bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipopolysaccharide Binds Platelet Toll-Like Receptor 4 and Mediates the Activation of Phagocytes in the Reticuloendothelial System (RES): A Novel Mechanism of Host Immunity.
Authors: Semple John W. , Aslam Rukhsana , Speck Edwin R. , Freedman John 
Published: April 2020

Abstract
Toll-like receptors (TLR) comprise a family of transmembrane proteins characterized by multiple copies of leucine-rich repeats in the extracellular domain and an IL-1 receptor motif in their cytoplasmic domain. The TLR family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition and the stimulation of adaptive immune responses. Recently, our laboratory demonstrated that TLR4 expression on platelets was responsible for lipopolysaccharide (LPS)-mediated thrombocytopenia and tumour necrosis factor-α production in vivo (Aslam et al. Blood107:637, 2006). To understand the mechanism of how platelet TLR4 may mediate RES activation, platelets from wild type (WT) and TLR4 knockout mice were incubated with various concentrations of LPS in vitro, washed extensively and transfused into WT mice. Results suggest that only the WT platelets could significantly stimulate TNF-α production in vivo. In vitro flow cytometric analysis of phagocytosis by the monocytic cell line THP-1 demonstrated that platelet TLR4 could efficiently present LPS to THP-1 cells and stimulated them to engulf the LPS-coated platelets. The phagocytosis of the platelets was correlated to elevated levels of intracellular TNF-α production in the THP-1 cells. These results suggest that platelets, via TLR4 expression, can act as initial sentinels of the innate immune system by presenting bacterial products such as LPS to phagocytes of the RES.
Two Mutations (1717 T〉C, GP IX;124del145, GPIb beta) Occur Frequently among Patients with Bernard Soulier Syndrome in India.
Authors: Jacob Rintu Rebecca , Jayandharan G. , Viswabandya Auro , Baidya Shoma , Nair Sukesh C. , George Biju , Mathews Vikram , Chandy Mammen , Srivastava Alok 
Published: April 2020

Abstract
Bernard Soulier syndrome (BSS) is a rare (1:1000000) hereditary bleeding disorder of platelet adhesion, caused by a qualitative or quantitative defect of glycoprotein (GP)Ib/IX/V complex. We report here the molecular basis of BSS in eight unrelated patients from India. The diagnosis was based on low platelet count, presence of giant platelets and aggregometry studies (absence of response to ristocetin) in eight unrelated patients who presented with mucocutaneous bleeding at 17.9 years of age (range:9 months–60yrs). Flow cytometry to assess surface GPIb/IX/V complex using anti CD42b antibodies showed reduced (7.7%–21.1%, n=6) expression. Genomic DNA was screened for mutations in the GPIb alpha, GPIb beta, GP IX genes by a novel PCR and conformation sensitive gel electrophoresis method. Mutations were identified in all the 8 patients. Six different mutations, including missense (n=4) mutations, a deletion and a duplication were identified of which 5 are novel. Three novel missense mutations were identified in a patient (CD42b:21.1%) of which, Tyr492His in GPIb alpha gene was in heterozygosity and the Glu129His, Leu132Pro mutations in GPIb beta was detected in a double homozygous condition. All these substitutions occur at a highly conserved codon in the transmembrane region of GPIb alpha and beta chains, a region critical for anchoring the glycoprotein subunits into the platelet membrane. A novel homozygous deletion of 22 bp in exon 2 of GPIb beta (124del145, p.Arg17CysfsX14) gene was identified in 3 different patients (CD42b: range, 10.6–15.9%). This deletion occurs at a sequence flanking the leucine rich repeat at the NH2-side of GPIb beta protein results in a premature termination of translation (PTC) and a truncated mutant with 30 amino acids. A novel homozygous duplication of 38 nucleotides (2119_2120 dup38) in the transmembrane region resulted in a frameshift (142AlafsX76) in one of the patient. This frameshift predicts termination after the addition of 75 amino acids. A previously reported homozygous transition (1717 T &gt;C) in the GP IX gene resulting in a Cys8Arg substitution was identified in the remaining 3 patients (CD42b:10.02%, 7.7%). The frequent presence of GP IX 1717 T&gt;C and GPIb beta 124del145 in 6 out of 8 (75%) patients studied, suggests that this could be due to a founder effect. This data adds significantly to the mutation database of this condition and will be useful for genetic diagnosis of this condition in India.
The αIIbP258S, a Newly Identified Mutation, and the αIIbG349D Substitutions Cause Glanzmann Thrombasthenia by Impairing αIIb-β3 Association in the Endoplasmic Reticulum.
Authors: Artoni Andrea , Gelain Federica , Afrasiabi Abdolreza , Margaglione Maurizio , Mannucci Pier M. 
Published: April 2020

