bluuebunny/crossref_metadata_2025_split huggingface dataset:
An RNA Interference Model of RPS19 Deficiency in Diamond Blackfan Anemia Recapitulates Defective Erythropoiesis and Rescue by Dexamethasone: Identification of Dexamethasone Responsive Genes by Microarray.
Authors: Ebert Benjamin L. , Lee Michele , Pretz Jennifer , Golub Todd R. , Sieff Colin 
Published: October 2019

Abstract
Diamond Blackfan Anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation, but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but the relevance of this ubiquitously expressed ribosomal gene to erythroid differentiation is unclear. Glucocorticoids are the primary pharmacological therapy for patients with DBA, but the molecular basis for the activity of glucocorticoids in erythroid differentiation has not been identified. Through retroviral expression of short hairpin RNAs (shRNAs), we demonstrate that targeted degradation of the RPS19 gene in cultured human CD34+ cells blocks the proliferation and differentiation of erythroid progenitor cells. Decreased RPS19 expression limited production of erythroid colony formation on methylcellulose, decreased cell surface expression of glycophorin-A, and decreased cellular proliferation. Treatment of RPS19 deficient cells with dexamethasone restored erythroid differentiation to normal levels. We investigated the molecular basis of pharmacologic therapies for DBA using oligonucleotide microarrays to survey gene expression in CD34+ cells treated with combinations of erythropoietin, dexamethasone, IL-3, and SCF. None of these agents had a direct effect on the expression of RPS19 or on the coordinate expression of other ribosomal genes. Instead, dexamethasone activated a genetic program that includes a set of key hematopoietic regulatory genes, including Flt-3 and PLZF, that increase proliferation of hematopoietic progenitor cells. Genes specific to erythroid progenitor cells were up-regulated by dexamethasone, while genes specific to non-erythroid lineages were powerfully down-regulated. Deficiency of RPS19 therefore blocks proliferation of immature erythroid progenitor cells, and dexamethasone activates proliferation of the same cell population through mechanisms independent of RPS19. Identification of the key regulatory genes affected by dexamethasone may aid in the development of novel therapeutics that de-couple the beneficial hematopoietic effects of dexamethasone from detrimental non-hematopoietic side effects.
Monoubiquitinated FANCD2 Is Both Necessary and Sufficient for Mitomycin C Resistance in the Absence of a Functional Fanconi Anemia Core Complex.
Authors: Shimamura Akiko , Leary Rebecca , Stotsky Maria , Moreau Lisa A. , D’Andrea Alan D. 
Published: October 2019

Abstract
Monoubiquitination of the Fanconi anemia (FA) protein, FANCD2, is necessary for resistance to MMC/DEB-induced chromosomal breakage, which is the diagnostic feature of Fanconi anemia. The upstream Fanconi proteins FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, and FANCL are necessary for FANCD2 monoubiquitination and Fanconi pathway function. We posed the question whether FANCD2 monoubiquitination is sufficient to restore the Fanconi pathway function in the absence of the upstream FA proteins. We constructed a chimeric monoubiquitinated FANCD2 protein by fusing the FANCD2 cDNA sequence contiguously (in-frame) with a ubiquitin coding sequence. This FANCD2-ubiquition fusion protein (D2-ubi) was stably expressed in FANCD2−/−, FANCA−/−, FANCC−/−, and FANCG−/− human patient-derived cell lines. D2-ubi expression corrected the MMC hypersensitivity of these FA cell lines in the absence of a functional upstream FA core complex. By western blot analysis, the D2-ubi protein migrated at a slower rate than the endogenous monoubiquitinated FANCD2 protein, consistent with its higher molecular mass. An even slower migrating D2-ubi form (D2-ubi-L) was detectable in cells harboring an intact Fanconi core protein complex (i.e., FA-D2 cells) but was absent in cells lacking functional Fanconi proteins FANCA, FANCC, or FANCG. These results indicate the D2-ubi can be further monoubiquitinated on lysine 561, but only in the presence of an intact FA complex. Furthermore, D2-ubi-L was upregulated in response to DNA damage in transfected FA-D2 and wild type cells. Like endogenous FANCD2, D2-ubi formed nuclear foci in the presence of the upstream Fanconi core complex. In the absence of the FA core complex, D2-ubi was localized diffusely throughout the nucleus, but unlike endogenous FANCD2, D2-ubi was retained within the nucleus following detergent permeabilization of FA-A, FA-C, and FA-G cells. These results further support the epistatic relationship of the FA genes in a linear pathway. In summary, these data indicate that a constitutively monoubiquitinated FANCD2 polypeptide is able to bypass defects in the upstream FA core complex to restore Fanconi pathway function. These studies identify FANCD2 ubiquitination as a potential therapeutic target for patients lacking the upstream FA core complex.
The E1841K Mutation in MYH-9 of a Patient with May-Hegglin Anomaly Inhibits the Disassembly of Non-Muscle Myosin IIA (MYH-9) Responsible for the Phenotype of the Disease.
Authors: Miyazaki Koji , Komatsu Satoshi , Watanabe Mariko , Nakadate Naoya , Ikebe Mitsuo , Higashihara Masaaki 
Published: October 2019

