bluuebunny/crossref_metadata_2025_split huggingface dataset:
PML Mutation in PML-Rarα Alters PML Nuclear Body Organization and Induces ATRA Resistance in Acute Promyelocytic Leukemia
Authors: Zhu Hong-Hu , Wu Congying , Sun Yujie , Hu Jiong 
Published: October 2019

Abstract
Acute promyelocytic leukemia (APL) has become a highly curable disease using target drugs including all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) with or without chemotherapy. However, APL patients with acquiredPML mutationsin PML-RARα had dismal outcomes and showed resistance not only to ATO but also to ATRA.In this study, we confirmed in the clinic that APL patients with mutations in the PML moiety of PML-RARα showed different resistanceto ATRA (cross-resistance). We demonstrated that critical mutations are responsible for this "cross-resistance". Super-resolution microscopy and electron microcopy were employed to examine the fine structural changes of PML/PML-RARα nuclear bodies (NBs) in response to therapy. We observed drug-induced acceleration of NB movement, which was absent in the mutant cells. Furthermore, NB doublets were revealed, and fusion events were captured upon drug treatment. We proposed that SUMOylation contributed to increased NB dynamics, leading to more fusion incidents and thustaking functional NBs for oncoprotein degradation. Different protein density in the mutant NBswas identified by single molecule quantification, indicating reduced permeability and recruitment of clients. Taken together, our results revealed that critical sites in the PML moiety of the PML-RARα fusion gene disrupted NB scaffold organization and perturbed fusion protein degradation in response to ATRA.Our work will largely improve the mechanistic understandings of APL pathology and therapy and will shed new light on unveiling the structure and function of PML NBs.

Disclosures
No relevant conflicts of interest to declare.
Decreased Factor V Ameliorates Bleeding Diathesis in Patients with Combined Deficiency of Factor V and Factor VIII
Authors: Shao Yanyan , WU Wenman , Xu Guanqun , Xuefeng Wang , Qiulan Ding 
Published: October 2019

Abstract
Combined factor V (FV) and factor VIII (FVIII) deficiency (F5F8D) is a rare autosomal recessive bleeding disorder caused by mutations in LMAN1 and MCFD2 gene. Though characterized with deficiencies of both FV and FVIII, it failed to lead to more severe bleeding than single defect of FVIII in similar degree. Our study on F5F8D patients revealed that mild decrease of FV and FVIII had counteractive effect on blood coagulation. Out of 5 subjects investigated, three showed potent thrombin generation as observed in thrombin generation assay (TGA), with peak heights ranging from 138 nM to 166 nM, compared with 81 nM in normal plasma when coagulation was initiated with 1pM TF. The same trend was seen when the TF concentration was increased to 5 pM.
Supplement of FV and FVIII, however, demonstrated distinct impact in TGA, with FVIII addition inducing more thrombin generation whereas higher FV level failed to further boost thrombin generation despite the shortened lag time.
FV is a Janus-faced protein and suppresses blood coagulation by protecting TFPI from degradation.Similar to patients with severe FV deficiency, both total and free TFPI levels also decreased slightly in F5F8D patients. Total TFPI levels decreased from healthy donors (65.95±8.35 ng/mL) to F5F8D patients (65.79±12.99 ng/mL), to severe FV-deficient patients (51.30±11.15 ng/mL, P=0.037). A more pronounced trend was observed for free TFPI levels, as free TFPI in healthy controls were 12.68±2.91 ng/mL, higher than those in F5F8D (4.73±1.31 ng/mL, P=0.004), and in severe FV-deficient patients (3.81±1.03 ng/mL, P=0.004).The lowered TFPI in patients with F5F8D may neutralize the impact of FV and FVIII deficiency on blood coagulation.
Desmopressin (DDAVP) increases endogenous FVIII and is used in the management of mild hemophilia A patients. In current study, 4 patients with F5F8D were treated with DDAVP. The administration of DDAVP saw no elevation of FV yet the increase of FVIII. The mean basal FV and FVIII levels were 11.5±3.4% and 15.1±4.8% respectively. The peak value of FVIII levels rose to 66.1±20.5% 30 minutes after the administration, and was sustained at two-folds of the baseline four hours post administration.
The distinctive changes brought by combined FV and FVIII deficiencies on blood coagulation functions indicates the lone FVIII replacement without FV supplement might be sufficient in bleeding management of patients with F5F8D. DDAVP can be considered as a potential substitute for FVIII concentrate in the treatment of F5F8D patients.

