bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inside-Out Signaling through ADAP Is Required for VWF-GPIb-IX-V Mediated Integrin AIibb3 Activation.
Authors: Kasirer-Friede Ana , Moran Barry P. , Nagrampa-Orje Jennifer , Swanson Ken , Schraven Burkhart , Neel5 Ben , Ruggeri Zaverio M. , Shattil Sanford J. 
Published: April 2020

Abstract
The interaction between von Willebrand factor (VWF) and platelet GPIb-IX-V is a key biological mechanism in hemostasis. In a previous study of signal transduction between GPIb-IX-V and integrin α Iibβ 3, we observed that platelet adhesion to a dimeric VWF A1 domain fragment via GP Ib-IX-V induced the tyrosine phosphorylation of the adapter protein, ADAP (Slap-130, FYB). While this protein has been implicated in cell proliferation and integrin clustering in leukocytes, its role in platelet function has not been determined. Here we used ADAP knockout mice to address this issue. Initiation of signaling in platelets adherent to murine dimeric VWF A1 domain caused α Iibβ 3 activation in wild-type, ADAP+/+ platelets, as determined by the binding of FITC-fibrinogen or POW-2, an anti-α Iibβ 3 antibody Fab specific for high-affinity α Iibβ 3. However, α Iibβ 3 activation through GP Ib-IX-V was diminished by 70–80 % in ADAP−/− platelets (P &lt;0.01), despite normal platelet adhesion under these static conditions. α Iibβ 3 activation by other agonist pathways, such as those stimulated through ADP or Par4 thrombin receptors was also decreased in ADAP−/− platelets (P &lt; 0.05 and 0.01 respectively). In addition, following adhesion of ADAP−/− platelets to VWF A1, tyrosine phosphorylation of numerous proteins, including the ADAP binding partner, SLP-76, was markedly reduced. Surprisingly, deletion of ADAP also affected the expression of another ADAP binding partner, Skap-HOM, and transient transfection of CHO cells confirmed that ADAP expression regulated Skap-HOM protein levels. Platelets from Skap-HOM−/− mice, which have normal ADAP levels, showed normal α Iibβ 3 activation, indicating that the inside-out signaling defect in ADAP−/− platelets was specifically due to deficiency of ADAP and not Skap-HOM. ADAP−/− mice displayed more re-bleeding from tail wounds compared to ADAP+/+ mice (64% vs. 16%, p &lt; 0.01). Under shear flow conditions over a combined matrix of VWF A1A2 and fibronectin to test interactions involving GP Ib-IX-V and α Iibβ 3, respectively, ADAP−/− platelets displayed reduced α IIbβ 3-dependent adhesion. These studies establish that ADAP is a necessary component of the inside-out signaling pathways downstream of GP Ib-IX-V and other receptors in platelets that regulate α IIbβ 3 affinity.
Hypertensive Encephalopathy in Children with Sickle Cell Disease.
