ALBERT

Description: In the western part of the Singhbhum Shear Zone (SSZ), East Indian Shield, borosilicate-bearing veins of variable thickness (tens of micrometers to 1 m thick) are hosted in kyanite-quartzite and kyanite-mica schist. The veins have been classified into three types, which are, from oldest to youngest, generation I (tourmaline), II (dumortierite + tourmaline), and III (tourmaline) veins. Alkali- and Mg-rich tourmaline [XMg = Mg/(Mg + Fe) = 0.68 {+/-} 0.09; X = Na, Ca, K, {square} (vacancy) = 0.40 {+/-} 0.12] is the sole borosilicate in generation I veins, which have been folded in response to regional deformation. Generation II veins were emplaced along shear bands (1 mm to 1 m thick) developed parallel to the axial planes of these folds. Long axes of fibrous dumortierite and prismatic tourmaline of generation II veins are oriented along the shear bands and have been bent around lenticular remnants of host kyanite-quartzite. Generation III veins have a dendritic pattern, crosscut generation II veins and show aggregates of fibrous to acicular tourmaline. Prismatic tourmaline in generation II veins is optically zoned with a green tourmaline core that is variably replaced and rimmed by blue tourmaline. Fibrous to acicular tourmaline in generation III veins is comprised up of blue tourmaline with compositions similar to the rim composition of prismatic tourmaline in generation II veins. Green and blue tourmaline is aluminous (Al total 〉7 apfu) and alkali-deficient (X = 0.71 {+/-} 0.08). High YAl content, high X, low XMg (0.19 {+/-} 0.10), and excess cation charge indicate tourmaline in generation II veins is rich in an "oxy-foitite" component. Foitite-rich tourmaline in generation III veins has tetrahedral Al and a slightly lower Mg-content and X than those of generation II veins. Optical zoning in prismatic tourmaline corresponds to an abrupt compositional change with paragenetically older green tourmaline having higher Al and XMg, but lower alkali content in the X-site than the blue tourmaline rim. The compositional variation in green and blue tourmaline can be explained by a combination of coupled substitutions represented by AlO[R(OH)]-1 and Al(NaR)-1, where R = (Fe2+ + Mg). Pseudosections in the system Na2O-K2O-Al2O3-SiO2-H2O constructed from bulk chemical compositions of the studied rocks and the P-T slopes of two isochors computed from brine-rich inclusions trapped in quartz grains indicate that borosilicate formation in generation II and III veins occurred within 4.1 {+/-} 0.5 kbar and 377 {+/-} 21 {degrees}C. The mineral assemblages and textures suggest that the borosilicate-bearing veins formed from infiltration-driven alteration of host kyanite-quartzite and kyanite-mica schist along structurally controlled conduits by more than one batch of chemically distinct boron-rich aqueous fluids.

Description: The Archaean Sittampundi Layered Magmatic Complex (SLC) of south India is interpreted as a part of the oceanic crust that formed in a suprasubduction zone setting. The assemblage corundum + anorthite + amphibole (magnesiohornblende to tschermakite to pargasite) + clinozoisite (C 1 PACz) developed in highly calcic anorthosite of the SLC at the culmination of a ca. 2.46 Ga tectonothermal event. Changing physicochemical conditions during this early Paleoproterozoic (Siderian) event produced spinel + anorthite + second generation amphibole through destabilization of corundum + first generation amphibole. Spinel retains the shape of the corundum that it replaces (spinel pseudomorphing corundum, SCP) and is surrounded by a rind of plagioclase that separates spinel from the matrix amphibole. Development of the assemblage chlorite + clinozoisite + secondary corundum after spinel + anorthite + amphibole marks the terminal metamorphic event in this area. Mass-balance calculations on pseudomorphs and modeling of preserved reaction textures show that Na, Mg, Ca, and silica were mobile during the formation of the SCP. Al and Fe were mobile at the grain scale but remained immobile in the scale of a thin section. Activity adjusted partial petrogenetic grid in the systems Na 2 O-CaO-Al 2 O 3 -SiO 2 -H 2 O (NCASH) and Na 2 O-CaO-MgO-Al 2 O 3 -SiO 2 -H 2 O (NCMASH) along with the mineralogy of the rocks that were co-metamorphosed with the anorthosite show that (1) the assemblage C 1 PACz was formed during high-pressure metamorphism (11 ± 1 kbar and 725 ± 25 °C), (2) SCP and the plagioclase rind around it were formed during exhumation of these higher pressure rocks to lower pressure (7 ± 1 kbar, 675 ± 50 °C) along a steeply decompressive retrograde P-T path, and (3) post decompression cooling and hydration at near isobaric condition triggered the formation of the chlorite–clinozoisite–secondary corundum assemblage (6–8 kbar, 〈620 °C). The inferred P-T path is consistent with the view that the studied area that represents Archaean oceanic crust of SSZ affinity was subducted and subsequently exhumed during the early Paleoproterozoic orogeny.