Disclosures
No relevant conflicts of interest to declare.
Prophylaxis of Trimethoprim-Sulfamethoxazole for Pneumocystis Pneumonia in Diffuse Large B-Cell Lymphoma Treated with R-CHOP Therapy: A Single Center Experience
Authors: Ha Joo Young , Lee Kyoungmin , Hong Jung Yong , Yoon Dok Hyun , Kim Sung-Han , Jang Seongsoo , Park Chan-Sik , Huh Jooryung , Ryu Jin-Sook , Lee Sang-wook , Suh Cheolwon 
Published: October 2019

Abstract
Background: Recent studies reported increased risk of Pneumocystis pneumonia (PCP) in patients treated with rituximab-containing chemotherapy and its incidence ranged from 2% up to 13%. However, there are no standard guidelines for PCP prophylaxis in patients with diffuse large B-cell lymphoma (DLBCL). Herein, we investigated the efficacy and safety of trimethoprim/sulfamethoxazole (TMP/SMX) as primary prophylaxis for PCP in patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP).
Method: We retrospectively review the patients with DLBCL who received R-CHOP every 21 days (R-CHOP-21) and received PCP prophylaxis with daily single-strength TMP/SMX (80mg/400mg) in Asan Medical Cencer, Seoul, Korea. We included patients only who completed at least 4 cycles of R-CHOP-21 and PCP prophylaxis. At first, we searched for patients with a positive test result for an immunofluorescence assay for PCP using bronchoalveolar lavage (BAL) samples. As a confirmative test of PCP, a direct immunofluorescence assay to detect P.jirovecii was performed with a commercially available kit (Light Diagnostics TM Pneumocystisjirovecii DFA Kit, Millipore, Billerica, MA, USA). Secondly, we searched for patients who received therapeutic doses of TMP/SMX for following clinical conditions; 1) symptoms such as fever, cough, or dyspnea, 2) radiologic findings compatible to PCP on chest X-ray or chest computed tomography, 3) no other definite cause of pneumonia.
Result: We identified 176 patients with DLBCL who received at least 4 cycles of R-CHOP-21 and concurrent PCP prophylaxis between June 2016 and December 2017. The median age was 59 (range, 22-84), 79 patients (44.8%) had advanced stage (stage III/IV) and 163 patients (92.6%) received 6 or 8 cycles of R-CHOP. The median follow-up duration of 13.6 months (range, 4.4 - 24), and TMP/SMX prophylaxis median cycles was 6 (range, 4-8). Among 176 patient, we identified 2 patients who had undergone bronchoscopy and the immunofluorescence assay for PCP using BAL samples in suspicious of PCP but no patients revealed positivity for the test. We also found that no patients received therapeutic doses of TMP/SMX during the chemotherapy. Grade 3/4 neutropenia and thrombocytopenia occurred in 30.6% and 8.1% of patients, respectively.
Conclusion: There was no PCP event in patients with DLBCL treated with R-CHOP-21 and TMP/SMX prophylaxis with daily single-strength TMP/SMX (80mg/400mg). Side effect profiles seems to be comparable with R-CHOP-21 alone.

Disclosures
No relevant conflicts of interest to declare.
Risk Factors Predisposing Development of Priapism in Sickle Cell Disease Patients
Authors: Alkindi Salam , AlMufargi Said , Pathare Anil 
Published: October 2019

Abstract
Background and Purpose: Penile erection and detumescence are complex physiologic processes, which require delicate neurohormonal and cardiovascular response. Priapism is defined as a persistent and painful erection lasting longer than four hours without sexual stimulation. Stuttering priapism is characterized by a self-limited, recurrent, and intermittent erection, frequently occurring in patients with sickle cell disease(SCD). The aim of this study was to identify the clinical and laboratory features in SCD patients with priapism in Oman.
Methodology: In this retrospective case-control study, medical records of all patients with SCD who developed priapism were retrieved from the hospital information system and compared in a 1: 1 ratio with age and gender matched SCD patients who did not ever had priapism. Addition information extracted include hematological and laboratory parameters, treatment, and complications. Data obtained were analyzed using IBM SPSS version 23. The study was conducted following approval from the hospital medical research and ethics committee.
Results & Discussion: Amongst the forty-one SCD patients evaluated, in the 21 patients with priapism (mean age 24.7 yrs), there was a significantly higher WBC, platelet, retic counts, LDH and serum total bilirubin as compared to controls (p<0.05, student's t test). However, there was no significant differences in the incidence of vaso-occlusive crisis, acute chest syndrome, pulmonary hypertension or cerebrovascular accident (p >0.05, Chi Square test). Patients with priapism were managed with exchange transfusion (100%), aspiration (52%) and shunting (5%). Interestingly, none of these patients showed impotence, although 10% manifested with infertility.
Conclusions: Priapism in this SCD cohort is a disease of the young, and seen predominantly in the hemolytic phenotype of the disease. Patients with priapism showed high WBC's, Platelets, Bilirubin, LDH and retics with a lower hemoglobin as compared to the controls. Favorable outcome is dependent on early intervention and almost half of these patients needed surgical intervention.