Authors: Minniti Caterina P. , Weinstein Steven L. , Khademian Zarir 
Published: April 2020

Abstract
Cerebrovascular (CVA) accidents are a serious complication of Sickle Cell Disease (SCD). Acute cerebral infarction occurs in approximately 10% of children with SCD under the age of 16 years, with silent infarcts identified in an additional 22%. Children however can present with acute neurologic deterioration mimicking a CVA but demonstrate a picture consistent with hypertensive encephalopathy (HTNE). The incidence of HTNE in children with SCD is unknown with few annedoctal reports available in the literature. We retrospectively identified 83 children with SCD, who received their care at Children’s National Medical Center in Washington, DC, from 1992 to 2005, who presented with neurologic complaints that prompted Magnetic Resonance Imaging (MRI). There were 37 females and 46 males, age 13 months to 17 years, with mean age of 5 years and 8 months. Clinical and neuro-imaging data identified 8 children (7 females and 1 male) with clinical picture compatible with HTNE (BP &gt; 2 std deviation for age), prior to neuroimaging. At the time of the hypertensive episode, the ages ranged from 6 to 16 years and 8 months, with an average of 14 years and 2 months. All patients had Hb SS. Neurologic complaints included seizure, sudden onset headache, confusion, loss of consciousness, and urinary retention. Elevated blood pressures were aggressively treated and all patient received an exchange transfusion. No specific etiology for the hypertension was determined. Initial MRI, obtained within 24–48 hours of presentation, did not show acute or prior infarction in this group. However, there was absence of diffusion restriction on T1 and T2 weighted images, and presence of cerebral cortical edema, which differentiate HTNE from acute infarction. The magnetic resonance angiogram (MRA) did not demonstrate evidence of arteriopathy. The hallmark of HTNE is a complete resolution of abnormalities of both the neurologic exam and the imaging studies at follow up. There was complete resolution of cerebral edema in all patients and mild interval prominence of the cerebral sulci, which can indicate cerebral volume loss, in three out of the 8 patients, in follow up studies obtained 10 days to 4 months later. No recurrences of the symptoms have been reported, with follow up ranging from 5 months to 8 years. None of the patients with HTNE has received chronic transfusions. We conclude that HTNE should be considered in the differential diagnosis of an acute neurologic event in a child with SCD. Accurate recording of vital signs and prompt correction of hypertension is indicated. MRI can accurately distinguish between HTNE and acute infarction, even when clinical symptoms are similar. This distinction helps physicians to establish proper treatment such as chronic transfusion following infarction.
Neonatal Cyanosis Due to a Novel Fetal M-Hemoglobin: Hemoglobin F-M Circleville (Gγ63 His→Leu).
Authors: Kutlar Ferdane , Shell Richard , Atkin Joan , Elam Dedrey , Holley Leslie , Davis Deborah , Harbin Jeanette , Kutlar Abdullah 
Published: April 2020

Abstract
Neonatal cyanosis can result from a multitude of acquired and inherited causes. Cyanosis resulting from fetal M-Hemoglobin variants is a rare cause of this condition. In fact, to date, only two Gγ variants causing methemoglobinemia and cyanosis in the newborn have been reported. These are Hb F-M Osaka (Gγ63 His→Tyr) and Hb F-M Fort Ripley (Gγ92 His→Tyr). We report a novel fetal M-hemoglobin presenting with neonatal cyanosis. The propositus was a 1-day old Caucasian male admitted to the hospital because of cyanotic episodes. The patient had an O2 saturation of 85% on room air and required supplemental O2 during his 10-day hospitalization. The family history was significant for a 4-year old sister having a 5-day hospitalization after delivery for cyanosis. The sister required supplemental O2 for 4–5 months to maintain a satisfactory O2 saturation. Both parents were healthy and had no recollection of neonatal cyanosis or O2 requirement. The newborn screening for hemoglobinopathies reportedly showed Hb F, A, and a variant migrating between Hb S and Hb F. Cation exchange HPLC performed at 1-month of age revealed 68.4% Hb F; 17.5% Hb A, and 14.0% Hb X, eluting between Hb F and Hb A. Reverse phase HPLC showed only βA, αa, Gγ, and Aγ chains without any detectable abnormal chains. O2 affinity showed a P50 of 24.2mmHg (control 23.4mmHg). Sequencing of the PCR amplified Gγ gene revealed heterozygosity for a CAT→CTT mutation (His→Leu) at codon 63. (GenBank Acession # AY662983). This novel variant was named Hb F-Circleville [GγE7(63)His→Leu]. The M-hemoglobins arise from mutations in the conserved proximal (F8) or distal (E7) histidine residues, which are important heme contacts. Substitution of these residues lead to methemoglobinemia and cyanosis. Fetal Hb variants causing methemoglobinemia are rare causes of neonatal cyanosis. Hb F-Circleville is only the third fetal M-Hb reported to date. The prognosis of cyanosis is excellent in these cases, as Hb F declines post natally and is replaced by normal Hb A. Supplemental O2 may be required for several months during the postnatal period as was the case with this patient. Some fetal M-Hbs have been shown to arise as de novo mutations. In our case, an older sister and the mother were both proven to carry the Gγ63 His→Leu mutation consistent with autosomal dominant inheritance.
