bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Distinct Domains Mediate Self-Assembly of Knob Protein in Plasmodium Falciparum-Infected Human Erythrocytes.
Authors: Chen Huiqing , Li Xuerong , Oh Steven S. , Chishti Athar H. 
Published: October 2019

Abstract
Plasmodium falciparum associated mortality is believed to originate from the adhesion of infected erythrocytes to the endothelial cells. The adhesion of mature parasite infected erythrocytes is mediated by the surface protrusions called knobs. A dominant constituent of knobs is the parasite derived knob-associated histidine-rich protein termed KAHRP or knob protein. Gene deletion experiments have shown that the knob protein is essential in malaria pathogenesis. Without the knob protein (KAHRP), adherence of malaria-infected erythrocytes is not established under physiological shear stress. The adhesive properties of infected erythrocytes are propagated by the direct interaction of KAHRP, a peripheral membrane protein, with the cytoplasmic domain of the P. falciparum erythrocyte membrane protein-1 (PfEMP1). Unlike the highly polymorphic extracellular domain, the cytoplasmic domain of PfEMP1 is conserved across species, hence the interest in delineating the mechanism of its interaction with KAHRP and components of the erythrocyte membrane. Our studies have observed recombinant KAHRP to self-associate into large aggregates in vitro. Under transmission electron microscopy, both knobs isolated from malaria-infected erythrocytes and self-assembled recombinant KAHRP in vitro have revealed a unique spiral shaped morphology. Based on these observations, we hypothesized that the unique knob spiral is formed by the self-assembly of KAHRP monomers and therefore must be encoded by distinct sequence elements within KAHRP. To investigate this speculation, we devised a solution binding assay and engineered a series of GST (glutathione S-transferase) and Trx (thioredoxin) fusion peptides to encompass the entire length of KAHRP. Binding measurements using heterologous combination of defined fusion peptides reveal a core histidine-rich amino terminal segment associating with a lysine-rich segment located within the carboxyl half of KAHRP. These findings suggest a novel head-to-tail interaction strategy employed by the intracellular parasite to assemble the spiral-shaped scaffold of KAHRP under the surface of electron dense knobs. Significantly, our results identify a novel self-interaction site within KAHRP that could be amenable to therapeutic intervention against cerebral malaria.
Red Cell Pyruvate Kinase Deficiency: Molecular Characterization of 10 New Variants.
Authors: Fermo Elisa , Bianchi Paola , Vercellati Cristina , Cotton Frederic , Zanella Alberto 
Published: October 2019

Abstract
PK deficiency is the most common glycolytic enzyme defect associated with chronic non-spherocytic hemolytic anemia. To date about 150 different mutations have been identified in the PK-LR gene. Among them only one large deletion has been described in Gipsy resulting in the loss of exon 11. We report 10 new variants of LR-PK gene in 8 families with pyruvate kinase deficiency. The entire coding region and intronic flanking regions were analyzed by direct sequencing. The results of the molecular analysis are reported in the table:
Pt Origin Hb (g/dL) Tx (n.) PK Activity (IU/gHb) Mutation Aminoacidic substitution SD Italy 15.1 0 10.6 107G / ? Ala36Gly/? TT Australia 8.9 0 nd 409A / del5006bp( IVS3-nt 1431) Ala137Thr / del ex 4-11 NR Italy 9 >50 5.5 661A /1209A Asp221Asn /Met403Ile NA Italy 9 >50 5.3 661A /1209A Asp221Asn /Met403Ile SA Italy 12 0 5.5 1456T/ 1209A Arg486Trp/ Met403Ile SC Italy nd nd 5.6 1529A/ 859C Arg510Gln/ Phe287Leu CM Italy 9.5 0 13.6 1456T/ 958A Arg486Trp/ Val320Met PS Italy 13.2 0 8 1094T /? Lys365Met /? VR Italy 10.5 0 10.7 1706A / ? Arg569Gln /? GR Guinea 14.4 0 7.6 1269A/ IVS9+43c Splice site/? Ref. Values 12.2–16 11.1–15.5
Mutations reported in bold are new. By comparing the amino acids sequences among several species (cat M1, chicken M, rat L, yeast and human), we found that mutations 661A, 859C, 958A, 1094T and 1209A involve highly conserved residues. Mutation 1209A when present in association with 661A (cases NA and NR) results in a severe clinical pattern with need of transfusion support, whereas in compound heterozygosity with 1456T (case SA, mother of NA and NR) is associated with a less severe clinical pattern. The variant 1706A was found in a patient carrying the polymorphism 1705C at the homozygous level; the mutation in association with the polymorphism determines the aminoacidic substitution Arg596Gln.
A deletion of 5006 nucleotides extending from intron 3 to the last 3 nucleotides of exon 10 has been found in an Australian baby dead at birth (case TT); the mutation results in a large cDNA deletion encompassing exon 4 and exon11 included. This is the largest abnormality so far detected in LR-PK gene.
Genome-Wide Identification of Cis-Regulatory Sequences Controlling Blood and Endothelial Development.
Authors: Göttgens Berthold , Donaldson Ian J. , Chapman Michael , Kinston Sarah , Landry Josette Renée , Knezevic Kathy , Piltz Sandie , Buckley Noel , Green Anthony 
Published: October 2019