Abstract
It has been demonstrated that MYH-9 (non-muscle myosin IIA) is responsible for the hereditary macrothrombocytopenia, such as May-Hegglin Anomaly (MHA), Fechtner syndrome (FS) and Sebastian syndrome (SS). We identified the E1841K mutation of MYH-9 gene of a patient with May-Hegglin Anomaly. Immunofluorescent staining of her peripheral blood smear samples revealed that non-muscle myosin IIA and actin filaments were co-localized at the Döhle-like inclusion bodies of neutrophils. To investigate the mechanism by which the E1841K mutation causes the phenotype of MHA, we first expressed GFP-tagged wild type and E1841K mutant MYH-9 cDNA in NIH3T3 cells. The mutant was able to form filaments and some inclusion bodies such as Döhle-like bodies, although it seemed to make no influences on the shape of the fibroblasts. Next we expressed the GFP-tagged wild and E1841K mutant MYH-9 in HEL cells and induced differentiation with TPA. HEL cells expressing the E1841K mutant failed in full differentiation. They could not produce the proplatelet-like projections as the cells overexpressing wild type of MYH-9 did. These results demonstrate that overexpression of this mutant can induce some phenotype similar to MHA. To clarify what molecular characteristics of the mutant myosin can cause the MHA phenotype, we expressed the recombinant wild and mutant proteins of non-muscle myosin IIA using Baculo-virus system. The mutant myosin failed to dissolve even in high ionic strength, suggesting this mutant can assemble in cytoplasm even under the condition, in which wild type myosin changes the conformation and exchanges dynamically assembly to disassembly. Next we induced myosin filaments in HEL cells by over-expressing the mutant of myosin light chain (DD mutant), which mimics the double-phosphorylated form. The DD mutant of myosin light chain also inhibited the proplatelet-like projections. Moreover silencing of MYH-9 gene with siRNA could have no inhibitory effects on the differentiation of HEL cells, producing rather more proplatelet-like projections than the control. These results indicated that filament formation of myosin is not critical for proplatelet-like projections, but disassembly of myosin is rather essential. Collectively, the E1841K mutant inhibits the disassembly of myosin to prevent the proplatelet-like formation of HEL cells, and the assembled myosin forms the Döhle-like inclusion bodies probably with actin filaments.
A Novel and Disease Specific Gene in Chronic Lymphocytic Leukemia.
Authors: Buhl Anne Mette , Jurlander Jesper , Pedersen Lone B. , Josefsson Par , Geisler Christian H. , Leffers Henrik 
Published: October 2019

