ALBERT

Description: ABSTRACT Enhanced warming in semi-arid regions has received much attention since it was first proposed, but the primary driver of this phenomenon remains unknown. This study applied dynamical adjustment to surface air temperature and partitioned the warming into two separate components: a thermodynamically forced component and a dynamically induced component. The results show that the mean amount of thermodynamic warming in the Northern Hemisphere in the study period 1902–2011 was 1.36 °C/109 years and that the amount of dynamic warming was 0.14 °C/109 years. In the mid-latitude zones of Asia, Europe, and North America, the thermodynamic warming was 1.60, 1.19, and 1.32 °C/109 years, respectively, and the corresponding dynamic warming was 0.26, 0.14, and 0.09 °C/109 years. Obviously, higher thermodynamic temperature warming was observed in semi-arid regions, suggesting that the enhanced semi-arid warming (ESAW) is the result of local thermodynamic effects. Thus, different local thermodynamic effects are responsible for the warming discrepancies in the semi-arid regions of Asia, Europe, and North America. Moreover, the considerable bias of Coupled Model Intercomparison Project Phase 5 (CMIP5) ensemble mean temperature trend appeared in semi-arid regions rather than other regions indicate that the simulation of semi-arid regions is particularly complex and difficult. (a) The surface air temperature trend during the cold season in 1902–2011 as a function of the climatological annual precipitation for the thermodynamic (red dots) and dynamic (blue dots) temperatures over the mid-latitudes of the NH (20°–60°N). (b) The regionally averaged temperature trend in (a). Surface air temperature change in Northern Hemisphere (NH) in 1902–2011 is characterized by enhanced warming in semi-arid regions. By applying the dynamical adjustment to temperature, this study separated the raw temperature into thermodynamic and dynamic components. It is found that the thermodynamic temperature exhibited uneven warming in the different climate regions, with particularly enhanced warming in semi-arid regions; whereas, the dynamic temperature exhibited a nearly consistent trend over all climate regions. This enhanced semi-arid warming is dominated by thermodynamic temperature change.

Description: Divalent metal ion transporter 1 (DMT1) functions in iron absorption at the apical surface of the duodenal enterocyte and in iron transport out of the endosomal compartment of the erythroblast, however, DMT1 is also expressed in cells which have no apparent role in iron metabolism. At least 4 isoforms of DMT1 mRNA have been identified that contain alternative 5′ (1A, 1B) and 3′ (IRE+, IRE-) exons. Recently we reported the first human mutation of the DMT1 gene in a patient with severe hypochromic/microcytic anemia. This patient was homozygous for a mutation in the ultimate nucleotide of exon 12 which resulted in preferential skipping of exon 12 during processing of pre-mRNA. Amplification of DMT1 mRNA from peripheral blood cells of this patient revealed multiple products; hematopoietic cells from normal controls had a similar, but not identical, pattern. No such additional PCR products were seen when DMT1 mRNA from other tissues or cultured cells was evaluated. To further investigate this observation, we amplified full length (1700–1800 bp) DMT1 transcripts (1A/IRE+, 1A/IRE-, 1B/IRE+ and 1B/IRE-) from a variety of tissues types, cell lines, and from hematopoietic cells. Expression of the 4 isoforms differed between the different cell types. EBV lymphocytes expressed primarily 1B/IRE+ and 1B/IRE- isoforms. Notably, EBV lymphocytes derived from the mutant patient expressed DMT1 mRNA which was 120 bp smaller than DMT1 PCR products from controls, corresponding to skipping of exon 12. Platelets, granulocytes and reticulocytes expressed primarily the 1B/IRE+ isoform, however, unlike the other cell types examined, more than one mRNA species was present (see figure). Full length high molecular weight PCR products derived from these cells were isolated and subcloned. Plasmid DNA was isolated, subjected to digestion with EcoR1, and the DMT1 inserts were sequenced. All inserts began with exon 1B and ended with exon 16 (IRE+); however, none contained “normal” full length DMT1 sequence. The majority contained either truncated portions of exon 3 or inserted portions of intron 3. Utilization of cryptic splice sites within exon 3 or within intron 3 resulted in early stop codons. We propose that alternative splicing of DMT1 mRNA either via skipping of entire exons or via the use of cryptic splice sites may provide a manner for regulating DMT1 activity in hematopoietic cells. Further studies are underway to elucidate this possibility and to evaluate the signals for alternative splicing. Figure Figure

