bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic Patterns of Migration and Expansion of Hematopoiesis during MGMT Mediated Drug Selection.
Authors: Lin Yuan , Cheung Perrin , Wilson David L. , Gerson Stanton L. 
Published: October 2019

Abstract
While hematopoietic engraftment kinetics are well appreciated after lethal irradiation in the mouse, most observations have been limited to blood samples or terminal examination of marrow or spleen. The development of non-invasive bioluminescence in vivo imaging technology allows a dynamic picture of engraftment and clonal expansion to be defined. We have extended this technology to the process of drug resistance gene therapy. We hypothesized that drug selection would profoundly affect the extent and dynamics of hematopoietic stem cells (HSC) engraftment and clonal expansion after lentiviral mediated gene transfer of the P140KMGMT gene into murine HSC. In previous studies, we have shown that P140KMGMT gene containing retroviral and lentiviral transduced bone marrow cells provided significant protection against chemotherapeutic drugs BCNU and TMZ given with BG (O6-Benzylguanine), in vitro and in vivo. We generated a bicistronic lentiviral vector containing P140KMGMT gene and firefly luciferase gene linked by 2A sequence of FMDV(Foot-and-Mouth Disease Virus), which will cleave itself during ribosomal translation. Whole bone marrow cells was collected from BALB/c mice 4 days after 5-FU treatment and transduced with P140KMGMT-luc lentiviruses at MOI of 1.4. Transduced bone marrow cells were transplanted into lethally irradiated or non-myeloablated syngeneic recipient mice at different cell numbers. Initial bioluminescent signal emerged 6–8 days after transplantation in both lethally irradiated and non-myeloablated recipients. The onset of bioluminescent foci after transplantation occurred in a cell dose dependent manner. The initial signal emitted predominantly from bone marrow, especially femurs, humeri and vertebrae during the early stage of clonal expansion. Intense signal appeared in spleen at days 12–14 and became weaker or even disappeared by days 20–28. Clonal expansion and engraftment greatly increased after a single course of BG+TMZ treatment and initiated strong hematopoiesis in non-myeloablated recipients. Total body bioluminescence intensity of drug treated mice increased 24 fold and 7 fold compared to non-treated mice in both non-myeloablated and lethally irradiated recipients, respectively. A transient phase suggesting migration through the lymphatic system and in the spleen occurred in most mice and was exacerbated by drug selection, but this was less clear in lethally irradiated mice, where engraftment was more confined to the marrow spaces. Bioluminescence in vivo imaging reveals active migration between the bone marrow and the spleen during hematopoiesis. Drug selection has a significant impact on the patterns of engraftment and clonal expansion of HSC and progenitor cells after transplantation.
Role of G-Protein Coupled Inwardly Rectifying Potassium (GIRK) Channels in ADP-Mediated Thromboxane A2 Generation.
Authors: Shankar Haripriya , Prabhakar Janani , Lakhani Parth , Kunapuli Satya P. 