Abstract
One of the best predictors of poor outcome in CLL is the absence of somatic hypermutations in the Ig-genes of the leukemic cells. This prognostic dichotomy, between cases of mutated and unmutated CLL, enabled us to screen for a gene associated with poor outcome CLL using differential display RT-PCR. We identified a novel transcript from chromosome 12q22, which by RT-PCR and Northern blotting seemed to be selectively over-expressed in CLL patients with poor prognosis. No matching genes or ESTs were annotated in the region, but screening of a cDNA library from unmutated CLL patients resulted in cloning of 7 cDNAs, which probably derived from alternative splicing of a common transcript. The majority of the transcripts had no significant reading frames, but one splice variant may encode a protein of 121 amino acids. Despite very low primary sequence similarity, structure modelling revealed that the peptide potentially can fold into a structure remarkably similar to human IL-4. By RT-PCR the various transcripts were undetectable in a panel of normal tissues and cell lines. Using quantitative RT-PCR (QRT-PCR), we could however detect minute amounts of the mRNAs in normal B-cells; the level was similar or slightly higher in good prognosis patients and much higher in poor prognosis patients. The expression level of the two major mRNAs was 24–50 fold higher in patients with unmutated Ig-genes compared to patients with somatic hypermutation (p<0.0001), and 20–30 fold higher in ZAP-70 positive patients compared to ZAP-70 negative patients (p<0.0001). The median time to treatment for patients with overexpression of the mRNAs (n=26) was 11 months compared to 104 months for patients with normal expression (n=31) (p=0.0005). The median overall survival for patients with overexpression was 97 months while patients with low expression had not reached a median overall survival at their last follow up (p=0.03). This prognostic power was comparable to that provided by mutational status, and stronger than that provided by ZAP-70 or CD38 expression. Finally, in 56 patients the gene dosage as defined in QRT-PCR was inversely proportional with time to treatment (p= 0.006). This novel gene therefore has several features that make it a candidate to be the first disease specific gene identified in CLL.
VEGFR-1 (Flt-1) Tyrosine Kinase Signaling Enhances Hematopoiesis, Proliferation/Differentiation and Immunity of Monocyte/Macrophage from Bone Marrow Hematopoietic Stem Cells, and Promotes Rheumatoid Arthritis.
Authors: Murakami Masato , Iwai Shinobu , Hirasuka Sachie , Iwakura Yoichiro , Maru Yoshiro , Shibuya Masabumi 
Published: October 2019

Abstract
VEGF and its receptor family including VEGFR-1(Flt-1) are well known to be a crucial regulatory system for normal development and pathological angiogenesis. Rheumatoid arthritis(RA) is a chronic systemic disease characterized by an inflammatory erosive synovicitis, which show marked neovascularization, inflammatory cell infiltration and synovial hyperplasia, then produce a pannus of inflammatory vascular tissue and lead to irreversible cartilage and bone destruction. We have already shown VEGFR-1 is expressed not only in vascular endothelial cells but also in inflammatory cells, especially in monocyte/macrophage. A recent report suggests the involvement of VEGFR-1 in RA by using collagen induced RA mouse model. To examine whether the signaling from VEGFR-1 is important for the pathological process of RA, we used VEGFR-1 tyrosine kinase(−/−) mice which cannot generate the signaling from this receptor, and an arthritis mouse model system carrying Human T-cell leukemia virus(HTLV-1) pX transgene. VEGFR-1 TK(−/−) mice with pX gene clearly showed a reduction in the incidence and the degree of clinical symptom of arthritis. Furthermore, the heterozygote VEGFR-1 TK(+/−) with pX transgene showed a partial decrease in the degree of clinical as well as pathological scores. To explain the reason of reduction of clinical symptoms, we investigated involvement of VEGFR-1 TK signal in lineage of bone marrow hematopoietic stem cell(HSC) to monocyte/macrophage proliferation and differentiation and their immunity. VEGFR-1 TK activities are not associated in number of HSC in bone marrow. However, VEGFR-1 TK(−/−) HSC toward multi-lineage proliferation is suppressed in colony-formation. In addition, failures of monocyte/macrophage faculties are observed in immunological reaction, phagocytosis, cytokine secretion(IL-6, VEGF) and migration. Furthermore, expressions of hematopoiesis and inflammation related genes in VEGFR-1 TK(−/−) macrophage are downregulated by microarray analysis. Next we treated with small molecule inhibitors of VEGF receptor(VEGFR-1 and VEGFR-2) of tyrosine kinase, KRN951, in RA model(pX transgenic model and type II collagen Ab cocktail model) for treatment. Treatment with KRN951 strongly attenuated the disease symptom through inhibiting recruitment of BM hematopoietic cells into peripheral inflammatory cells. These observations indicate that VEGFR-1 signals play an important role in both RA mouse model. The tyrosine kinase activity and the signaling of VEGFR-1 enhances hematopoiesis, proliferation/differentiation and immunity of monocyte/macrophage from bone marrow HSC, and promotes rheumatoid arthritis, which may be a new possibilities for the treatment of RA in humans.
Figure Figure
Myeloma Cell Lines Are Not Sensitive to Modulation of Cyclin D1 Level.
Authors: Nickerson Helen D. , Chesi Marta , Bergsagel Peter Leif 
Published: October 2019

