bluuebunny/crossref_metadata_2025_split huggingface dataset:
Splicing Mechanisms That Generate Distinct Isoforms of Protein 4.1R During Terminal Erythroid Differentiation.
Authors: Parra Marilyn K , Mohandas Narla , Conboy John G. 
Published: October 2019

Abstract
Abstract 4036
Poster Board III-972
The protein 4.1R gene is a large complex gene with two translation initiation sites (AUG1 and AUG2) that encode protein isoforms with distinct N-terminal structure and function. Expression of these isoforms is regulated by alternative splicing at either of two splice acceptor sites that flank exon 2' (E2'), in which AUG1 is located. In late erythroblasts E2' is excluded, ensuring translation at AUG2 and synthesis of 80kDa protein 4.1R isoforms. Our earlier studies (EMBO J. 27:122-31, 2008) described a two-step intrasplicing pathway that enforces this splicing outcome exclusively in 4.1R pre-mRNA initiated at exon 1A (E1A), the major transcription start site in late erythropoiesis. The downstream exon 1B (E1B) first splices to the proximal acceptor site at E2' to generate an intermediate structure, which is then re-processed by splicing of E1A to the internal acceptor at E2, removing E1B as well as E2'/AUG1. Experiments with minigenes suggested that intrasplicing is likely independent of specific promoter elements at E1A, but absolutely requires 5' splice site and branch point motifs associated with E1B. Here we sought evidence for functionality of these latter elements in the more physiological context of the endogenous 4.1R gene. The intrasplicing model predicts that morpholino oligonucleotides complementary to key regulatory motifs will block the first step of the pathway in natural 4.1R pre-mRNA transcripts, and yield inappropriate splicing of E1A to the first acceptor at E2'. Antisense morpholinos directed against the E1B branchpoint or E1B 5'splice site were transfected into cells and 4.1R splicing was examined 48hrs later by RT-PCR analysis. Both anti-4.1 morpholinos, but not a control morpholino, resulted in a concentration-dependent shift of E1A splicing to the proximal E2' acceptor site. In other studies we explored whether intrasplicing could occur internally within a gene, using a model pre-mRNA in which a constitutive exon was engineered between E1A and E1B. Analysis of this experiment suggested that any exon upstream of active E1B would follow the intrasplicing pathway and delete E2'. We speculate that internal E1B-like elements in other genes could be selectively activated or silenced by splicing regulatory motifs in order to control downstream splice acceptor choice. Preliminary experiments indicate that weakening the E1B 5' splice site and its upstream pyrimidine tract might permit such regulation as observed for other alternative exons. Together these results provide additional support for the intrasplicing model and suggest that it could function more widely in human genes to coordinate splicing events and generate multiple protein isoforms with distinct functions.

Disclosures:
No relevant conflicts of interest to declare.
Non-Competitive Protein Phosphatase-1 Inhibitors Prevent Sickling of SS RBCs.
Authors: Hairston Jerod , Combi Keon , Foster Altreisha , Kim Bak , Gordeuk Victor R. , Nekhai Sergei 
Published: October 2019

Abstract
Abstract 4038
Poster Board III-974
Protein phosphatase-1 (PP1) has been implicated in the regulation of KCC (K:Cl) transporters, which transport K+ and Cl- ions from red blood cells (RBCs) and in the setting of sickle cell disease may contribute to RBC dehydration and sickling. We have studied host cell protein phosphatase-1 (PP1) in the context of HIV-1 replication and designed novel small molecule non-competitive inhibitors of PP1 that are efficient in the inhibition of HIV-1 but not toxic for cultured cells. We analyzed the effect of our novel non-competitive PP1 inhibitors and the conventional competitive PP1 inhibitor, ocadaic acid, on the sickling of hemoglobin SS RBCs in vitro. We cultured hemoglobin SS RBCs overnight at 1% O2 in the presence of the PP1 inhibitors and then photographed the RBCs and counted the percentage of sickled RBCs. We found that the non-competitive PP1 inhibitor, 1E7-04 prevented RBC sickling by 40% at 10 mM concentration. The 1E7-04 was not toxic at 10 mM concentration for cultured CEM T cells as determined by trypan blue exclusion assay using an automatic cell counter. Our study suggests that small molecular inhibitors of PP1 might be candidates for the future design of anti-sickling drugs.
Acknowledgments.
This work was supported by NHLBI grant U54HL090508-02; NHLBI grant R25 HL003679-08 from the National Institute of Helath and The Office of Research on Minority Health and by U.S. Civilian Research & Development Foundation grant.