Published: October 2019

Abstract
Platelet activation is associated with platelet shape change, fibrinogen receptor activation, platelet aggregation, secretion of granule contents and phospholipase A2 (PLA2) activation. Agonist-induced PLA2 activation is important for liberation of arachidonic acid from membrane phospholipids, which is then subsequently converted to thromboxane A2 (TXA2) via the sequential effects of cyclo-oxygenase and thromboxane synthase respectively. Thus the generated TXA2 plays a very predominant role in potentiating the effects of other physiological platelet agonists like collagen, thrombin and ADP. ADP-induced TXA2 generation requires co-stimulation of the P2Y1 and P2Y12 receptors, fibrinogen receptor activation and subsequent outside-in signaling. We recently demonstrated that G-protein gated inwardly rectifying potassium channels (GIRKs) are important for P2Y12 receptor-mediated platelet functional responses, namely platelet aggregation, dense granule secretion and Akt phosphorylation. In this study, we evaluated the role of GIRK channels in ADP- mediated TXA2 production in human platelets. ADP-mediated TXA2 generation was inhibited in the presence of SCH23390 and U50488H in a concentration-dependent manner, with maximal inhibition occurring at 10 μM. At these concentrations of GIRK blockers, none of the other P2Y12 receptor-dependent platelet functional responses including aggregation, Akt phosphorylations were affected. We confirmed our findings by performing similar experiments with 2-MeSADP, a potent and selective P2Y1 and P2Y12 agonist. We observed that GIRK channel blockers inhibit 2-MeSADP-mediated TXA2 generation. These results suggest the existence of two different populations of GIRK channels;-one that plays an important role in TXA2 formation (sensitive to low concentrations of the GIRK blockers) and the other population that plays an important role in platelet aggregation (sensitive to higher concentrations of GIRK blocker). Experiments done in the presence of SKF38393, a structurally similar but inactive analog of SCH23390 did not have any effect on ADP or 2-MeSADP-induced TXA2 production in the non-aspirinated platelets. Finally, we also observed that arachidonic acid-induced TXA2 production is not affected by 10 μM concentrations of SCH23390 and U50488H. These data suggest that GIRK channel blockers inhibit TXA2 formation by interfering with agonist-induced PLA2 activation. Based on these findings we conclude that there exist different populations of GIRK channels, one that contributes to ADP- and 2-MeSADP-induced TXA2 and the other that contributes to P2Y12 receptor-dependent platelet functional responses. Also, each of the two populations of GIRK channels has varying susceptibilities to the GIRK channel blockers.
Arg333 and Arg334 in the C-Terminus of the Human Platelet P2Y1 Receptor Are Crucial for Gq Coupling.
Authors: Ding Zhongren , Tuluc Florin , Bandivadekar Kavita R. , Zhang Lili , Jin Jianguo , Kunapuli Satya P. 
Published: October 2019

Abstract
ADP-induced platelet aggregation plays an important role in hemostasis and thrombosis. Human platelets express two ADP receptors: the P2Y1 and P2Y12 receptors. The Gq-activating P2Y1 receptor plays an important role in ADP-induced platelet shape change, aggregation, and thromboxane A2 generation. In this study, we investigated the role of the carboxyl terminus of the human P2Y1 receptor in Gq activation. Human P2Y1 receptors, either wild type (P2Y1-WT) or a mutant in which the C-terminus was truncated (P2Y1-ΔT330-L373), were stably expressed with an HA-tag at the N-terminus in CHO-K1 cells. Stimulation of P2Y1-WT cells with 2-MeSADP caused inositol phosphate production and mobilization of calcium from intracellular stores. In contrast, P2Y1-ΔT330-L373 completely lost its response to 2-MeSADP, indicating that the C-terminus of the human P2Y1 receptor is essential for the activation of Gq. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 carboxyl terminus failed to elicit Gq functional responses, indicating that the P2Y1 carboxyl terminus is essential but not sufficient for Gq activation. Radioligand binding demonstrated that both the P2Y1-WT- and P2Y1-ΔT330-L373- expressing cells have almost equal binding of [3H]MRS2279, a P2Y1 receptor antagonist, indicating that C-terminus truncation did not drastically affect the conformation of the receptor. Two additional truncation mutants in the C-terminus, P2Y1-ΔR340-L373 and P2Y1-ΔD356-L373, were expressed in CHO-K1 cells, and responded to 2-MeSADP with functional responses. These results indicate that the 10 amino acids (330TFRRRLSRAT339) in the C-terminus of the human P2Y1 receptor are essential for Gq coupling. Finally, the cells expressing a double mutant P2Y1 receptor (R333A and R334A), in the conserved BBXXB region in the C-terminus of the Gq-activating P2Y receptors, completely lost its functional ability to activate Gq. We conclude that the two Arg residues (R333R334) in the C-terminus of the human platelet P2Y1 receptor are essential for Gq coupling and subsequent platelet activation.
Di-Annexin, a Novel Tool To Identify and Protect Phosphatidylserine Exposing Cells.
Authors: Kuypers Frans A. , Larkin Sandra K. , Emerson Renee K. , Allison Anthony C. 