Description: Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease (MPD) initiated by p210-BCR-ABL–mediated transformation of hematopoietic stem cells (HSCs). Inhibition of the ABL kinase alone is not sufficient to eradicate leukemic stem cells (LSCs). We have previously shown that the deficiency of Rac2 GTPase signaling, but not Rac1, in p210-BCR-ABL–transduced hematopoietic cells prolonged survival of mice with MPD. Here we demonstrate that absence of Rac2 GTPase prolongs survival of HSC-initiated, inducible Scl/p210-BCR-ABL (Scl/p210) binary transgenic mice, it induces apoptosis, and, unlike in normal HSC and progenitor (HSC/P), impairs LSC and progenitor (LSC/P) proliferation in vivo. As a result, Rac2 deficiency causes functional exhaustion of the LSC pool in vivo. This defect is not due to impaired interaction with the hematopoietic microenvironment as reflected by its unaltered adhesion, migration, and homing to recipient organs. In summary, Rac2 deficiency exhausts the LSC pool in vivo through impairment of oncogene-induced proliferation and survival signals.

Description: The characterization and targeting of Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL)–initiating cells remains unresolved. Expression of the polycomb protein Bmi1 is up-regulated in patients with advanced stages of chronic myelogenous leukemia (CML). We report that Bmi1 transforms and reprograms CML B-lymphoid progenitors into stem cell leukemia (Scl) promoter-driven, self-renewing, leukemia-initiating cells to result in B-lymphoid leukemia (B-ALL) in vivo. In vitro, highly proliferating and serially replatable myeloid and lymphoid colony-forming cultures could be established from BCR-ABL and Bmi1 coexpressing progenitors. However, unlike in vivo expanded CML B-lymphoid progenitors, hematopoietic stem cells, or multipotent progenitors, coexpressing BCR-ABL and Bmi1 did not initiate or propagate leukemia in a limiting dilution assay. Inducible genetic attenuation of BCR-ABL reversed Bmi1-driven B-ALL development, which was accompanied by induction of apoptosis of leukemic B-lymphoid progenitors and by long-term animal survival, suggesting that BCR-ABL is required to maintain B-ALL and that BCR-ABL and Bmi1 cooperate toward blast transformation in vivo. Our data indicate that BCR-ABL targeting itself is required to eradicate Ph+/Bmi1+ B-ALL–initiating cells and confirm their addiction to BCR-ABL signaling.

Description: Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.