Signalling by the Platelet C-Type Lectin Receptor CLEC-2 Is Mediated by a Novel Mechanism Involving Syk and a Single YxxL Motif.
Authors: Pearce Andrew C. , Fuller Gemma L.J. , Suzuki-Inoue Katsue , Tomlinson Michael G. , Watson Steve P. 
Published: April 2020

Abstract
We have recently identified the C-type lectin-like receptor CLEC-2 as a novel receptor for the snake toxin rhodocytin on platelets. CLEC-2 is a 32 kDa type II transmembrane protein with a single cytoplasmic tyrosine residue in a YxxL consensus sequence. It is the first C-type lectin receptor that has been shown to activate platelets. The aim of the present study was to investigate the mechanism of signalling by CLEC-2 using model cell lines and to establish the role of the YxxL motif in this process.
We have expressed CLEC-2 in wild type DT40 B cells and Jurkat T cells and in mutant derivatives of these cell lines that lack key signalling proteins. We have assayed PLCγ activation using an NFAT-luciferase reporter assay. Site directed mutagenesis of the cytoplasmic tyrosine residue of CLEC-2 has been used to assess its role in CLEC-2 signalling. We have compared the signalling pathway of CLEC-2 with that of the ITAM-coupled collagen receptor GPVI by expressing GPVI in the mutant cell lines and monitoring responses to the GPVI specific agonist convulxin as above.
Cells transfected with CLEC-2 respond to rhodocytin. Mutation of the CLEC-2 YxxL motif to FxxL abrogates CLEC-2 signalling. CLEC-2 signalling is abrogated in cells deficient in Src or Syk family kinases or PLCγ . CLEC-2 signalling is partially dependent on Tec family kinases and the adaptors LAT and SLP-76/BLNK. Interestingly, GPVI signalling and CLEC-2 signalling show a differential requirement for the SLP-76 family adaptor proteins SLP-76 or Blnk; GPVI signalling is entirely dependent on SLP-76/Blnk, whereas CLEC-2 signalling is only partially dependent on these proteins.
These observations are consistent with a model whereby CLEC-2 recruits Syk via its YxxL motif and initiates a signalling cascade similar to the major platelet glycoproteins GPVI and integrin α IIbβ 3. CLEC-2 is the first platelet receptor shown to regulate Syk through a single YxxL motif. Importantly, despite the similarity in the signalling proteins involved in signalling by the ITAM receptor, the integrin receptor and the C-type lectin receptor, the signalling pathways are organised and initiated differently.
Pharmacological Inhibition of K-Cl Cotransport Alters In Vitro Maturation of Normal and β-Thalassemic Human Erythroid Progenitors.
Authors: De Franceschi Lucia , Ronzoni Luisa , Iolascon Achille , Cimmino Francesca , Alper Seth L. , Servedio Valeria , Turrini Franco , Cappellini Maria D. 