Abstract
Glanzmann Thrombasthenia is a recessively inherited hemorrhagic disorder caused by the quantitative or qualitative deficiency of integrin αIIbβ3, platelet fibrinogen receptor. We previously analyzed the phenotypic effects of the αIIbG236E substitution, which is located in a strategic point of the highly organized αIIb propeller. We demonstrated that the effect of αIIbG236E is to hamper the association with β3, impairing the correct assembly of the complex. In the present study we analyze the phenotypic effects of two other mutations in the same domain of αIIb. The first mutation, the αIIbP258S substitution, is a newly found missense substitution that was identified during a genetic screening of a cohort of patients with Glanzmann Thrombasthenia coming from Southern Iran. The presence of the mutation was confirmed in several family members affected by Glanzmann Thrombasthenia; no other additional mutations were identified. The effect of the substitution is to eliminate the key proline from blade 4 of αIIb propeller. The second mutation, the αIIbG349D substitution, has been previously described in an Italian patients with Glanzmann Thrombasthenia. It causes a charge substitution in strand D of blade 5 of the propeller. Both substitutions were inserted by site directed mutagenesis in pCDNA3.1 containing normal αIIb cDNA. Human Embryonic Kidney (HEK) cells were then transfected by electroporation with normal or mutated αIIb in conjunction with pCDNA3.1 containing normal β3. After 48 hours cells were analyzed by flow cytometry: the binding of 10E5-FITC (murine anti-human αIIbβ3) in cells expressing αIIbP258Sβ3 or αIIbG349Dβ3 was approximately at the same level of negative controls (cells transfected with αIIb only) and greatly reduced in comparison to the binding to cells transfected with normal αIIbβ3. HEK cells transfected with either αIIbβ3 or αIIbP258Sβ3 or αIIbG349Dβ3 were lysed; cells lysates were analysed by SDS-PAGE electrophoresis and immunoblotting. All the lysates were reacting at the same level with antibodies directed against β3. When immunoblotting was done in non-reducing conditions utilizing an antibody reacting with αIIb (mab1990) a band corresponding to αIIb was present in both lysates, although less intense in cells transfected with mutants αIIb. In reducing condition αIIb from cells transfected with αIIbβ3 was nearly all mature, while in cells transfected with αIIbP258Sβ3 or αIIbG349Dβ3 the ratio pro-αIIb: αIIb was 1:1. In conclusion we report the identification of a new mutation responsible for Glanzmann Thrombasthenia located in αIIb propeller. This mutation, in addition with αIIbG349D, causes Glanzmann Thrombasthenia interfering with αIIbβ3 complex formation in the endoplasmic reticulum as shown by the increase amount of pro-αIIb found in cells expressing these mutants.
A Specific Subtype of Sitosterolemia with Macrothrombocytopenia in a Chinese Family.
Authors: Su Yanghua , Wang Zhaoyue , Yang Haiyan , Liu Fang , Bai Xia , Ruan Changgeng 
Published: April 2020