Abstract
Dysregulation of D type cyclins is an almost universal event in multiple myeloma. Overexpression of cyclin D1 may occur by chromosomal translocation of t(11:14)(q13:32), and also in association with hyperdiploidy. To investigate the role of cyclin D1 in the growth of human myeloma cell lines, we used both shRNA plasmid constructs, and an siRNA Smartpool against cyclin D1 and compared to control cells transfected with anti-GFP or GAPDH constructs. siRNA was electroporated into multiple myeloma cell lines overexpressing cyclin D1 (KMS12PE, U266, H929) which gave a 50–60% transfection efficiency, assessed using a GFP plasmid.
Successful reduction in levels of cyclin D1 was confirmed by western blotting and normalization to a beta actin standard. Cyclin D1 protein in sh- or siRNA transfected cells was reduced on average to 32% of that in cells transfected with an anti-GAPDH or anti-GFP siRNA. In individual experiments reduction of cyclin D1 protein to as little as 10% of control was observed. However, we found no sign of increased apoptosis by Annexin V/Propidium iodide staining. Furthermore, cell cycle analysis by ethidium bromide staining and flow cytometry revealed no significant change in the cell cycle of sh- or siRNA transfected cells when compared to control cells. qPCR analysis revealed no acute compensatory increase in the RNA levels of other D-type cyclins (D2, D3) following reduction of cyclin D1 levels.
These results may be explained by the residual presence of sufficient cyclin D1 protein for cell cycle progression. An alternative explanation is that changes that frequently occur in myeloma to other cell cycle regulators, for example p16(INK4a) and Rb are able to circumvent cell cycle effects of reducing cyclin D1protein. These results suggest that the human myeloma cell lines tested are not acutely sensitive to cyclin D1 level.
The Transcriptional Program of Polycythemia Vera Revelas Altered Expression of Multiple Putative Tumor Suppressor Genes.
Authors: Berkofsk-Fessler Windy D. , Licht Jonathan D. , McConnell Melanie-Jane , Neuberg Donna S. , Bowler Timothy S. , Fruchtman Steven M. 
Published: October 2019

Abstract
Polycythemia vera (PV) is a myeloproliferative disease characterized by accumulation of erythrocytes and cells of the myeloid and megakaryocyte lineages. Although genes like PRV-1 and PTP-MEG2 have been implicated in the pathology of PV, there is no consensus on their importance in the disease process. Progenitor cells from PV patients can grow in the absence of erythropoietin, and are hypersensitive to a variety of other growth factors. This suggests that polycythemic hematopoietic progenitor cells possess a significantly different genetic program. We tested this idea by molecular profiling hematopoietic progenitor cells (CD34+) from PV specimens and normal donors. We purified CD34+ cells from the marrow of 10 PV patients and harvested total RNA. Biotinylated cRNA was made through two rounds of linear amplification, and hybridized to Affymetrix HGU133A genechips. CD34+ cells from marrow mononuclear cells of 5 normal controls were processed similarly. The resulting datasets were normalized to the median across chips and across genes. Unsupervised hierarchical clustering showed that PV samples had a distinct gene expression profile from the controls. We then performed supervised clustering using a non-parametric t-test (Wilcoxon rank sum test) using the Benjamini and Hochberg multiple testing correction held to a p-value of 0.01 to determine genes that were significantly different between disease and normal samples. Using these stringent criteria, there were 331 genes that reached significance. Strikingly most of these were decreased in expression compared with control CD34+ cells and only 34 genes were upregulated in PV. A 35 gene predictor set was discovered through the use of a k-nearest neighbor metric. This set was 100% accurate for the prediction of PV in a leave one out cross-validation approach. Among these genes are EVI1, a known oncogene and one of only two genes upregulated in PV on this list, and the putative tumor suppressor genes TUSC4 (NPR2), NDRG1 and KLF4. Also among the predictor genes is BAALC, a gene expressed in normal CD34+ cells and known to be a prognostic indicator gene for acute myeloid leukemia.
Early Tumor Detection Using Platelet Uptake of Angiogenesis Regulators.
Authors: Klement Giannoula , Kikuchi Lena , Kieran Mark , Almog Nava , Yip Tai-Tung , Folkman Judah 
Published: October 2019