Published: April 2020

Abstract
The K-Cl cotransporter family (KCC) plays a crucial role in cell volume regulation, and KCC1 and KCC3 have been reported to participate in cell growth events (Shen MR, PNAS98, 2001; Shen MR JBC278, 2003). Expression of KCC1, KCC3 and KCC4 has been reported in erythroid cells. In β-thalassemic red blood cells (RBCs), K-Cl cotransport activity is abnormally activated and contributes to red cell loss of water and K. This study evaluated the gene expression of two KCC gene products and the effects of the KCC inhibitor [(dihydroindoenyl)oxy]alkanoic acid (DIOA) on in vitro liquid-culture expansion of human normal and β thalassemic (β thal) erythroid precursors from peripheral blood CD34+ cells. Cells from normal subjects and from β thalassemia major patients (cod39cod39) were cultured for 7 days (to the pro-normobast stage) and 14 days (to the eythroblast stage) in the presence or absence of 10 mM DIOA, At each time point the following parameters were evaluated;
cells counts; cytospins stained with Wright-Giemsa to assess differential cell counts and morphology, cell cycle stage by fluorescence-activated cell sorting after propidium iodide staining; KCC protein expression by Western-blot analysis with antibody to the shared KCC carboxy-terminus; mRNA by real time-PCR analysis.
KCC protein expression increased during erythropoiesis in both normal and β thal cells, and was higher in β thal cells than in normal controls. KCC1 mRNA level was increased only in β thal cells at day 14, whereas KCC3 mRNA level was increased at day 14 in both normal and β thal cells. DIOA significantly reduced the number of both normal and β thal cells, parallelled by increases in the percentage of polychromatophilic normoblasts among normal progenitors and of basophilic normoblasts among b thal progenitors. We further investigated the inhibitory effects of DIOA on cell growth by FACS evaluation of cell cycle distributions and by determination ofCycD, p21, Casp3 and Casp8 gene expression. At day 14 DIOA exposure was associated with:
significant reduction in the percentage of β thal cells in S-phase compared to either untreated cells or DIOA-treated normal controls; up-regulation of CycD gene expression in both normal and β thal cells; down-regulation of p21 in β thal cells; up-regulation of casp3 and casp8 in both normal and β thal cells. These data suggest that KCC is involved in the late phase of erythropoiesis mainly in β thal cells, and support a novel role of KCC in erythroid cell growth.
Negative Hematopoietic Scaffold Lnk Upregulates Integrin Outside-In Signaling in Platelets.
Authors: Eto Koji , Takizawa Hitoshi , Nishikii Hidekazu , Seita Jun , Oda Atsushi , Tamura Noriko , Goto Shinya , Takatsu Kiyoshi , Takaki Satoshi , Nakauchi Hiromitsu 
Published: April 2020

Abstract
The adaptor molecule Lnk negatively regulates signaling downstream of cytokine receptors in self-renewal of hematopoietic stem cells, proliferation of B precursors and differentiation/maturation of megakaryocytes. The circulating number of platelets in Lnk-deficient mice is increased by 5-folds to control, presumably owing to its negative effects on TPO-elicited signaling. Lnk expression is maintained in platelets, however, the precise functions in platelets remain unknown. Here we report that Lnk deficiency results in the prolonged bleeding time (2-folds, p&lt;0.01) due to, at least in part, defective the outside-in signaling from α IIbβ 3 integrin. Lnk-null platelets exhibited less activation of PAK kinase, a Rac effector in fibrinogen-adherent platelets. These were concomitant with the defect of lamellipodial formation upon adhesion to fibrinogen in the absence of Lnk, compared to control platelets (p&lt;0.01). Defective Lnk-null platelet functions were also demonstrated by the reduced three-dimensional growth (height) of platelet thrombi on the collagen surface under whole blood flow condition with the adjusted same platelet number at high wall shear rate of 1,500 s-1 (8.1±1.4 μ m in Lnk-deficient vs. 12.1±2.2 μ m in control, p&lt;0.001), while the two-dimensional surface coverage was not impaired. On the other hand, there was no difference between Lnk-null and control platelets in aggregation and α IIbβ 3 integrin activation in response to stimulation with thrombin or collagen. Taken together, implication of Lnk to sites of adhesion may explain the phenotype of Lnk-deficient platelet function. The findings indicate that Lnk is a unique “dual-functioning” scaffolding molecule, which upregulates thrombus formation, while suppressing cytokine-mediated signaling.