Abstract
Background and Objective. Sitosterolemia is a rare autosomal recessive inherited disorder of lipoprotein metabolism characterized by xanthomas, premature coronary artery disease, arteriosclerosis and increased plasma levels of phytosterols, while hematological abnormalities such as large platelets have seldom been described. Recently, we found a Chinese family with sitosterolemia; three children suffered from sitosterolemia, who presented with obviously large platelets, some as large as erythrocytes. Their platelet volume distribution curves were displaced rightward. The patients’ bleeding time (11,12 and 15 min) was longer than normal range (4–8 min). The platelets could normally aggregate in response to collagen and ADP, but failed to agglutinate in the presence of 1.2-mg/ml ristocetin. Platelet membrane glycoproteins (GP)IIb/IIIa and GPIb were normal. Their parents are consanguineous. In this study, we characterized the clinical, biochemical and molecular genetic features of a Chinese family with sitosterolemia that mainly manifested hematological abnormalities.
Design and Methods. The clinical features of the affected patients in this family were analyzed. Their plasma sterol levels were measured by HPLC method. ABCG5 and ABCG8 genes sequencing searched for the mutation.
Results. The main clinical feature of the three affected patients was macrothrombocytopenia; they had increased plasma sitosterol, stigmasterol and cholestanol levels, but maintained normal cholesterol levels. Sequence analysis of ABCG5 and ABCG8 genes revealed a C to T transition at nucleotide 18802 resulting in a nonsense mutation in ABCG5 exon 1, Gln22X. The affected patients were homozygous whereas their parents and other two family members were heterozygous for this defect. Allele-specific restriction enzyme analysis using BfaIdid not detect this mutation in 70 healthy individual.
Interpretation and Conclusions. A few patients with sitosterolemia may present with obvious macrothrombocytopenia. It suggests that the blood cells could be a “target” in the toxic effect of phytosterols in plasma, the hematological abnormalities might represent a specific subtype of sitosterolemia. Therefore, it is necessary to propose that sitosterolemia should be considered in the differential diagnosis of macrothrombocytopenia. A novel nonsense mutation in ABCG5 was reported.
Scott Syndrome Dogs Demonstrate a Failure of Coated-Platelet Formation.
Authors: Brooks Marjory B. , Catalfamo James L. , Friese Paul , Dale George L. 
Published: April 2020

Abstract
In 2002 Brooks et al. (Blood 99:2434) described a colony of German shepherd dogs (GSD) with a hereditary bleeding diathesis. The affected GSD (aGSD) had normal fluid phase coagulation as well as normal platelet aggregation and secretion, but their platelets failed to generate prothrombinase activity and did not express phosphatidylserine (PS) upon activation with calcium ionophore. These features are characteristic of Scott Syndrome. In the current study, we defined a broader platelet function defect in aGSD, i.e. a failure to produce significant levels of coated-platelets. Coated-platelets, a subclass of activated platelets formed upon dual-agonist activation, express not only surface PS but also retain several alpha-granule proteins, support a robust prothrombinase activity and release microparticles (J. Thromb. Haemost.3:2185, 2005). We activated platelets from control dogs and aGSD with convulxin plus thrombin and performed flow cytometric analyses to detect bound fibrinogen and membrane PS exposure. Post-stimulation, bound fibrinogen was detected on 34.6 ± 8.1 % of control dog platelets (mean ± 1SD; n=5) in contrast to 5.1 ± 2.5 % of aGSD platelets (n=5; p=0.006). In response to the same dual agonists, control dogs had 56.7 ± 10.3 % annexin-positive platelets while aGSD had only 4.7 ± 4.4 % (n=5; p=0.006). Control dogs and aGSD were also tested for their ability to release intracellular calcein upon dual agonist stimulation. As expected from studies with human platelets, control dog platelets released calcein from a sub-population of cells upon dual agonist stimulation; however, aGSD platelets demonstrated minimal loss of calcein [36.6 ± 6.4 % calcein negative control platelets (n=5); 3.5 ± 0.9 % calcein negative aGSD platelets (n=4; p=0.002)]. Additional activation events known to be associated with coated-platelet formation are currently being examined in aGSD to better define the basis of the platelet phenotype. Our results demonstrate that decreased production of coated-platelets is associated with abnormal bleeding and that a single hereditary defect is capable of abolishing prothrombinase activity and coated-platelet formation.
Ubiquitin-Proteolytic Control of HOX Homeodomain Proteins.
Authors: Zhang Jianxuan , Shieh Jae-Hung , Liu Liren , Plasilova Magdalena , Zhang Yue , Morrone Gianni , Moore Malcolm A.S. , Zhou Pengbo 
Published: April 2020