Description: The molecular basis for the divergence of the erythroid (red blood cell) and megakaryocyte (platelet) lineages from a common bipotent MEP (megakaryocyte-erythroid progenitor) remains undefined. We now demonstrate that Rgs18 (regulator of G protein signaling 18), a GAP (GTPase activating protein) factor and a transcriptional gene target of the Gfi1b transcriptional repressor complex, likely arbitrates this critical lineage decision downstream of Gfi1b. Rgs18 was identified in a chromatin immunoprecipitation (ChIP on chip) screen for Gfi1b/LSD1/Rcor1 targets in erythroid cells. Accordingly, Rgs18 expression was found to be up-regulated in LSD1 inhibited, and Gfi1b deficient erythroid cells confirming repression of this gene by Gfi1b and its co-factors in this lineage. In contrast, Rgs18 expression was comparable in megakaryocytic cells derived from wild type and gfi1b-/-hematopoietic progenitors indicating Gfi1b independent expression of Rgs18 in these cells. Manipulation of Rgs18 expression produced opposite effects in the erythroid and megakaryocytic lineages. Rgs18 inhibition retarded megakaryocytic differentiation while its ectopic over-expression promoted differentiation at the expense of proliferation. The reverse was observed in erythroid cells where Rgs18 inhibition produced an enhancement of differentiation while over-expression impaired erythropoiesis. Since Rgs signaling regulates the activity of downstream MAPK pathways we determined the status of these pathways in Rgs18 manipulated cells. Inhibition of Rgs18 stimulated ERK phosphorylation in megakaryocytes but diminished it in erythroid cells. In contrast, Rgs18 inhibition in erythroid cells elevated p38MAPK protein and phosphorylation levels. The opposite effects of Rgs18 manipulation on MAPK signaling in erythroid versus megakaryocytic cells while intriguing are consistent with the changes in differentiation and proliferation observed in each lineage, respectively. Although Rgs18 manipulation produced opposite effects in erythroid and megakaryocytic cells, the level and activity of this factor correlated similarly with those of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1) and KLF1/EKLF (Kruppel like factor1) in both cell types. In both lineages, Rgs18 protein levels correlated directly with Fli1, and inversely with KLF1, message levels. Since Fli1 promotes megakaryocytic, and KLF1 erythroid, development; these results demonstrate that Rgs18 promotes the emergence of megakaryocytic cells from bipotent MEPs by modulating MAPK signaling and altering Fli1/KLF1 stoichiometries. Although it is unclear why Gfi1b mediated repression of Rgs18 is erythroid specific even though the former is expressed in both lineages, these results demonstrate that repression of Rgs18 by Gfi1b in fetal liver MEPs limits megakaryopoiesis and augments erythropoiesis. However following megakaryocytic commitment, robust Gfi1b independent expression of Rgs18 drives differentiation of this lineage while continued repression of Rgs18 by Gfi1b in erythroid cells ensures their proper maturation. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4421 Factor VIIa (fVIIa) is one of the key proteins in the blood coagulation cascade. It activates factors IX and × on a negatively charged phospholipid surface in either a TF-dependent or TF-independent fashion (Silverberg et al, 1977; Bom et al, 1990). Monroe et al (1997) demonstrated that fVIIa binds to activated platelets independent of TF and partially restores thrombin generation in an in vitro model of hemophilia. Thus, it appears that interaction of fVIIa with platelet phospholipids plays an important role. We report that binding of 1,2-dihexanoyl-sn-glycero-3-phospholipids -L-serine (C6PS) and 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE) to fVIIa causes changes in its activity as well as structure. Titration with C6PS led to changes in intrinsic fluorescence indicative of two or more binding sites for this lipid. Similar titrations with C6PE indicated that it probably binds to a single site on the protein. Experiments are underway to test this initial conclusion. Both lipids bind with comparable affinity (kd ~ 165 and 160 μ M) when data were analyzed using a single site model. We also examined the effect of the soluble lipids on the activity of fVIIa. Both C6PS and C6PE binding increased fVIIa proteolytic and amidolytic activity, with the effect of C6PS being more pronounced. Based on current data, it appears that both lipids bind to a single weak site, but that binding of either to this site promotes binding of C6PS to a second, tighter, and C6PS-specific site, which seems to be crucial in regulating activity. Further experiments are underway to test this hypothesis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 113 The Kruppel-like factor 5 (Klf5) is a transcription factor that regulates cellular signaling and is involved in cell proliferation and oncogenesis. Recent studies have implicated Klf5 in the self-renewal and maintenance of pluripotent stem cells. In this project, we examined the role of Klf5 in hematopoietic stem/progenitor cells (HSC/P) activity. Using a murine model of inducible deficiency of Klf5 in hematopoietic tissue (Mx1-Cre/Klf5flox/flox, Klf5-deficient), we observed a severe engraftment deficiency in competitive repopulation assays in primary and secondary recipients, although it did not cause any significant hematopoietic defects basally or after chemotherapy stress in primary mice. Engraftment failure of Klf5-deficient HSC was associated with a severe defect in homing (1.8±0.8% vs 6.0±2.0% for Klf5-deficient and WT HSC/P, respectively, p