Disclosures:
No relevant conflicts of interest to declare.
ACUTE PROMYELOCYTIC LEUKEMIA (APL) IN PATIENTS AGED > 70 Years.
Authors: Finsinger Paola , Breccia Massimo , Minotti Clara , Carmosino Ida , Cannella Laura , Volpicelli Paola , Vozella Federico , Stefanizzi Caterina , Loglisci Giuseppina , Cimino Giuseppe , Foà Robin , Latagliata Roberto 
Published: October 2019

Abstract
Abstract 4160
Acute Promyelocytic Leukemia (APL) is a rare subtype of Acute Myeloid Leukemia (AML) more common in younger adults, with a median age of 45 – 50 years at onset. The use of All-trans Retinoic Acid (ATRA) as tailored treatment has made APL a very curable disease also in patients aged > 60 years; however, there are only few case reports in very elderly APL patients. To address this issue, we revised clinical data and treatment results in 12 patients aged > 70 years with newly diagnosed APL followed at our Institution from 1/91 to 12/2008. Clinical characteristics at onset were as follows: M/F 7/5, median age 74.7 years (range 70.0 – 80.8), M3/M3v 11/1, median WBC 1.3 × 109/l (range 1.0 – 7.4), median PLTS 53 × 109/l (range 12 – 302), BCR1/BCR3 6/6. According to Sanz risk score, 7 patients were at low-risk and 5 at intermediate-risk; 6/12 patients had arterial hypertension, 4/12 a concomitant cardiologic disease, 3/12 a cerebro-vascular disease and 2/12 a previous neoplasia. Induction therapy consisted of ATRA + Idarubicin in 8 patients (2/8 with reduced Idarubicin dosage) and ATRA alone in 4 patients; in this latter group, however, 2/4 needed to add chemotherapy (CHT) based on Mitoxantrone + AraC due to hyperleukocytosis during ATRA treatment. All patients achieved both morphological and molecular Complete Remission (CR) after a median time of 50 (range 29 – 65) and 105 (range 51 – 239) days, respectively. Infective complications were observed in 10/12 patients (4 episodes of FUO, 6 sepsis, 2 cystitis and 1 oral abscess) while ATRA syndrome occurred in 2/12 patients; in addition, there were 3 episodes of cardiac ischemia and 3 episodes of paroxystic atrial fibrillation. All but one patient received consolidation therapy (based on CHT alone in 7 patients, CHT + ATRA in 3 patients and ATRA alone in 1 patient), followed by maintenance treatment in 8 patients. Four patients had a relapse (hematological in 3 cases and molecular in 1 case) after 8, 11, 35 and 56 months respectively. At present, 6 patients are still alive, 4 died due to disease progression (3) or senectus while in CR (1) and 2 were lost to follow-up while in CR: mean event-free survival and overall survival were 89.2 months (95%CI 52.6 – 125.8) and 99.9 months (95%CI 65.0 – 134.7), respectively. In conclusion, ATRA-based treatment of APL is safe and effective also in very elderly patients, with long-lasting disease-free and overall survival.