Abstract
Haematopoiesis has long served as a paradigm for adult stem cell systems and studies over the last 20 years have established that transcriptional control is central to the specification and subsequent differentiation of haematopoietic stem cells (HSCs). With many of the key transcription factors known, haematopoiesis provides a powerful cellular system for the analysis of mammalian gene regulatory networks. The key missing ingredient, particularly for the stem and progenitor cell stages, is a set of experimentally validated gene regulatory regions together with a molecular understanding of their biological activity. Despite progress in lower organisms, genome-wide computational identification of mammalian cis-regulatory sequences has been hindered by increased genomic complexity and cumbersome transgenic assays. In order to identify transcriptional control regions active during early embryonic haematopoietic development, we have developed a strategy that is based upon a haematopoietic stem cell enhancer (+19 enhancer) identified downstream of the SCL transcription factor, a key regulator of haematopoietic stem cell formation. Starting with this well-characterised blood stem cell enhancer, we have developed computational tools which allow genome-wide identification of functionally related enhancers. This approach has been used to identify novel enhancers involved in the regulation of early blood and endothelial development, and which exhibit predicted biological activity in vitro and in transgenic mice. Transcription factors binding to these enhancers have been identified by chromatin immunoprecipitation. Our data allow the construction of an experimentally verified nascent transcriptional network, which controls the development of blood and endothelium, and which so far contains the SCL, GATA-2, Fli-1, Elf-1 and Hex transcription factors. Additional candidate members of the network have been identified and are being subjected to functional validation. This approach represents a widely applicable strategy for characterising the transcriptional networks controlling a broad range of mammalian developmental programmes.
Effects of Single Point Mutations within Proposed Copper Binding Sites on Specific Activity and Inter-Chain Affinity of Factor VIII.
Authors: Wakabayashi Hironao , Zhou Qian , Nogami Keiji , Fay Philip J. 
Published: October 2019

Abstract
Copper ions appear important for the structural integrity and the function of factor VIII. Reconstitution studies have shown that Cu2+ increases specific activity of factor VIII as well as increases affinity between heavy chain (HC) and light chain (LC). Based on the ceruloplasmin homology, the existence of three Cu2+ binding sites has been proposed. These include a type 1 site in HC (A1 domain) coordinated by C310, H315, H267, and M320, a type 1 site in LC (A3 domain) coordinated by C2000, H1954, H2005, and M2010, and type 2 site spanning A1 and A3 domains coordinated by H99 and H1957 with possible other additional residues. We rationalized that point mutations within a putative Cu2+ coordination sites would diminish Cu2+ binding at that site, as detected by changes in factor VIII specific activity and/or inter-subunit affinity. We produced several mutant factor VIII proteins bearing point mutation of C310S, H315A, C2000S, H1954A, H99A, or H1957A using a B-domainless human factor VIII vector. Each mutant was stably expressed and purified by SP-sepharose, which bound factor VIII primarily though LC. Western blotting indicated a reduction in the relative amount of HC in C310S and H99A factor VIII forms, suggesting that inter-chain affinity was reduced by these mutations. EDTA-treated factor VIII forms in the presence of Ca2+ were titrated with Cu2+ and activity was monitored using a factor Xa generation assay. The concentration of free Cu2+ was controlled by the presence of EDTA and Ca2+ based on the known values for Cu2+-EDTA and Ca2+-EDTA affinities. The activity regain observed for wild type factor VIII following titration with Cu2+ yielded a Kd = 11.5 ± 2.2 fM. All of the mutants tested retained a high affinity response to Cu2+, suggesting that single point mutations were not sufficient to eliminate Cu2+ binding. However, while Kd values for H1957A (10.3 ± 2.2 fM), H99A (2.1 ± 1.9 fM), H315A (6.7 ± 3.1 fM), and C310S (12.7 ± 3.4 fM) retained similar or slightly reduced affinity for Cu2+, the Kd values for C2000S (304 ± 105 fM) and H1954A (365 ± 105 fM) showed a significant reduction in Cu2+ affinity. When factor VIIIa subunits were titrated with Cu2+ and monitored by intrinsic fluorescence, only purified A1 subunit showed a saturable effect of the signal (Kd = 11.9 ± 4.9 fM), indicating that this subunit contained a high affinity Cu2+ binding site. Taken together, these results suggest that occupancy of the type 1 Cu2+ binding site in A3 contributes to the Cu2+-dependent increase in specific activity of factor VIII and that the type 1 site in A1 and/or the type 2 site contributes to HC-LC association.
Safety and Efficacy of Low Molecular Weight Heparins for Hemodialysis in Patients with End-Stage Renal Disease: A Meta-Analysis of Randomized Trials.
Authors: Lim Wendy , Cook Deborah J. , Crowther Mark A. 
Published: October 2019