Abstract
The HOX homeodomain proteins are key regulators of hematopoiesis. HOX genes are expressed in primitive hematopoietic cells, and their prompt downregulation is associated with hematopoietic differentiation and maturation. Although transcriptional inactivation of HOX genes during hematopoietic differentiation has been established, little is known about the biochemical mechanisms underlying the subsequent removal of HOX proteins. We have previously shown that the CUL-4A ubiquitin ligase controls the stability of HOXA9 by promoting its ubiquitination and proteasome-dependent degradation. Interfering with CUL-4A biosynthesis results in altered HOXA9 protein levels, which is mirrored by the impairment of myeloid progenitor cells to undergo proper terminal differentiation into granulocytes. Here we show that additional HOX proteins are also subjected to CUL-4A-mediated ubiquitination and destruction. Consistently, the HOX homeodomain, which is highly conserved among all HOX proteins, is responsible for controlling their stability. Silencing of CUL-4A by RNA-mediated interference in human umbilical cord blood CD34+ cells significantly perturbs their self-renewal, expansion, and differentiation properties. These results reveal a novel regulatory mechanism of hematopoiesis by ubiquitin-dependent proteolysis. Recent studies on the biochemical mechanisms underlying HOX ubiquitination and by which leukemogenic HOX fusions evade CUL-4A-dependent degradation will be presented.
Epo Modulation of Cell Cycle Regulatory Genes Via PY343/STAT5 and PI3K Pathways in Primary Erythroblasts.
Authors: Fang Jing , Menon Madhu P. , Sathyanarayana Pradeep , Bogacheva Olga , Bogachev Oleg , Kapelle William , Houde Estelle , Wojchowski Don M. 
Published: April 2020

Abstract
Erythropoietin’s essential role in late erythropoiesis is ascribed to survival effects on colony forming unit-erythroid-derived erythroblasts. However, several studies have pointed to possible Epo regulation of cell cycle factors, and proliferation. To investigate this issue, developmentally-staged KitposCD71high erythroblasts were prepared from murine marrow; were shown to proliferate in response to low-dose Epo (0.1U/ml); and were used in the Affymetrix 430 2.0 array-based discovery of Epo-modulated cell cycle regulatory genes. In keeping with certain prior studies, myelocytomatosis oncogene (c-Myc) was induced while cyclin-dependent kinase inhibitor 1B (p27) was inhibited several-fold. Beyond this, four select cell cycle progression factors were rapidly up-modulated up to five-fold by Epo: nuclear protein 1 (Nupr1), G1 to S phase transition 1 (Gspt1), early growth response 1 (Egr1) and Ngfi-A binding protein 2 (Nab2). B-cell leukemia 6 (Bcl6) in contrast, was down-modulated several-fold. Finally, among cyclins, D2 was up-modulated ~5-fold while G2 was selectively down-modulated 4- to 5-fold (All findings were confirmed by RT-PCR). Studies in erythroblasts expressing knocked-in minimal erythropoietin receptor (EpoR) alleles indicated EpoR proximal tyrosine-343 (PY343) regulation of cyclin D2 induction, and EpoR C-terminal regulation of cyclin G2 inhibition. Despite the capacity of a PY-null EpoR-HM allele to support steady-state erythropoiesis, EpoR-HM erythroblasts proved to be skewed in cell cycle phases, especially in G2/M (0.01% vs ~5% for wt-EpoR and EpoR-H alleles). For cyclin D2, cyclin G2, p27 and Bcl6, in silico models for promoter regulation are outlined which highlight Stat and Forkhead action. In summary, specific new evidence is provided for erythroblast cell cycle entry modulation by Epo (especially as elevated during anemia) via discrete EpoR subdomains, and nine selectively modulated cell cycle regulatory genes.
Endomitotic MKs Undergo Cortical Contraction during Anaphase but Do Not Complete Furrow Ingression.
Authors: Geddis Amy , Fox Norma , Sear Katherine , Tkachenko Eugene , Kaushansky Kenneth 
Published: April 2020