Published: October 2019

Abstract
Phosphatidylserine (PS) is found exclusively in the inner monolayer of plasma membranes. During programmed cell death this well-preserved asymmetry of phopholipid distribution is lost, and PS is exposed on the outer surface. PS exposure is also found in subpopulations of red blood cells (RBC) in disease states such as sickle cell disease (SCD). PS exposure leads to increased cell-cell interactions as well as recognition and removal by macrophages. PS serves as a binding site for factors involved in blood coagulation and provides a target for secretory phospholipase A2 (sPLA2) mediated membrane degradation. Measurement of PS-exposing cells (PSRBC) relies on the use of proteins that bind to PS and can be fluorescently labeled and visualized by flow-cytometry. The human protein annexin V (AV) has a high affinity for PS and has been shown to interfere with the physiologic processes induced by the exposure of PS. To improve detection of PS exposing cells, and develop a probe that may be used in vivo, we generated plasmids encoding a dimer of human AV (DAV), and expressed the protein in E.coli. We show that DAV has a 5–8 fold higher sensitivity than AV in binding to PSRBC, and effectively identifies PSRBC in SCD blood samples. Moreover, DAV protects PSRBC from sPLA2-induced hemolysis. While normal RBC show only 2% hemolysis after incubation with 100 ng/ml sPLA2 for one hour, sPLA2 induces more than 90% hemolysis in PSRBC. DAV, when added to PSRBC before incubation with sPLA2, protects more than 90% of the PSRBC from hemolysis. Our data show that DAV effectively competes with factor Va for PS binding and inhibits thrombin formation in the presence of the pro-thrombinase complex. At low concentrations, DAV was significantly more efficient in inhibiting thrombin formation than AV (P<0.001). The increased size of DAV gives the molecule a significantly increased survival time in the murine circulation compared to AV, and allows visualization of PS exposing membranes in vivo. Together this data shows that DAV is a superior probe to identify PS-exposing membranes, protects PSRBC from sPLA2-induced hemolysis, and interferes with cellular hemostasis. The use of DAV will allow more precise determination of PS exposure by flow-cytometry as compared to currently available commercial probes. Moreover, it will allow the measurement of PS exposing membranes in vivo, and may ultimately be proven to be useful as a therapeutic agent in SCD pathology by masking PS exposing cell membranes.
Functional Analysis of Two Mutants of Pyrimidine 5′ Nucleotidase Causing Nonspherocytic Hemolytic Anemia.
Authors: Chiarelli Laurent , Mattevi Andrea , Galizzi Alessandro , Fermo Elisa , Bianchi Paola , Zanella Alberto , Valentini Giovanna 
Published: October 2019

Abstract
Pyrimidine 5′-nucleotidase type-I (P5′N-1) catalyzes the dephosphorylation of UMP and CMP to their respective nucleosides. In red blood cells, the enzyme has a major role in the catabolism of nucleotides formed from RNA degradation. P5′N-1 possesses also phospho-transferase activity suggesting an additional role of the enzyme in nucleotide metabolism. P5′N-1 deficiency is an autosomal recessive disorder characterized by hemolytic nonspherocytic anemia, heavy basophilic stippling in the peripheral blood smear, and accumulation of pyrimidine nucleotides within the erythrocytes. P5′N-1 deficiency is the third most common RBC enzymopathy causing hemolysis after G6PD and PK deficiency. Fourteen different mutations have been identified at the DNA level to date including four missense mutations. To increase our understanding on molecular basis of the P5′N-1 deficiency, after mutants N190S and G241R (Chiarelli et al, Blood 2003, abstract; Chiarelli et al, The Hematology Journal 2004, abstract), we have undertaken the biochemical characterization of D98V and L142P enzymes, identified respectively in a Norwegian family and in Japanese patients. The proteins were produced in E. coli cells as recombinant forms, and purified to homogeneity. The L142P protein showed a drastic reduction in the thermal stability (t1/2 at 37°C about 6 min compared to a fully stable wild-type), and kinetic properties slightly altered (kcat values nearly halved and Km 3–5 times higher). D98V exhibited reduced heat stability (t1/2 at 37° about 25 min) and catalytic efficiency turned especially versus UMP (about 25 times) owing the increased Km values. Thus, the decreased activity observed in Japanese patients homozygous for the L142P mutation is essentially due to lowered enzyme levels caused by protein instability, whereas the D98V mutation of Norwegian patients alters both stability and catalytic efficiency. We suggest that substitution D98V affects an amino acid residue involved in substrates binding site.