Disclosures
No relevant conflicts of interest to declare.
Detection of the BCR-ABL Gene By the Real-Time PCR Method in Patients with Chronic Myeloid Leukemia in Rio Grande Do Norte, Brazil
Authors: Paiva Aldair Sousa , Paiva Hugo Diogenes De Oliveira , Cavalcanti Geraldo Barroso , Leão Gioconda DR , Leão Marcos Dias , Vasconcelos Roberto C , Fernandes Andrea LAC , Bahia Frank , Soares Victor lima , Gil Erica Aires , Januario Geisa , Santos Karla Cristina , Lemos Telma Araujo , Jardim Alessandra Suelen 
Published: October 2019

Abstract
Background: The Philadelphia chromosome is a cytogenetic change resulting from a reciprocal translocation of genetic material between ABL genes from chromosome 9 and BCR from chromosome 22 or t(9; 22) (q34; 11), forming the chimeric gene BCR- ABL, being associated with chronic myeloid leukemia (CML), acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). The p190 variant is usually associated with acute forms of leukemia, including AML and ALL, whereas the p210 variant is associated with the chronic phases of CML. Due to the high sensitivity and specificity, nucleic acid amplification techniques by real-time PCR have replaced the conventional cytogenetic techniques for the identification of the Philadelphia chromosome and its p190 and p210 variants. Molecular analysis has been indicated in the initial diagnostic phase and also for the therapeutic monitoring defining the percentage of neoplastic cells present in the patients during the different phases of the treatment (Minimum Residual Disease or MRD).The aim of this study was the transcript BCR-ABL identification in patients with suspected of CML and evaluation of the gene frequency in these patients. Methods: The presence of BCR-ABL gene was investigated in blood samples from 42 patients with suspected CML. The RNA extraction was performed by phenol/chloroform method. The cDNA was submitted to PCR, using specific primers for and BCR-ABL genes by Real time PCR. Results: From all studied patients, 16 (38.10%) were negative, and 26 (59.09%) positive for one of rearrangements: p210 b3a2 and b2a2 in 18 cases (40.91%) and p190 a1a2 in 2 cases (4,76%) and double positive p120/190 in 6 cases (14,28%). We observed that the most common rearrangement was the p210 b3a2, and the molecular results were compatible with clinical and hematologic suspicion. Conclusions: The Real-timePCR, because of its specificity and sensitivity, can be considered the most used technique in routine diagnosis and investigation of MRD of CML patients.

Disclosures
No relevant conflicts of interest to declare.
Recurrent Stroke: The Role of Thrombophilia in a Large International Pediatric Stroke Population
Authors: Deveber Gabrielle , Kirkham Fenella Jane , Shannon Kelsey , Brandao Leonardo R. , Sträter Ronald , Kenet Gili , Clausnizer Hartmut , Moharir Mahendranath , Kausch Martina , Askalan Rand , MacGregor Daune , Stoll Monika , Torge Antje , Dlamini Nomazulu , Ganesan Vijeya , Prengler Mara , Singh Jaspal , Nowak Goettl Ulrike 
Published: October 2019