Description: Introduction The increased risk of thromboembolic events under low oxygen environments such as high altitude regions is a well known phenomenon but with little knowledge about molecular events underlying its pathogenesis. We have recently reported the proteomic changes in hyperreactive platelets under hypoxia and demonstrated that increased activity of protease calpain is crucial for the induction of prothrombotic phenotype under hypoxic environment (Tyagi et al, 2014). However, considering the complex nature of hemostatic reactions, multiple aspects of the regulation of hemostatic balance under hypoxia need to be understood. Hypoxic environment creates oxidative stress in biological system which needs to be contained by counteractive factors. One of such factor, the Protein disulfide isomerase (PDI), which catalyzes the formation and rearrangements of disulfide bonds on proteins, has been found to be upregulated in multiple cell types and is believed to be protective under oxidative stress. The extracellular PDI has been recently reported to play a key role in initial thrombotic events in vivo and in vitro, but whether it affects the hemostasis under hypoxia remains unknown. We analyzed the role played by PDI in calpain regulated hypoxia induced prothrombotic phenotype by using specific PDI inhibitor Quercetin-3-rutinoside (Q-3R) in an animal model. Method:The Sprague-Dawley male rats were exposed to simulated hypobaric hypoxia in a specially designed animal decompression chamber maintained at pressure of 366 torr (equivalent to altitude of 6096m) for 3h duration. The exposed group of animals was infused with either Q-3R or vehicle, via tail vein injection prior to hypoxia exposure. The effect of PDI inhibitor on coagulation was analyzed by measuring clotting time and Prothrombin time. The PDI activity was measured in plasma by insulin reduction assay. The plasma calpain activity was measured by fluorescence based assay. The status of oxidative damage was assessed by measuring MDA levels which are hallmark of oxidative stress. We also analyzed the factor V activity in plasma of animals by standard clot based assay using factor V deficient plasma. Results: As previously reported by us, the animals exposed to hypoxic conditions demonstrated prothrombotic tendency as evident in significant shortening of clotting times (PT 84% of control, p 〈 .03) as compared to control animals. The exposed animals preinfused with PDI inhibitors demonstrated further reductions in clotting times. The preinfusion of Q-3R (0.25 to 1.5 mg/kg body wt) significantly shortened the Prothrombin times (71%, p 〈 .01) as compared to respective vehicle control. The hypercoagulative tendency triggered by Q-3R was also evident in drastically reduced activated clotting times in whole blood. Hypoxia induced the activation of PDI which was reflected by significantly elevated activity as compared to control animals. The dose dependent PDI inhibition in Q-3R preinfused animals was confirmed by the fallen activity of PDI in plasma from exposed animals. The calpain activity remained elevated in hypoxic animals (compared to controls) as observed previously. The oxidative damage as measured by MDA levels, was found to be much higher in exposed animals preinfused with Q-3R as compared to the vehicle group. Interestingly, the active factor V was observed to be significantly higher in case of PDI inhibition with largest activity increase in animals infused with highest dose of Q-3R. Conclusion:These results demonstrate the critical role of PDI in regulation of hypoxia induced prothrombotic state. The prevention of increase in activity of PDI in hypoxic animals by using specific PDI inhibitor accelerated the prothrombotic effect of hypoxia. These results appear to be in contrast with recently reported in vivo antithrombotic effect of PDI inhibition. However, as PDI upregulation is considered largely as a protective mechanism, this prothrombotic effect of PDI inhibition under hypoxia seems to be in part due to uncontrolled oxidative stress as shown by higher MDA levels. Also, as factor V activity was shot up by PDI inhibitors, this shows the activation of factor V to be under control of PDI, which can also be explained by role of disulfide linkages in activation of released factor V. Together, these results suggests a novel role of PDI, along with calpain, in regulating hypoxia induced prothrombotic phenotype. Disclosures No relevant conflicts of interest to declare.