Disclosures:
No relevant conflicts of interest to declare.
Fondaparinux for the Treatment of Recurrent Venous Thromboembolism During Low Molecular Weight Heparin Therapy: A Retrospective Case Series.
Authors: Ramasubbaiah Rashmi , Kiel Patrick J , Mehta Rakesh , Greist Anne , Jones Kellie L 
Published: October 2019

Abstract
Abstract 4179

Introduction:
Fondaparinux is a synthetic pentasaccharide that exerts its anticoagulant effects by indirectly inhibiting Factor Xa. Large randomized controlled multicenter trials have shown that daily administered subcutaneous fondaparinux at specified weight based dosing is as effective as unfractionated heparin (UFH) and low molecular weight heparin (LMWH) for the initial treatment of venous thromboembolic events (VTE), in conjunction with warfarin. In the case of recurrent DVT or PE, however, there is no standard of care and optimal anticoagulation still remains a challenge. Based on limited data, most patients on warfarin with recurrent VTE would be switched to LMWH or UFH, but the use of fondaparinux in this setting is limited to anecdotal experience.

Methods:
We report the Indiana University experience with the use of fondaparinux for recurrent DVT or PE, failing therapy with LMWH. This is a retrospective review of 13 patients with recurrent thromboembolism, studied in two hospital settings. The mean age was 37 years (range 23-65), there were 8 females and 5 males, and all the patients had failed enoxaparin (two patients had also progressed on tinzaparin). The underlying medical conditions that predisposed to thromboses varied: sickle cell disease (3), antiphospholipid syndrome (3), cancer (3), May Thurner syndrome (1), and 3 patients with inherited thombophilia (Antithrombin III deficiency, Factor V Leiden and Prothrombin 20210 Gene Mutation). Mean weight was 90.7 kg (range 52-150). Mean time to recurrence on LMWH was 330 days. Fondaparinux was prescribed at 7.5mg daily or 10mg daily, based on body weight. Anti-Factor Xa levels were measured in at least half of the patient population, and response was assessed by clinical improvement or progression.

Results:
Thirty one percent (4/13) of patients had recurrent thromboembolic episodes (response rate 69%). Time to progression during therapy with fondaparinux was available on 2/4 patients, and was 17 months (range 13-21). No adverse events including bleeding complications or death were reported.

Conclusions:
Fondaparinux may have efficacy in the treatment of recurrent VTE that has progressed on LMWH. These results are hypothesis generating and should be evaluated prospectively.

Disclosures:
No relevant conflicts of interest to declare.
Decreased Immunogenicity of Purified Topical Bovine Thrombin Preparations.
Authors: Zhu He , Hoppensteadt Debra , Wahi Rakesh , Fareed Jawed 
Published: October 2019

Abstract
Abstract 4209
It has been reported that severe coagulopathy following exposure to topical bovine thrombin may be attributed to the impurities in bovine thrombin preparations. Prepared through the activation of bovine prothrombin by thromboplastin, a crude thrombin preparation was further purified using ion-exchange chromatography and membrane filtration steps to yield thrombin 4A and 4B preparations. The aim of this study was to compare the relative purity of these bovine thrombin preparations by virtue of the detection of bovine prothrombin-related antigens. Bovine prothrombin was administered to three individual rabbits on days 0, 21, 42, 91, 123 and 151 using standard immunologic method. Blood was drawn on 30, 50, 105, 137 and 165 days and the pooled antisera from three rabbits were purified to obtain IgG using protein G affinity columns. Utilizing western blotting, serial diluted bovine crude thrombin, 4A and 4B preparations were probed using the prothrombin IgGs from each time point to explore prothrombin-related antigens in these preparations. Compared with the IgG collected on day 30, the prothrombin IgGs collected from day 50 to 165 showed a time-dependent increased detecting ability for the prothrombin antigens in the bovine thrombin preparations. The lowest amount of crude thrombin, 4A and 4B preparations that prothrombin IgG could detect was 0.125U, 10U and 20U, respectively. The rank order of the number of immunoreactive bands in three bovine thrombin preparations probed by the prothrombin IgGs collected from any given time point was consistant and reproducible: crude thrombin > thrombin 4A > thrombin 4B. The results indicate that thrombin 4B represents the most purified thrombin preparation and relatively non-immunogenic form among three thrombin preparations studied.