Anxiety, Self-Esteem and Social Functioning in Adolescents with Thalassemia Major.
Authors: Sachdeva Anupam , Yadav Satya , Gujral Satinder Kaur , Gupta Ashum , Khanna Varinder Kumar 
Published: April 2020

Abstract
Whereas it has been established that it is important to provide a therapeutic strategy in Thalassemia major that includes not only medical but also psychological and social interventions from childhood to adulthood, the modalities and instruments of intervention and methods of evaluation and verification still have to be defined. In the present study an attempt has been made to obtain compare thalassemic adolescents to that of healthy adolescents in the areas of psychological factors - anxiety, self-esteem and social functioning. In this study we prospectively studies 40 adolescents (age 13–19 years) of which 20 had thalassemia major and 20 were normal healthy adolescents. In order to compare the two groups on anxiety and self-esteem the t-test was used. A semi-structured interview was used to assess psychological factors. In order to find significant difference between two groups on this semi-structured interview, chi square test was used. The measure of anxiety in the present study is that of trait anxiety stable personality measure which is resistant to situational fluctuation. There was no difference found between two groups in the present study on standardized measure of trait anxiety (t-value 0.96). Most theoretical formulations have stressed that thalassemia can have effect on self-image. Specially, body changes and disruption of activities are seen as lowering the patient self worth however, no significant difference was found between two groups in present study on standardized measure of self-esteem. (T-value 0.70) In the social functioning the chi square test found a significant difference in the extent to which the two groups liked the school (p value &lt;0.05) as 35% of thalassemics like the school a lot against only 10% of healthy. In leisure activity thalassemic adolescents were more sedentary type (p value &lt;0.25). In daily functioning no significant difference was noted among two groups. In illness issues the only question which revealed significant difference were that “ are you able to run as much as others”(P value &lt; 0.01) Thalassemic adolescents responded to be less able to run and falling more often ill than the healthy adolescents. In regards to future 50% thalassemics responded to be cured in future whereas 45% of healthy adolescents had responded to having friends as the future hope. In the views on employment prospects significant difference was found (p value &lt;0.01) where 30 % of thalassemics were pessimistic and rest 70% optimistic whereas 100% of healthy adolescents were optimistic. In the field of marriage and parenthood majority of adolescents in each group anticipated marriage and parenthood. No significant difference was noted. In conclusion this study shows that, in general, the self-concepts of thalassemics were normal.
Kinetic of Iron Absorption and Expression of Iron Related Genes in Beta-Thalassemia.
Authors: Breda Laura , Gardenghi Sara , Guy Ella , Amariglio Ninette , Adamsky Konstantin , Weizer-Stern Orly , Rachmilewitz Eliezer , Rechavi Gideon , Giardina Patricia J. , Grady Robert W. , Rivella Stefano 
Published: April 2020

Abstract
We generated the first transplantable adult mouse models of beta-thalassemia intermedia and major by infusing mouse hematopoietic-fetal-liver cells (HFLC) heterozygous or homozygous for a deletion of the beta-globin gene (respectively with th3/+ and th3/th3 cells) into lethally irradiated congenic C57BL/6 mice. Six to 8 weeks post transplantation, mice transplanted with th3/+ HFLCs show 7 to 9 g/dL of hemoglobin levels, splenomegaly, abnormal red cells and increased iron overload. Mice transplanted with th3/th3 HFLCs, unless blood transfused, die 8 to 10 weeks after engraftment showing profound anemia, massive splenomegaly and very rapid and dramatic iron overload. For this reason, we began a systematic study to compare iron content and the expression level of iron related genes in normal and thalassemic mice of varying ages and sex in different organs (liver, duodenum, spleen, kidney