Abstract
Background: The anticoagulant management of patients with thromboembolic disease who also have end-stage renal disease is a frequently encountered and unresolved clinical problem. Low molecular weight heparins (LWMHs) are the preferred initial treatment for these disorders but are renally excreted, and generally avoided in patients with renal failure due to concerns of bioaccumulation and increased bleeding risks. Unfractionated heparin (UFH) remains the primary anticoagulant used in this patient population despite the efficacy and convenience of LMWHs. Although LMWHs are typically not used to treat thrombosis in patients with end-stage renal disease, they are frequently used during hemodialysis to prevent thrombosis of the extracorporeal dialysis circuit. While there are no randomized data evaluating the safety and efficacy of LMWHs in these patients, randomized trials have been performed using LMWHs in the setting of hemodialysis. We performed a systematic review and meta-analysis of these randomized trials summarizing this data.
Purpose: To evaluate the safety and efficacy of LMWHs compared to UFH for preventing thrombosis of the extracorporeal dialysis circuit.
Data sources: We identified studies using MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and FirstSearch, reviewed reference lists and contacted pharmaceutical companies.
Study selection: We included randomized, controlled trials that compared a LMWH with another anticoagulant during hemodialysis in patients with end-stage renal disease, and reported at least one of the following outcomes: bleeding, extracorporeal circuit thrombosis, or anti-Xa levels.
Data extraction: Two reviewers independently extracted data on methodologic quality, study design, clinical outcomes, and anti-Xa levels.
Data synthesis: We included 17 trials in this systematic review, 11 of which were included in the meta-analysis. We found that LMWH did not significantly affect the number of bleeding events (relative risk [RR] 0.96, 95% CI, 0.27 to 3.43), bleeding assessed by vascular access compression time (weighted mean difference −0.87, 95% CI, −2.75 to 1.02) or extracorporeal circuit thrombosis (RR 1.15, 95% CI, 0.70 to 1.91) as compared with UFH.
Conclusions: LMWHs appear to be as safe as UFH in terms of bleeding complications, and as effective as UFH in preventing extracorporeal circuit thrombosis. However, inferences from these trials assessing anticoagulation for patients undergoing hemodialysis will continue to be weak until larger, more rigorous randomized trials are conducted.
Naturally Processed Peptides Eluted from Acute Myeloid Leukemia Cells : A Potent Antigen Source for Immunotherapy.
Authors: Delluc Stephanie , Tourneur Lea , Boix Charlotte , Michallet Anne-Sophie , Varet Bruno , Guillet Jean-Gerard , Buzyn Agnes 
Published: October 2019