Disclosures:
No relevant conflicts of interest to declare.
Stem Cell Mobilization Using 5 Pm Dosing of Plerixafor.
Authors: Cooper Dennis L , Baker Julie , Medoff Erin , Pratt Kathryn , Conkling-Walsh Ann , Snyder Edward L. , Seropian Stuart , Foss Francine 
Published: October 2019

Abstract
Abstract 4233

Introduction
Based on the superiority of plerixafor plus G-CSF (PG) compared to G-CSF (G) alone for mobilizing autologous stem cells, PG has been approved for stem cell mobilization in patients with multiple myeloma and NHL. PG may also be useful for mobilizing stem cells in patients who have failed mobilization with G alone or chemotherapy plus G. Although pivotal studies in myeloma and NHL employed evening (10 pm) dosing of P in order to capture peak mobilizing activity of PG 10-11 hours later, this schedule is inconvenient. Methods: A regimen using 5 pm dosing of P was employed in 17 patients (9 NHL, 7 myeloma, 1 germ cell tumor) including 10 patients who received PG and 7 pts who transitioned to PG after chemo plus G. Apheresis was performed at 8:30 am after P. Four patients had failed previous mobilization with chemo plus G and 4 pts were expected to be poor mobilizers because of prior Rx (lenalidomide 3, fludarabine 1). In nine patients, P was added to G (2 pts) or chemo plus G (7 pts) during the mobilization episode because of inadequate stem cell collections.

Results
An engraftable dose (> 2.5 × 10E6CD34/kg) of stem cells was achieved in all eight pts who either failed prior mobilization or were anticipated to be poor mobilizers because of past Rx and in 6 of 9 pts who had P added during suboptimal mobilization with either G (2/2) or chemo plus G (4/7). In successfully mobilized pts (14/17), the median number of CD34 cells collected was 6 × 10E6/kg in two aphereses. In the latter group, the median CD34 count/μl on the first day after P was 29 (R: 11-161) and had increased a median of 4.5 fold (R: 2.75 – 40.2) compared to one day earlier. Six pts have had transplant with a median WBC and platelet recovery of 10 and 11 days, respectively. Conclusions: PG with 5 pm dosing of P and apheresis at 8:30 the next morning was effective in pts who failed prior mobilization and in pts expected to be poor mobilizers because of prior Rx. Further studies are required to determine the efficacy and optimal scheduling of P when added during suboptimal chemo plus G mobilization.

Disclosures:
Off Label Use: Plerixafor is a stem cell mobilizing agent that is approved for multiple myeloma and NHL. One patient with a germ cell tumor has been included in this study of an alternative mobilization strategy. Foss:Eisai : Speakers Bureau.
High-Dose Etoposide with Granulocyte Colony-Stimulating Factor for Mobilization of Autologous Peripheral Blood Stem Cells in Patients with Hematologic Malignancies.
Authors: Zhang Xiao-yan , Li Jianyong , Qian Sixuan , Qiu Hongxia , Wu Han-xin , Lu Hua , Xu Wei , Hong Ming 
Published: October 2019

Abstract
Abstract 4235
We hoped to explore the efficacy and safety of high-dose of etoposide with granulocyte colony-stimulating factor (G-CSF) for mobilization of peripheral blood stem cells. 43 patients (25 males and 18 females, 15-71years old) with hematologic malignancies, including 17 patients with multiple myeloma, 16 with non-Hodgkin's lymphoma, 1 with Hodgkin's lymphoma, 8 with acute non-lymphocytic leukemia, 1 with acute lymphocytic leukemia, received an etoposide dose of 1.6g/m2. One of the patients had failed to be mobilized by cyclophosphamide and G-CSF before. The total dose of undiluted etoposide was given on day 1 as a continuous intravenous infusion via a central vein for 10 hours. G-CSF 5μg/kg was used on day 3 and given daily subcutaneously until leukopheresis was completed. The results showed that leukopheresis was started at days 10 (range 9-13 days) following etoposide therapy. the mean number of CD34 positive cells collected in all 43 patients was 9.71×106/kg (range 0.5-53.26×106/kg),by an average of 2.15 (range 1-5) leukophereses times. Mobilization procedure that produced yields of greater than 2 .0×106/kg were achieved in 40 patients (including the one failed to be mobilized by cyclophosphamide and G-CSF). Toxicity showed oropharyngeal mucositis, faucitis, urethritis and upper respiratory tract infection in some patients. It is concluded that high-dose etoposide with G-CSF is an effective and safe mobilizing regimen for autologous peripheral blood stem cells in majority patients with hematologic malignancies.