Abstract
We report a new function for platelets: selective sequestration of tumor-derived angiogenesis regulatory proteins above the concentration of these molecules in plasma.
Iodinated VEGF in a Matrigel pellet (from 100 to 600 ng/100 microl), implanted subcutaneously in mice, accumulates almost exclusively in platelets in a dose-dependent manner over a period as long as 2–3 weeks, without raising plasma levels of VEGF. Similarly, platelet VEGF increases in the presence of a single microscopic VEGF-secreting human tumor of up to only 1 mm3 in SCID mice without any increase of VEGF in plasma. In addition to VEGF, other factors such as bFGF, PDGF, BDNF, endostatin and other regulators of angiogenesis are taken up by platelets in a selective and quantifiable manner which is dependent on tumor generation of these molecules. Our data show that these proteins are not simply associated with the platelet surface, but are internalized. Furthermore, they are protected from degradation within the platelet, and are not released by classical degranulating agents, such as thrombin, ADP or epinephrine. Incubation of human platelets with endostatin at above physiological levels results in decrease of the majority of platelet-associated VEGF and bFGF in a concentration-dependent manner.
Using SELDI-ToF mass spectroscopy of platelet extracts, we have found that this novel property of platelets enables the detection of microscopic tumors that undetectable by any presently available diagnostic method. The platelet angiogenic profile is more inclusive than a single biomarker because it can detect a wide range of tumor types and tumor sizes. Relative changes in the platelet angiogenic profile permit the tracking of a tumor throughout its development, beginning from an early in situ cancer.
Conclusions: (i) While the half-life of mouse platelets is approximately 3 days, the platelet angiogenic profile persists for as long as the tumor (or Matrigel pellet) is present. This indicates that platelets may continuously scavenge proteins which regulate angiogenesis. (ii) The fact that the presence of a human tumor can now be detected at microscopic size, suggests that it may not be necessary to know the type and location of a tumor before initiating treatment, especially since it is feasible to use anti-cancer therapies of little or no toxicity.
Figure Figure
Antiphospholipid Antibodies, Platelet and Endothelial Activation in Acute Thrombotic Thrombocytopenic Purpura (TTP).
Authors: Bidot Carlos J. , Jy Wenche , Jimenez Joaquin J. , Horstman Lawrence , Ahn Eugene , Lander Gabriela , Yaniz Miriam , Fontana Vincenzo , Ahn Yeon S. 
Published: October 2019

Abstract
BACKGROUND. Although the etiology of TTP remains controversial, some notable associated conditions have been identified. These include genetic predisposition, infections, drugs, pregnancy, systemic lupus erythematosus (SLE) and deficiency of ADAMTS13. These associations underscore the suspected immunological nature of this disorder. Some authors have reported the presence of antiphospholipid antibodies (APLA) with onset of fulminate severe TTP. Also, platelet and endothelial activation has been described in TTP but their possible relation with APLA is unknown.
METHODS. We measured APLA in 19 patients with acute TTP and 17 in remission. Fifteen were female and four were male, mean age of 37 ± 15. All met the diagnostic criteria of TTP and were treated with standard therapy including exchange plasmapheresis. The severity of TTP was classified based on duration of hospitalization and numbers of plasmapheresis needed. Recurrence of TTP was assessed by history and review of medical records. CBC, platelet counts, blood chemistry and LDH were monitored. We investigated APLA antibodies IgG and IgM against 6 antigens (β2GP1, FVII/VIIa, CL, PC, PS, PE) by ELISA. To investigate platelet activation, we measured by flow cytometry: CD62P on platelets, and platelet microparticles (PMP) by CD41 (PMP41). We also measured endothelial microparticles (EMP) using CD62E.
RESULTS.
Lab study : In acute TTP, 42% (8/19) were APLA positive for at least one IgG antibody. The most frequent was aPS (33%) followed by aCL(28%), aPC(28%), aβ2GP1 (28%), FVII/VIIa (21%) and PE (21%). IgM antibodies were not detected. APLA was two-fold more prevalent in acute TTP (42%) than in remission (21%). In acute TTP multiple antibodies (two or more) were seen in 75% (6/8) while in remission only one (12.5%) had multiple antibodies. Platelet activation measured by CD62P was higher in APLA+ than in APLA− (102 ± 25 vs 33 ± 8 fluorescence units, p<0.02, X ± SEM). PMP41 was higher in APLA+ than in APLA− but this difference was not significant. We also found that EMP62E was significantly higher in APLA+ than in APLA− patients (p<0.01).
Clinical observations : When comparing the two groups (APLA+ vs APLA−), APLA+ group had higher incidence of recurrence of TTP than APLA− (75% vs 36%), p<0.05. Platelet counts, LDH, severity and incidence of CNS syndrome were not significantly different between APLA+ and APLA− groups.
CONCLUSIONS: APLA are frequently seen in acute TTP (42%) and they tend to decline in remission. Their presence is associated with increased activation of platelets and endothelial cells, and also with clinical recurrence of TTP. APLA may play a role in excerbation and recurrence of TTP.