and heart). In liver, we observed that iron content increases proportionally with the level of anemia, age and if the blood transfusion is included. We are currently analyzing the other organs. The expression of hepcidin, ferroportin, Hfe, ferritin, transferrin, transferrin-receptor 1 and 2, ceruloplasmin, divalent metal transporter 1 and hemojuvelin are being tested also in all these organs. In particular, we observed that hepcidin is dramatically downregulated in liver of beta-thalassemic animals. Our hypothesis is that low expression of this gene leads to high iron content in these animals. We intend to demonstrate that administration or increasing hepcidin levels of this peptide can prevent iron absorption in beta-thalassemia. We developed two alternative strategies to test our hypothesis. In the first one, we synthesized the active form of the mouse hepcidin peptide that will be administered intraperitoneally to mice affected by beta-thalassemia. In the second, lentiviral vectors have been generated in order to constitutively secrete hepcidin in the bloodstream of animals affected by beta-thalassemia. These vectors were introduced into hematopoietic stem cells derived from mouse embryos of normal and mice affected by beta-thalassemia and engrafted in myeolablated normal mice. The engrafted mice express hepcidin 6 weeks post transplantation by RT PCR. These animals, along with the animals in which hepcidin will be administrated intraperitoneally, will be analyzed at the endpoint of the experiment (&gt; 4 months) for their hematological values and iron content to see if the use of hepcidin can be used to prevent excessive iron absorption in beta-thalassemia.
Elevated Levels of Homocysteine Compromise Blood-Brain Barrier Integrity in Mice.
Authors: Chauhan Anil K. , Kamath Atul F. , Kisucka Janka , Dole Vandana S. , Loscalzo Joseph , Handy Diane E. , Wagner Denisa D. 
Published: April 2020

Abstract
Elevated levels of plasma homocysteine (Hcy) correlate with increased risks of cardiovascular and Alzheimer’s diseases. We studied the effect of elevated Hcy on the blood-brain barrier (BBB) to explore the possibility of a vascular link between the two diseases. On a methionine-enriched diet, cystathionine beta-synthase (CBS) heterozygous mice develop hyperhomocysteinemia (HHcy). To evaluate the extent of HHcy induced by an 8-week methionine-enriched diet (Diet), we determined plasma Hcy concentration. Concentrations were 23.5 ± 5 μM for wild type (WT) and 98.4 ± 22 μM for CBS+/− on Diet and 4.1 ± 0.20 μM for WT on normal chow. Inflammation can lead to BBB breakdown, therefore we first determined whether we could detect systemic endothelial activation/increased leukocyte adherence in the CBS+/− mice as an indicator of inflammation. We observed mesenteric venules (200–300 μm) in WT and CBS +/− mice fed Diet and compared them with WT on chow. Leukocyte rolling velocity was slower in CBS+/− than in WT mice indicating an increase in the density of adhesion molecules on the endothelium. In CBS+/− mice, 60% of leukocytes rolled at a velocity of &lt; 10 μm/s as compared with only 8% in WT mice on Diet (P&lt;0.002) and 3% WT on chow. The slower leukocyte velocity was due at least in part to higher expression of P-selectin on the endothelium. Infusion of fluorescent beads coated with antibody to P-selectin showed a several fold increase in their binding to venules of CBS +/− mice with HHcy as compared with WT on Diet (P&lt;0.05). We also observed increased leukocyte adherence in CBS +/− mice (P&lt;0.05). We then compared BBB function in mice after the 8-week Diet. We injected Evans blue (EB) dye intraperitoneally. EB binds to albumin and does not readily cross the BBB. The CBS+/− mice exhibited 25% greater EB in cortex compared with WT. EB leakage in WT mice was 3.19 ± 0.09, while CBS+/− leakage was 3.99 ± 0.29 (P &lt; 0.03). WT mice on Diet showed slightly higher plasma Hcy levels than on normal chow; but this increase was not sufficient to break the BBB, as the BBB leakage in the WT mice on Diet was similar to that of WT mice on normal chow. Our study suggests an important toxic effect of elevated Hcy on brain microvessels and implicates Hcy in the disruption of the BBB.