Abstract
Acute myeloid leukemia (AML) is a heterogenous group of diseases characterized by a clonal proliferation of myeloid progenitors. Its poor prognosis with conventional chemotherapy justifies seeking for adjuvant immunotherapeutic approaches to eliminate minimal residual disease. We evaluated an immunotherapeutic strategy that bypass the need for epitope identification and the limitation due to HLA restriction. Naturally processed peptides were extracted by acid elution from AML cells at diagnosis, and loaded on mature dendritic cells (mDCs) derived from autologous monocytes obtained when the patients were in complete remission (CR). We evaluated i) the feasibility to elute naturally processed peptides from AML cells at diagnosis, ii) the capacity of mDCs loaded with eluted peptides (mDC/EP) to stimulate specific T cell lines in vitro. We showed that stimulation by mDC/EP was able to generate anti-leukemic T cells lines from PBMC of 6 AML patients in CR. CD4+ and CD8+ T cells were isolated from T cell lines of 5 patients and analyzed for their proliferation, INF-γ production and cytotoxicity in response to autologous or allogeneic AML targets, or to normal autologous PBMC. We showed that both CD4+ and CD8+ leukemia-specific T cells were generated in vitro by mDC/EP stimulations since proliferation of CD4+ T cells, IFN-γ secretion by CD4+ and CD8+ T cells and cytotoxicity mediated by CD8+ T cells were induced in response to stimulation with autologous AML cells. Furthermore, we could not detect auto-immune recognition of autologous normal PBMC, consistent with the specificity of the T cell response induced by mDC/EP. These results provide the proof of concept for using mDC/EP to vaccinate patients with poor-risk AML, and will soon be evaluated in a phse I/II clinical trial.
Phase 2 Study of the Anti-Angiogenesis Agent AG-013736 in Patients with Poor Prognosis Acute Myeloid Leukemia (AML) or Myelodysplastic Syndrome (MDS).
Authors: Giles Francis J. , Steinfeldt Heidi , Bellamy William T. , Bycott Paul , Pithavala Yazdi , Reich Steven D. , List Alan F. 
Published: October 2019

Abstract
Background: AG-013736 is a potent and selective inhibitor of VEGF, PDGF and cKit receptor tyrosine kinases with broad preclinical activity in solid tumors and an AML model. To investigate the anti-leukemic effects of AG-013736 in patients with poor prognosis AML and MDS, we performed a multicenter Phase II Trial.
Methods: Eligible patients (pts) had poor prognosis AML (defined as age of pt ≥65 years, secondary AML, or unfavorable cytogenetics) or poor prognosis MDS (defined as ≥10% myeloblasts in marrow or blood). A 2-stage Simon design was employed with an “unimportant” response rate set at 10% and an “important” rate at 30%. The α and β rates were set at 0.1 with 1 response (complete or partial) in 11 pts in the first stage required before proceeding to the second stage. A successful trial required at least 5 responses in 25 pts. AG-013736 was dosed orally at 5 mg twice a day. Marrow and blood samples were taken for various markers of angiogenesis and microvessel density.
Results: 12 pts (8 AML; 4 MDS) were enrolled including 1 pt who replaced a patient who was withdrawn for lack of compliance after 1 dose. Average age was 77 (range 58–88) years; 7 men, 5 women; 10 white and 2 Asian. All pts were ECOG performance status 0 (5 pts) or 1 (7). 8 pts required blood and 9 platelet transfusions. 8 pts discontinued for lack of efficacy, 3 for non-compliance, and 1 for refusal to participate further. AG-013736-related Grade 3 and 4 adverse events (AEs) included hypertension or blood pressure (BP) elevation (4), mucosal inflammation (1), and deep vein thrombosis (1). Common (>10%) mild-moderate AEs included hoarseness (8 pts), diarrhea (5), BP increased or hypertension (4), proteinuria (3), and mucosal inflammation (2). 9 (75%) pts required at least 1 antihypertensive medication. There were no responses, and therefore enrollment was stopped at 12 patients. Patients received study drug for a median of 53 (range 1–190) days, and 2 pts, one with AML and one with MDS, had stable disease for 132 and 190 days, respectively. Treatment-related changes in biomarkers are being assessed.
Conclusion: AG-013736 was generally well tolerated in this small cohort of elderly pts, 2 of whom had stable disease of significant duration. The combination of AG-013736 with other targeted therapies or cytotoxic agents appears feasible and may be warranted in future studies in patients with poor prognosis AML/MDS.
Generation of a Novel Engineered Murine Apyrase in Pichia Pastoris. Enzymatic Profile and Inhibition of Human Platelet Reactivity.
Authors: Olson Kim E. , Drosopoulos Joan H.F. , Broekman Marinus J. , Islam Naziba , Pulte Dianne , Marcus Aaron J. 
Published: October 2019