Disclosures:
No relevant conflicts of interest to declare.
Finding of Kinase Domain Mutations at Diagnosis IN PATIENTS with Chronic PHASE Chronic Myeloid Leukemia (CP-CML) MAY Identify Those at High RISK of Disease Progression.
Authors: Carella Angelo Michele , Cirmena Gabriella , Catania Gioacchino , Pica Gianmatteo , Beltrami Germana , Ballestrero Alberto , Patrone Franco , Garuti Anna 
Published: October 2019

Abstract
Abstract 4251
Kinase domain (KD) mutations are associated with resistance to Imatinib in CP-CML but their incidence and prognostic significance before any treatment are unclear. To assess if KD mutations at diagnosis may have prognostic significance, we have recently reviewed (before treatment with Imatinib) the mutation status of 45 patients,of whom low risk: 24 patients; intermediate risk: 8 patients; high risk: 13 patients, according to Sokal/Euro. We found that a) no patient with low and intermediate risk showed KD mutations at diagnosis; b) 4/13 high risk patients showed the following mutations at diagnosis: S265R, E255K, F359Y and E255V/E258V; other 5 patients developed mutations during Imatinib treatment (E255V, T315I, E255V/E258V, H396R and D248-274). All patients with low-intermediate risk are alive and well at a median of 44 months (range, 15-71 months). On the contrary, 3/4 high risk patients with KD mutations at diagnosis progressed and died of blastic evolution at 23, 33 and 69 months despite Nilotinib and Dasatinib therapy. Other 3/5 high risk patients who developed mutations under Imatinib, died of blastic evolution at 22, 23 and 43 months despite Nilotinib and Dasatinib treatment. Seven high risk patients are alive under Imatinib between 4 and 51 months. In conclusion, the KD mutations at diagnosis were frequent in high Sokal/Euro risk group supporting the concept that such mutations could be related to the basic biology of the disease. These data, if confirmed, could modify our approach to high risk patients with KD mutation at diagnosis, i.e. utilizing second/third TKI generation earlier during the disease and, in selected cases, reconsidering allografting earlier before leukemic evolution make such procedure useless.

Disclosures:
No relevant conflicts of interest to declare.
LOW SPARC EXPRESSION IN CML and ITS Upregulation DURING Imatinib TREATMENT.
Authors: Palumbo Giuseppe Alberto , Giallongo Cesarina , Cava Piera La , Tibullo Daniele , Stagno Fabio , Romano Alessandra , Conticello Concetta , Chiarenza Annalisa , Raimondo Francesco Di 
Published: October 2019