Abstract
OBJECTIVE:
Risk factors for arterial ischemic stroke (AIS) in children are multiple and include cardiac disease, vasculopathy, and prothrombotic risk factors (PR). The relevance of these factors to a second AIS event is incompletely understood.
METHODS:
We conducted a multicenter cohort study to assess the rate of symptomatic stroke recurrence following initial AIS, pooling data on recurrent AIS from the databases held in Canada, Germany, and UK. We followed 894 patients aged 1 month to 18 years (median 6 years) at initial AIS for median 35 months.
RESULTS:
160 of 894 patients (17.9%) had recurrence from 1 day to 136 months (median 3.1 months) after first AIS. Recurrence was significantly more common in children with (hazard ratio (HR) 2.5, 95% confidence intervals (CI) 1.92-3.5, p<0.001) compared to children without vasculopathy. After adjusting for vasculopathy, antithrombin deficiency, elevated lipoprotein (a), and the presence of any combined PR were independently associated with recurrence. Recurrence rates calculated per 100 person-years were 10 (95%CI: 3-24) for antithrombin deficiency, 6 (95%CI 4-9) for elevated Lp(a), and 13 (95%CI 7-20) for combined PR.
CONCLUSIONS:
Identifying children at increased for recurrent AIS events is important in intensifying preventative measures. Among 894 Canadian, English and German pediatric stroke patients, 17.9% experienced recurrent AIS at a median of 3.1 months after the index stroke. The presence of more than one prothrombotic risk factor is associated with AIS recurrence in children

Disclosures
No relevant conflicts of interest to declare.
Prostaglandin E2 Glyceryl Ester Contributes to Hyperalgesia in a Humanized Model Pf Sickle Cell Disease through P2Y6 Receptors
Authors: Simone Donald , Gupta Kalpna 
Published: October 2019

Abstract
Sickle Cell Disease (SCD) is the most common monogenic red blood cell disorder characterized by a remarkable phenotypic complexity. Common complications of SCD are unpredictable episodic and chronic pain. Transgenic homozygous HbSS-BERK mice expressing >99% human sickle hemoglobin and exhibiting both pain and neurochemical changes similar to humans, and HbAA control mice expressing normal human hemoglobin, were used to determine the contribution of the prostaglandin E2 glyceryl ester (PGE2-G), to mechanical and heat hyperalgesia and the mechanism of its pro-nociceptive effect. Mechanical hyperalgesia was defined by a decrease in paw withdrawal threshold, and heat hyperalgesia was defined by a decrease in paw withdrawal latency to radiant heat applied to the plantar hind paw. Considering that prostaglandin E2-G (PGE2-G) is the product of oxidation of 2-arachidonylglycerol (2-AG) by cycloxygenase-2 (COX-2), the expression of COX-2 protein and COX-2 activity was measured in dorsal root ganglia (DRG). The levels of 2-AG and PGE2-G in DRG were determined by HPLC-MS. Mechanical and heat hyperalgesia in HbSS mice were associated with a decreased level of 2-AG and increased level of PGE2-G in DRGs of HbSS mice due to elevated level of COX-2 protein and COX-2 activity. In HbAA control mice, intraplantar (i.pl.) injection of PGE2-G (1 µg/10 µl) produced mechanical hyperalgesia in the injected paw independently from PGE2 receptors. Also, PGE2-G (1 µg/10 µl, i.pl.) produced mechanical and heat hyperalgesia in the injected paw in C3H mice. The hyperalgesia was blocked by co-injection of PGE2-G with MRS 2578, the purinergic P2Y6 receptor antagonist (1.4 mg/10 ml, i.pl.). Importantly, MRS 2578 (4.7 mg/50 ml, i.p.) also attenuated hyperalgesia in HbSS mice. Thus, increased expression of COX-2 in DRG neurons of HbSS mice results in the oxidation of 2-AG and production of PGE2-G, a pronociceptive mediator. PGE2-G produced mechanical and heat hyperalgesia in HbAA and C3H mice, and these effects were mediated by P2Y6 receptors.
Acknowledgments: Supported by the National Heart, Lung and Blood Institute (HL135895-16).

Disclosures
Gupta: Novartis: Honoraria; Tau tona: Consultancy.
The Role of Thrombin Deficiency on Skeletal Health
Authors: Taylor Hillary J , Goldscheitter Galen , Taylor Jason A 
Published: October 2019