Abstract
Megakaryocytes (MKs) develop high degrees of polyploidy in the course of normal maturation through a process called endomitosis. Previous studies have shown that endomitotic MKs abort anaphase after sister chromatid separation, and studies using fixed cells have shown that a midzone is formed in anaphase but that a well-developed cleavage furrow is not. However, studies in fixed cells may miss transient dynamic events that are critical for evaluating potential mechanisms of failed cytokinesis in endomitosis. Therefore we have used live cell imaging as a tool to further define the ability of primary endomitotic MKs to furrow during anaphase. We imaged mouse marrow MKs expressing tubulin fused to YFP, acquiring brightfield and fluorescent images every 2 minutes. Endomitotic cells were identified by the formation of a multipolar spindle, and anaphase was defined as the point at which separation of spindle poles was visible. The extent of cell shape change following anaphase was determined visually and also by measuring the circularity of the cell, with a perfectly round cell having a circularity value of 1. We found that 56% of endomitotic MKs underwent some shape change (n=25), visible as localized indentation, protrusion or elongation of the cell cortex, with an average reduction in circularity of 0.043 occurring a mean of 14.6 minutes after the initiation of anaphase. After an average of 7.8 minutes the cells generally reverted to a round shape without completing furrow ingression. In contrast, a typical mitotic control cell showed a reduction in circularity of 0.443 occurring 8 minutes after the start of anaphase and resulting in the formation of two cells (see Figure 1, representative cells). To determine if the shape change seen in endomitosis after anaphase is dependent on RhoA, we imaged MKs in the presence of the RhoA inhibitor C3 toxin. Although 2 μg/ml C3 toxin blocked cytokinesis in diploid control cells, it did not measurably alter the frequency (44%, n=16) or degree of shape change (0.039) in endomitotic MKs following anaphase, although treatment did appear to delay shape change (onset occurring a mean of 24.8 min after anaphase) and prolong the period of cortical contractility. Therefore although endomitotic MKs exhibit a phase of increased cortical contractility following anaphase, it is not solely dependent on the activity of RhoA and it does not result in sustained furrow ingression. Live cell imaging represents a powerful new tool for dissecting the mechanisms of endomitosis.
Figure 1. Endomitotic MKs undergo shape change following anaphase Figure 1. Endomitotic MKs undergo shape change following anaphase
Protein Phosphatase 2A Plays an Essential Role in Migration of CD34+ Cells to SDF-1/CXCL12.
Authors: Basu Sunanda , Ray Nicole T. , Atkinson Simon J. , Broxmeyer Hal E. 
Published: April 2020

Abstract
Although cord blood contains significant numbers of hematopoietic stem and progenitor cells (HSPC), its applicability has remained largely in children (1Broxmeyer and Smith, 2004). Homing of hematopoietic stem progenitor cells (HSPC) to bone marrow is a critical determinant for success of transplantation. Enhancement of homing of HSPC from cord blood has the potential to increase the applicability of cord blood transplantation in adults. Therefore, it is important to understand the molecules that underlie directional movement of HSPC. Stromal derived factor-1 (SDF-1)/CXCL12 is the most potent chemoattractant for both mouse and human HSPC and has been shown to play an important role in the homing and retention of HSPC in bone marrow. Earlier studies have shown that PI-3 kinase plays a critical role in chemotaxis by attracting various pleckstin homology (PH) containing domain proteins at the leading edge and spatial localization of PI3Kinase and PTEN is critical for maintaining the leading edge. Controlled activation of Akt/PKB, one of the various PH containg domains, has been described to be required for efficient chemotaxis. In this study we have demonstrated that protein phosphatase 2A (PP2A), a serine-threonine phosphatase, plays an important role in chemotaxis of cord blood CD34+ cells towards SDF-1, primarily by modulating Akt activity. Inhibition of PP2A by okadaic acid (OA) or siRNA impairs chemotaxis; this involved impairment in the ability of CD34+ cells to polarize and reduced speed of movement. This was associated with robust and prolonged Akt phosphorylation. Indeed, over expression of constitutive active Akt in CD34+ cells impaired SDF-1 directed chemotaxis. Co-immunoprecipitation experiments demonstrated increased association of Akt with PP2A following SDF-1 stimulation and this increased association of Akt and PP2A-catalytic subunit was observed at the plasma membrane of SDF-1 stimulated CD34+ cells by confocal microscopy. The importance of PP2A in maintaining a critical level and duration of activated Akt was also supported by our finding that inhibition of PI-3kinase by low dose of LY294002, partially recovered chemotactic activity in CD34+ cells pretreated with OA. Interestingly, glycogen synthase kinase-3 (GSK-3) associated with Akt following SDF-1 stimulation, although it was found to be constitutively associated with PP2A-C. Inhibition of GSK-3 using GSK-3 IX inhibitor impaired chemotaxis, raising the possibility that GSK-3 is a downstream target for Akt in SDF-1 directed chemotaxis. The physiological relevance of our in vitro findings is established from the observation that OA pretreatment impaired engraftment potential of CD34+ cells in NOD-SCID mice. Our findings contribute to the growing understanding of molecules that affect directional movement and may have the potential implications for homing and engraftment of HSPC.