Efficient Inhibition of Notch3 and Notch4 Family Members In Vivo by a Dominant Negative Mutant of Mastermind.
Authors: Pross Seth , Millholland John M. , Sai Hong , Weng Andrew P. , Aster Jon C. , Pear Warren S. , Maillard Ivan 
Published: October 2019

Abstract
The four mammalian Notch receptors play multiple roles in the hematolymphoid system. Notch1 is critical for the generation of hematopoietic stem cells (HSCs) during embryonic development and for commitment to the T cell lineage, while Notch2 regulates marginal zone B cell development. In addition, important roles for Notch are starting to be identified in peripheral T cells, in which all four Notch family members are upregulated upon T cell activation. Canonical Notch signaling involves proteolytic cleavage of Notch receptors and translocation of intracellular Notch (ICN) to the nucleus where it interacts with the transcription factor CSL/RBP-J and recruits Mastermind-like proteins (MAMLs) for transcriptional activation. By inhibiting Notch1 and Notch2-dependent developmental decisions with a dominant negative mutant of MAML1 (DNMAML1), we have previously demonstrated that MAMLs critically regulate Notch signaling in vivo (Maillard et al., Blood 2004). However, it is unclear if Notch3 and Notch4 are equally dependent on the activity of MAMLs in vivo, and if DNMAML1 can thus be used as a true pan-Notch inhibitor.
To address this question, we used a modified fetal thymic organ culture (FTOC) system and Notch-mediated T lineage commitment as a readout for Notch function. Fetal liver cells (FLCs) were transduced with retroviruses expressing ICN1, ICN3 or ICN4 and a retrovirus expressing DNMAML1. Irradiated fetal thymic lobes were reconstituted with transduced FLCs and cultured in the presence of gamma secretase inhibitors to inhibit the activation of endogenous Notch receptors. In this manner the effect of a specific ICN could be evaluated in the absence of endogenous Notch signals. Gamma secretase inhibitors resulted in a profound block in T cell development and in the generation of intrathymic B cells, consistent with Notch1 inhibition. ICN1, ICN3 and ICN4 were all able to rescue commitment to the T cell lineage and to block B cell development, indicating that each ICN can deliver appropriate signals for T lineage commitment in the absence of endogenous Notch1 signaling. In cells doubly transduced with specific ICNs and DNMAML1, the rescuing effect of ICN1, ICN3 or ICN4 was blocked, leading to the generation of B cells. In this competitive situation, DNMAML1 only partially inhibited ICN1, the most potent of all four Notch family members, but it was able to completely inhibit ICN3 and ICN4. We conclude that DNMAML1 can efficiently inhibit Notch3 and Notch4 in vivo. The results validate the use of DNMAML1 as a bona fide pan-Notch inhibitor in multiple settings, including studying the role of Notch in HSCs and peripheral T cells.
Hemoglobin Levels and Bone Density in Older Persons: Results from the Inchianti Study.
Authors: Cesari Matteo , Pahor Marco , Lauretani Fulvio , Penninx Brenda W.H.J. , Bartali Benedetta , Russo Roberto , Cherubini Antonio , Woodman Richard C. , Bandinelli Stefania , Guralnik Jack M. , Ferrucci Luigi 
Published: October 2019

Abstract
Background: Hypoxemia has been recognized as a risk factor for bone loss. The aim of the present study is to investigate the relationship of anemia and hemoglobin levels with bone mass and density measures in a large sample of older community-dwelling persons.
Methods: The present study is based on data from 950 participants enrolled in the “Invecchiare in Chianti” (Aging in the Chianti area, InCHIANTI) study. All the analyses were performed considering continuous hemoglobin levels as well as the dichotomous anemia variable (defined according to WHO criteria as hemoglobin <12 g/dL in women and <13 g/dL in men). A peripheral quantitative computerized tomography (pQCT) scan of the right calf was performed in all participants to evaluate total bone density, trabecular bone density, cortical bone density, and the ratio between cortical and total bone area. Linear regression analyses were used to assess the multivariate relationship of pQCT bone measures with anemia and hemoglobin levels after adjustment for demographics, chronic conditions, muscle strength and biological variables.