Abstract
To study thrombogenesis in wild-type and transgenic mice we engineered a recombinant, soluble form of murine ENTPDase1/CD39 for use as a therapeutic agent. ENTPDase1 contains N- and C-terminal intracellular and transmembrane regions flanking an extracellular domain containing the 5 enzymatically active apyrase-conserved regions. The soluble portion of murine CD39 was amplified using PCR with 5′-XhoI and 3′-XbaI linked primers and cloned into the XhoI/XbaI-digested yeast expression vector pGAPZα. The vector, which features the constitutive GAP promoter and α factor secretion signal, was modified by elimination of the Ste13 cleavage sites and by introduction of a 5′-6His tag separated from the murine solCD39 coding sequence by an enterokinase cleavage site. Stably expressing clones were selected. Aliquots of 30-fold concentrated conditioned medium, obtained following 72 h culture of a yeast clone, were assayed for ADPase and ATPase activity and for the ability to inhibit platelet reactivity. Nucleotidase activity was monitored by native gel electrophoresis and staining using ATP metabolism to phosphate and conversion of Ca-phosphate into PbS, as well as by radio-TLC analyses. Platelet aggregation was quantitated using ADP and collagen as stimuli in heparinized and citrated human platelet-rich plasma (PRP). SolCD39 produced in mammalian cells (CHO, or COS-1) yields an active enzyme that is heavily N-glycosylated. Similarly, Pichia pastoris-produced (murine) solCD39 yields a comparably active protein that is also heavily glycosylated, albeit with the yeast-specific high mannose-type N-glycans. Its ADPase to ATPase activity ratio was ~1.2. Yeast-derived murine solCD39 was treated with PNGase F, an amidase that cleaves between the innermost N-acetyl glucosamine of complex oligosaccharides and asparagine residues of glycoproteins. This removed all saccharides from solCD39, as shown by PAS staining, and by mobility shifts by Western blot analysis and Pb staining in native gels. Interestingly, the deglycosylated protein retained ~60% of both enzymatic and biological activities as compared to the parent molecule. The figures shows platelet aggregation to 5 μM ADP in citrated PRP for control (no murine solCD39), isolated solCD39, and equal quantites of solCD39 that was deglycosylated, as well as subjected to the deglycosylation reaction in the absence of PNGase F. These data are similar for the present murine yeast-produced molecule and COS-produced human solCD39. Our data indicate the possibility of high scale production of solCD39 in yeast, with excellent yield, stability and antithrombotic activity.
Figure Figure
Significant Differences in Neutralization of Heparin and Its Low Molecular Fragments by Protamine Sulfate: An In Vivo Study.
Authors: Crowther Mark A. , Petr Klement , Peng Liao , Frank Chen , Leslie Berry B. , Anthony Chan K.C. 
Published: October 2019

Abstract
In clinical practice, patients receiving low molecular weight heparin (LMWH) occasionally suffer bleeding. Protamine sulphate (PS) is often used to reverse the anticoagulant effect of LMWH in such cases. However, the optimal regimen of PS for complete neutralization of LMWH fragments has not been established. Results from our previous in vitro studies indicate that the ability of PS to neutralize LMWHs is inversely related to the charge of the low molecular weight heparin molecule; more heavily charged LMWHs (such as Tinzaparin) are more readily neutralized than less charged LMWHs (such as Enoxaparin). The aim of the current study was to confirm these findings using an in vivo model. Twenty minutes after administration of either saline, unfractionated heparin [UFH, 100U/kg], Tinzaparin [100U/kg] or enoxaparin [100U/kg], 50% of anesthetized rabbits received either saline or PS [1 mg/100 U of heparin or LMWH]. The efficacy of PS neutralization was assessed by serial measurements of anti-factor Xa heparin plasma levels. Results are presented as mean of the anti-factor Xa heparin activities normalized to the level at 10 minutes and summarized in the table below. As expected, PS completely neutralized the anti-factor Xa effect of UFH. However, PS was significantly less effective for neutralization of Tinzaparin (about 66%) and Enoxaparin (about 44%) at the dose tested. We conclude that when tested in an in vivo model LMWHs vary in their protamine neutralizability. More highly charged LMWHs (e.g. Tinazaparin) are more neutralizable than less highly charged products (e.g. Enoxaparin).
Residual Anti-Xa heparin effect
Time Enoxaparin Tinzaparin UFH Saline + PS Enoxaparin +PS Tinzaparin + PS UFH + PS 10 min 1.00 1.00 1.00 0 1.00 1.00 1.00 20 min 0.85 0.86 0.70 0 0.80 0.85 0.89 Protamine 25 min 0.71 0.75 0.69 0 0.45 0.29 0.01 35 min 0.68 0.64 0.48 0 0.40 0.28 0 50 min 0.48 0.32