Abstract
Abstract 4258
SPARC, also known as osteonectin, is a highly conserved between species phosphoprotein that has been recently identified as a tumor suppressor gene. Besides its involvement in bone metabolism, where it mediates collagen deposition, it has been shown that SPARC plays several roles, ranging from cell adhesion to inhibition of cell-cycle progression and VEGF activation. In mice, it has been reported that implanted tumors grow faster in SPARC-null animals. In humans, patients affected by MDS 5q-syndrome responsive to lenalidomide, have an increase of SPARC expression.
Chronic myeloid leukemia (CML) is a disease characterized by uncontrolled proliferation and adhesion defect. In our study we investigated the expression of SPARC mRNA by quantitative PCR in 22 CML patients. mRNA SPARC relative expression in mononuclear cells isolated from peripheral blood at diagnosis was significantly lower (range 0.004-0.98, mean 0.3, median 0.21, SD 0.3) than in normal control subjects assumed as a reference (range 0.31-2.5, mean 1, median 0.8, SD 0.8).
During imatinib treatment, SPARC mRNA increased of 2 logs at 3 months and of 3 logs at 1 year. In one patient that had to stop imatinib treatment a decrease of expression to levels comparable to normal subjects, around 1 log higher than pretreatment levels, was noticed; when the patient restarted treatment with the tyrosine kinase inhibitor, SPARC level raised of 2 logs again.
Bcr/Abl+ K562 cell line has a low level of SPARC mRNA (0.4 relative expression compared to pooled normal controls), probably due to promoter hypermethylation. Treatment of K562 with an hypomethylating agent, such as 5-azacytidine, for 72 h resulted in an increased expression of SPARC mRNA of 1 log. Moreover, K562 proliferation at 72 h was inhibited by adding esogenous SPARC to colture medium with an IC50 of 50 mcg/ml. In the presence of imatinib 1 μM, SPARC IC50 was lowered to 30 mcg/ml.
Taken all together these data suggest that SPARC deficiency in CML could play a role in excess growth, cell adhesion and angiogenesis impairment. Furthermore, its upregulation after imatinib treatment may be related to the alteration of bone metabolism that is observed in CML patients treated with this tyrosine kinase inhibitor.

Disclosures:
No relevant conflicts of interest to declare.
Preliminary Study On Influence of Imatinib-Induced Apoptosis in Chronic Myeloid Leukemia Cells Co-Culture with Fibronectin Adhesion.
Authors: Xiaoli Liu , Xu Na , Du Qingfeng , Liu Zhi , Li Rong , Yang Jun , Wang Shuang , Zhong Min , Wang Yu 
Published: October 2019

Abstract
Abstract 4264
The bone marrow microenvironment supports growth, differentiation and proliferation of normal hematopoietic cells and can also contribute to malignant growth. Recently,it is considered that except for the point mutant of BCR-ABL kinase contribute to imatinib-resistant therapy for patients with chronic myeloid leukemia(CML), environment-mediated drug resistance (EM-DR) is a potential factor in imatinib resistance. Our previous studies found that Integrin, focal adhesion kinase(FAK), RhoA(a small GTPase) are important adhesion molecules,and related to imatinib resistance. But how and what they crosstalk with each other is still open to debate.
In order to simulated bone marrow microenvironment, we used the major components of bone marrow microenvironment- Fibronectin (Fn) co-cultured with human leukemia K562 cells.and then K562 cells were inoculated with Fn, collagen-coated plate(Co) and suspended cultures as control(mask) group,and then treated with 0.4μM,0.8μM,1.6μM,3.2μM,6.4μM imatinib for 24h,48h and 72h, detected cell apoptosis and proliferation by MTT and AnnexinV-PI assay, examined p-FAK and Rho-GTP by Wersten Blotting and Pull down-Wersten Blotting. The data showed that compared to the Co and mask groups, the cells growth inhibition and apoptosis in Fn co-culture group was significantly reduced. The protein expression of p-FAK and Rho-GTP was higher in the Fn group,and in time-dependent manner. When K562 cells in Fn group were transfected with 150nM siRNA-RhoA for 48h, there was no significant difference compared with the Co and mask groups. Furthermore, the above groups treated with anti-integrin monoclonal antibody (anti-CD29 mAb), we found that p-FAK was significant lower compared with without anti-CD29 mAb in the Fn group; but there was no significant difference of Rho-GTP compard with without anti-CD29 mAb in Fn group. These results indicate that Fn adhesion co-culture could reduce imatinib-induced cell growth inhibition and apoptosis, and this mechanism may be correlated to Rho-GTP activity,and anti-integrin monoclonal antibody could not completely block the integrin binding to Fn on K562 cells, or there was other pathway activated RhoA. The mechanism of EM-DR is complex,and which is well worth us to speculate and study.

Disclosures:
No relevant conflicts of interest to declare.