Description: The congenital polycythemic disorders with elevated erythropoietin (Epo) have been until recently an enigma, and abnormality in the hypoxia-sensing pathway has been hypothesized as a possible mechanism. The tumor suppressor von Hippel-Lindau (VHL) participates in the hypoxia-sensing pathway, as it binds to the proline-hydroxylated form of the hypoxia-inducible factor 1α (HIF-1α) and mediates its ubiquitination and proteosomal degradation. The loss of VHL function may result in the accumulation of HIF-1α and overproduction of HIF-1 downstream target genes including Epo. VHL syndrome is an autosomal dominant disorder predisposing to the development of tumors, due to inherited mutations in the VHL gene. Some rare patients with VHL syndrome have polycythemia, which has been attributed to Epo production by a tumor. It was recently found that homozygosity for theVHL Arg200Trp mutation is the cause of Chuvash polycythemia, an autosomal recessive polycythemic disorder characterized by elevated serum Epo and hypersensitivity of erythroid cells to Epo. We evaluated the role of VHL in 8 children with a history of polycythemia and an elevated serum Epo level and found 3 different germline VHL mutations in 4 of them. One child was homozygous for the Arg200Trp VHL mutation, and another compound heterozygous for the Arg200Trp and the Val130Leu mutations. Two children (siblings) were heterozygous for an Asp126Tyr mutation, one of them fulfilling some criteria of VHL syndrome. We propose that mutations of the VHL gene represent an important cause of pediatric sporadic polycythemias with an inappropriately high serum Epo concentration.

Description: Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD, IDS, and MPP1 genes, which together were informative in about 65% of female subjects. To increase our ability to detect clonality, we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these, all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis, whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly, interferon α therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET, and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus, these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels.

Description: Divalent metal transporter 1 (DMT1) is a transmembrane protein crucial for duodenal iron absorption and erythroid iron transport. DMT1 function has been elucidated largely in studies of the mk mouse and the Belgrade rat, which have an identical single nucleotide mutation of this gene that affects protein processing, stability, and function. These animals exhibit hypochromic microcytic anemia due to impaired intestinal iron absorption, and defective iron utilization in red cell precursors. We report here the first human mutation of DMT1 identified in a female with severe hypochromic microcytic anemia and iron overload. This homozygous mutation in the ultimate nucleotide of exon 12 codes for a conservative E399D amino acid substitution; however, its pre-dominant effect is preferential skipping of exon 12 during processing of pre–messenger RNA (mRNA). The lack of full-length mRNA would predict deficient iron absorption in the intestine and deficient iron utilization in erythroid precursors; however, unlike the animal models of DMT1 mutation, the patient is iron overloaded. This does not appear to be due to up-regulation of total DMT1 mRNA. DMT1 protein is easily detectable by immunoblotting in the patient's duodenum, but it is unclear whether the protein is properly processed or targeted.