Results: Participants were 75.0 (SD 6.9) years old. In our sample, 101 participants (10.6%) were anemic. In women, coefficients from adjusted linear regression analyses evaluating the association between pQCT bone measures (per SD increase) and hemoglobin levels/anemia showed significant associations of anemia with total bone density (ß=−0.335, SE=0.163; p=0.04), and cortical bone density (ß=−0.428, SE=0.160; p=0.008). Relationships with borderline significance were found for the associations of anemia with trabecular bone density and the ratio between cortical and total bone area. Significant associations were found between hemoglobin levels and trabecular bone density (ß=0.112, SE=0.049; p=0.02), total bone density (ß=0.101, SE=0.046; p=0.03), cortical bone density (ß=0.100, SE=0.046; p=0.03), and the ratio between cortical bone and total area (ß=0.092, SE=0.045; p=0.04). In men, significant associations were found for the associations of hemoglobin levels with total bone density (ß=0.076, SE=0.036; p=0.03) and cortical bone density (ß=0.095, SE= 0.41; p=0.02). A borderline significance was reported for the association between anemia and cortical bone density.
Conclusion: Anemia and low hemoglobin levels are negatively and independently associated with bone mass and density. The bone loss associated with hemoglobin levels mainly occurs in the cortical bone. Women with lower hemoglobin levels demonstrate a higher bone loss than men.
Multigenic Dissection and Prognostic Modeling of Overt Stroke in Sickle Cell Anemia.
Authors: Sebastiani Paola , Ramoni Marco F. , Nolan Vikki , Baldwin Clinton T. , Steinberg Martin H. 
Published: October 2019

Abstract
Stroke is a major vascular complication of sickle cell anemia, more frequent in patients under the age of 20 years. Transcranial Doppler (TCD) flow studies can predict the likelihood of stroke in children with sickle cell anemia, but only 10% of individuals with abnormal TCD values will have stroke, and stroke will occur in some individuals with normal TCD. Therefore, more precise means of prognosis would be welcome so that prophylactic treatments like transfusions or hydroxyurea can be targeted to individuals at highest risk. Using Bayesian networks (BNs), we analyzed 235 single nucleotide polymorphisms (SNPs) in 80 candidate genes in 1398 unrelated subjects with sickle cell anemia enrolled in the Cooperative Study of Sickle Cell Disease. Bayesian networks are a novel generation of multivariate models that represent the complex structure of interactions between many variables by a network of interrelated modules. These modules can be learned from data and then can be used to describe how changes in some variables affect other variables and ultimately the risk for the phenotype of interest. Bayesian networks provide a coherent framework within which genotypes, phenotypes and environmental factors can be seamlessly integrated into a comprehensive genomic landscape. We found that 25 SNPs on 11 genes and 4 clinical variables - including α thalassemia and fetal hemoglobin - interact in a complex network of dependency to modulate the risk of stroke. This network of interactions includes three genes (TGFBR2, TGFBR3, BMP6) with a functional role in the TGF-beta pathway and one gene (SELP) already associated with stroke in the general population. We validated our results in a different population by predicting the occurrence of stroke in a set of 114 subjects not included in the original study: 7 stroke patients and 107 control patients, a proportion consistent with the phenotype distribution in the original cohort study. Our model predicted the correct outcome for all 7 stroke patients, and for 105 of 107 non-stroke patients, with 100% true positive rate and 98.14% true negative rate, and an overall predictive accuracy of 98.2%. Our results support the hypothesis that stroke in sickle cell anemia patients is a complex trait caused by the interaction of multiple genes, and the predictive accuracy of our model is a step toward the development of additional prognostic tests to allow us to more precisely identify sickle cell anemia patients at risk for stroke. The presence in our model of genes already associated with stroke, such as SELP, suggests that some genetic factors predisposing to stroke are shared by both sickle cell anemia patients and stroke victims in the general population, and that our model may offer some insights into the genetic basis of the third leading cause of death in the United States.