Abstract
Introduction
Persons with hemophilia have decreased bone mineral density (BMD) and increased risk of fracture. Our group has demonstrated that mice with factor VIII deficiency have decreased BMD and decreased biomechanical strength. The underlying mechanism for the decreased skeletal health remains undetermined. This study uses small interfering(si) RNA to knockdown prothrombin gene expression to test the hypothesis that thrombin deficiency results in decreased skeletal health.
Method
Seventeen 3-week old male C57BL/6J wild-type mice were given subcutaneous injections of siRNA against α-prothrombin (1 mg/Kg) and compared to an equal number of mice injected with saline. Mice were injected at 3, 4, 5, 7, 9, 11, 13, 15, 17, and 19 weeks of age with a volume of 200 ul and measurements taken at peak bone mass at 20 weeks of age. In initial dose response studies, this regimen results in >85% knockdown of prothrombin gene expression (data not shown). Studies included whole body and femoral BMD, thromboelastography (TEG) of plasma, micro computed tomography (µCT), and three-point breaking.
Results
TEG studies show significant differences between mice treated with siRNA (n=17) and mice treated with normal saline (n=6) with R, K and α-angle being p < 0.02, p < 0.014, and p < 0.005 respectively. These results confirm that the thrombin is knocked down by the siRNA against α-prothrombin. Comparative analysis of the whole body and femoral BMD between the treatment and saline groups demonstrate no significant differences (p = 0.64 and p = 0.74). There was no significant difference seen in µCT scans of the right femur, including femoral cortical area (p=0.19), cortical thickness (p= 0.63), and cancellus area (p= 0.25). Three-point break data show no significant difference in ultimate force (p = 0.268), strength (p = 0.546), or modulus (p = 0.07).
Conclusion
This study seeks to understand the underlying mechanism for bone disease in hemophilia. The results indicate that >85% prothrombin knockdown in wild-type mice starting at 3 weeks of age does not significantly alter skeletal health. This suggests that either 1) thrombin deficiency is not involved in the mechanism of decreased skeletal health in hemophilia or; 2) the role of thrombin deficiency occurs early in bone development (prior to 3 weeks of age). To evaluate these two potential explanations, thrombin deficiency needs to be evaluated earlier in development. Thrombin deficient mice are embryonically lethal. Therefore our laboratory is currently using a series of transgenic mice to characterize the role of thrombin receptors present osteoblasts and their role in bone metabolism.

Disclosures
Taylor: Alnylam: Research Funding; Shire: Research Funding; CSL: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees.
The 3.2 Angstrom Crystal Structure of a Bioengineered Factor VIII Construct Illustrates Two Conformations of the C2 Domain
Authors: Smith Ian W , d'Aquino Anne , Parker Ernest T. , Doering Christopher B , Spiegel Paul C 
Published: October 2019

Abstract
Treatment for hemophilia A consists of repeated intravenous infusions of blood coagulation factor VIII (fVIII), either recombinant or derived from pooled plasma cryoprecipitate. While several recombinant versions are clinically available, all existing fVIII replacement products express at low levels, harbor short half-lives, and are cost intensive to produce for prophylaxis and therapeutic intervention. Here, we report the 3.2 Å structure of a bioengineered construct of fVIII, termed ET3i, which is a human/porcine chimeric B domain-deleted heterodimer with an improved expression profile and slower A2 domain dissociation following proteolytic activation of fVIII (Figure 1A). Previous structural knowledge of fVIII is at 3.7 Å resolution or lower, and these structures display inconsistencies in their atomic details. The structure reported herein shows remarkable conservation amongst the core folds and domain arrangement of the fVIII protein as well as provides a more detailed view of the interface between the A2 domain and the remaining fVIII structure. With two fVIII molecules in the asymmetric unit of the crystal, we independently observe two distinct conformations of the C2 domain relative to the remaining fVIII structure. While one model (chain A) superimposes with previously-determined fVIII structures without significant deviation, the second model (chain B) indicates a 'barrel tilt and roll' conformational change of the C2 domain that may have implications for how proteolytically activated fVIII binds to activated platelet surfaces (Figure 1B). The improved model and stereochemistry of the ET3i A chain was subsequently used as a scaffold to generate an improved, refined model of human fVIII. In the case of both original datasets to 3.7 Å (pdb#: 2R7E) and 3.98 Å (pdb#: 3CDZ), our new model based on the ET3i structure resulted in improved refinement statistics for human fVIII. These improved structures further illustrate the conserved nature of the ET3i protein as well as provide a more confident model of fVIII for next generation engineering efforts to develop fVIII therapeutics with longer half-lives, higher expression levels and lower immunogenicity.
Figure 1 Figure 1.

Disclosures
Doering: Expression Therapeutics, LLC: Equity Ownership, Research Funding.