Description: Hemostasis at site of the vessel injury is initiated by platelets being tethered to exposed subendothelium. The tethering events are mediated by interaction between subendothelial von Willebrand factor (VWF) and its platelet receptor GP Ib-IX-V complex. If the ligand-receptor interaction, when it occurs in intact vascular lumen or at the site of ruptured atherosclerotic plaques, results in life-threatening thrombosis found in patients with thrombotic thrombocytopenic purpura and myocardial infarction. The development of these thrombotic events suggests that the VWF-GP Ib interaction may be a potential target for therapeutic intervention. To explore such potentials, we have developed and examined the effects of a G-quartet oligonucleotide (T30923, GGGTGGGTGGGTGGGT) on the VWF-GP Ib interaction. G-rich DNA sequences, from which the G-quartets derive, were originally identified in the telomeres DNA and have the ability to form rigid G-quartet structures that interact with proteins. The T30923 structure was designed and determined by nuclear magnetic resonance to form an intramolecular G-quartet that is composed of two identical G-quartets in the center and two GTGT loops on the top and bottom of stacked G-quartets, respectively. Platelet aggregation was induced by four agonists: ristocetin, botrocetin, collagen, and ADP in the presence of either 20 mM of T30923 or the control oligonucleotide. We found that T30923 specifically and dose-dependently blocked platelet aggregation induced by ristocetin, but not by other agonists, with a maximal inhibition achieved at 20 mM. Computer-simulation based on the crystal structure of the VWF A1 domain and the N-terminal VWF-binding domain of GP Iba suggested that the G-quartet binds to multiple sites on VWF-A1. Since the action of ristocetin mimics that of fluid shear stress, we tested the ability of T30923 to block VWF-GP Ib interaction under flow condition. Shear-induced platelet aggregation at 100 dyn/cm2 was blocked 59.3%±11.8% by 20 mM of T30923, but not a control oligonucleotide. Furthermore, the G-quartet T30923 blocked over 80% of platelet adhesion to immobilized VWF, but not collagen under a constant wall shear stress of 30 dyn/cm2. These results demonstrate the potential of T30923 to serve as a VWF antagonist. Furthermore, alterations of DNA sequences and tertiary structure provide a large number of G-quartet variants to be generated and then screened for specific phenotypes. The approach may lead to a new class of potential G-quartet based antithrombotic agents.

Description: Collagen exposed on the subendothelial matrix supports platelet adhesion and aggregation at the site of vessel injury. Its interaction with the platelet membrane receptor GP V/FcRγ chain initiates intracellular signals that recruit the hematopoietic tyrosine kinase Syk to immunoreceptor tyrosine activation motifs (ITAMs) within the FcRγ cytoplasmic tail, leading a cascade of intracellular signal events that eventually activate platelets. In the present study, we demonstrated that collagen and the collagen-related peptide (CRP) stimulation also dose-dependently (2.5–10 mg/ml, 10 min) phosphorylated the signal transducers and activators of transcription 3 (STAT3) at the tyrosine residue 705 (Tyr705) in platelets, indicating that STAT3 participates in collagen-induced platelet activation, likely through a non-transcriptional mechanism. To test this hypothesis, we determined whether a STAT3-blocking G-rich oligonucleotide (T40214, GGGCGGGCGGGCGGGC) blocks platelet activation and aggregation induced by collagen or CRP. T40214 was designed based on G-rich DNA sequences, which were originally identified in the telomeres DNA to have the ability to form the protein-binding rigid G-quartet structures. It was formed through four guanine (G) bases into cyclic Hoogsteen H-bonding. NMR determined the tertiary structure of T40214 as two stacked identical G-quartets in the center and two G-C-G-C loop domains on the top and bottom, respectively. It has previously been shown to inhibit STAT3 phosphorylation in cancer cell lines and to induce tumor apoptosis in vivo. T40214 was delivered into platelets using nano-particle polyethylenimine (PEI) as the carrier. The treatment of washed platelets with T40214 inhibited STAT3 phosphorylation induced by collagen (10 mg/ml, 10 min), while the scramble oligonucleotides did not. Incubation of washed platelets and platelets-rich plasma (PRP) with T40214 inhibited collagen-induced platelets aggregation. Finally, thrombus formation on immobilized collagen was significantly reduced when whole blood treated with T40214 (10ug/ml) under a wall shear stress of 30 dyn/cm2. Results demonstrate a novel role of STAT3 in regulating the collagen-induced intracellular signal and platelet functions. The STAT3-blocking G-quartet offers a new class of potential platelet-regulating reagents derived from screening tertiary structure of the G-rich DNA sequences that form protein-binding G-quartets.