic development at day 6+10 consisting in 5% CD90 positivity declining at later time points in unstimulated EBs. Interestingly the fraction of CD90 positive cells could not be augmented by G-CSF alone. Upon IL-3 stimulation the fraction of CD90 positive EBs increased twofolded as compared to unstimulated controls. This remained for the entire duration until day 6+30. Double stimulation with IL-3 and G-CSF abrogated this stimulatory effect. We selected a number of genes of interest in hematopoietic development based on published gene expression profiling studies. Under G-CSF stimulation there is a continuous minor upregulation of some of these genes. Interestingly, under IL-3 stimulation most of these genes (GATA-2, SCL, PU.1, Epo and HoxB4) are markedly downregulated while CD90 positive cell fractions are stimulated. This downregulation is most prominent at day 6+10 and 6+15.
Conclusion:
G-CSF activates key genes of hematopoietic development in E14 ESCs without causing differentiation. IL-3 activation of hematopoiesis in EBs parallels downregulation of important genes known as activators of adult hematopoiesis. This could mean that causally determined effects of growth factors may differ at early and committed progenitor cell levels.
Effects of Darbepoetin Alfa Administered Once Per Cycle for the Prevention of Anemia on the Incidence of Transfusions, Neurocognitive Functions, and Symptom Assessment.
Authors: Vadhan-Raj Saroj , Trent Jon , Patel Shreyaskumar , Collard Mindy , Bueso-Ramos Carlos E. , Cleeland Charles , Benjamin Robert , Meyers Christina 
Published: April 2020

Abstract
Background: Chemo-naïve sarcoma patients (pts) experience severe anemia during front-line chemotherapy (CT) with doxorubicin and ifosfamide (AI). In our prior study with AI, vast majority of pts (65 of 68, 93%) who received &gt;1 cycle of CT, required PRBC transfusions. Anemia has significant negative impact on pts quality of life (QOL) and neurocognitive (NC) functions. Prior studies have shown that cancer pts receiving CT experience decline in NC functions and QOL. Preclinical studies suggest that treatment with erythropoietic stimulating agents (ESA) may protect NC functions. The purpose of the present study was to evaluate the effects darbepoetin alfa (DA) administered once per cycle as prophylaxis on the prevention of anemia, need for transfusions, symptom burden and NC functions in sarcoma pts receiving AI.
Methods: DA (initial dose 500 mcg SC) was administered once per cycle before CT (day 0) starting from cycle-1(n=26) or cycle 2 (n=23) and continued up to max 6 cycles. Pts underwent a complete battery of neuropsychological tests and assessment of symptoms via the MD Anderson Symptom Inventory (MDASI) and quality of life with the Functional Assessment of Cancer Therapy-Anemia (FACT-An) Scale at baseline, during CT (after 3 cycles) and at the end of study. At baseline, the median hemoglobin (Hb) was 13.2 g/dL, and only 5 of 49 pts were anemic to a level &lt;11 g/dL. However, significantly more NC deficits or symptoms, including memory loss (29% of pts), fatigue (24%), pain (21%), distress (16%), and sadness (16%) were seen in pts at baseline as compared to normal populations. Pts with higher baseline Hb values had significantly less sadness (p=0.008) and less shortness of breath (p=0.003). Of the 45 pts that received &gt; 1 cycle of CT (median, 4; range, 2–6 cycles), 38% avoided PRBC transfusions. Patients who avoided transfusions had less distress (p=0.032) and less sadness (p=0.01) during treatment. Patients with higher Hb nadir during treatment had less numbness and tingling (p=0.006). Overall, there was a significant decline in verbal fluency, non-dominant hand dexterity (p&lt;0.001), and memory recall (p=0.018). Interestingly, there was a significant improvement in several symptoms including distress (p=0.001), sadness (p=0.032), and pain (p=0.004) during treatment as compared to baseline. These findings suggest that cancer pts can have NC deficits prior to CT. Prevention or early intervention with ESA may avoid the need for transfusions, reduce symptom burden and NC deficit.