Polar cap patches are localized enhancements in ionospheric density which originate from solar EUV ionization on the dayside, enter the polar cap at the dayside cusp, convect anti-sunward at km/s velocities, and exit the polar cap near midnight to merge with sunward returning flow patterns. Plasma irregularities associated with polar patches are the leading cause of scintillations in L-band satellite signals such as GPS, and fast shear flows near the dayside cusp are thought to be integral to patch formation. In this paper, we report on the on the characteristics of polar cap patches and fast flows derived via analysis of the spectra of GPS scintillations recorded at Longyearbyen, Svalbard, following the CME impact on 22 January 2012. Following the interaction of the CME with the high latitude ionosphere, elevated GPS TEC values indicate the passage of patches through the cusp between 11-15 MLT, accompanied by significant GPS phase scintillations (σφ ~ 0.5 radians) but minimal amplitude scintillations (S4 <; 0.05). Examination of the scintillation spectra reveal that amplitude fluctuations were present, but not easily detected in the S4 observations because the fluctuation power was concentrated at high frequencies. In fact, these amplitude spectra can be explained in terms of Fresnel filtering of the path integrated irregularity spectrum with a relatively high cutoff frequency (8 Hz). This filtering is consistent with weak scatter of the satellite signals by irregularities scanning past the ray path with an effective velocity ~ 3 km/s. Since the velocity of the satellite penetration point is negligible, by comparison, this scan velocity is attributed to fast plasma flow, presumably associated with shear flows near the cusp. To exploit the Fresnel filtering effect, we developed a technique to derive the flow velocity by reconciling the phase and amplitude spectra with weak scatter theory. We applied this approach- to investigate the noontime entrance of patches into the dayside cusp and the midnight exit of patches from the polar cap. We find clear evidence of strong phase scintillations with reduced S4 values in the presence of fast flows near the cusp, when the increasing Fresnel break frequency effectively suppresses the low frequency content in the amplitude fluctuations. The scan velocity increased from about 500-1000 m/s following the initial CME impact at ~6:00 UT, to sustained velocities between 1500-3000 m/s measured by GPS satellites whose ray paths intersected fast plasma flows near the cusp. In this sector, the phase spectral index (p) generally ranged between 2.4-2.8, with a tendency for somewhat larger values when the flow is faster. Weaker irregularities were detected in the outflow sector between 20-24 MLT, when p generally ranged from 2.6-3.0. The scan velocities measured in the outflow sector were slower, generally between 400-600 m/s. These velocity estimates compare favorably with ion drift measurements made by the DMSP satellites. Since our analysis technique is automated, it could potentially enable continuous monitoring of flow patterns in the polar cap using a relatively inexpensive GPS scintillation monitor. These measurements could then complement measurements from space-based platforms that sample the polar cap only intermittently and incoherent scatter radars which provide excellent diagnostics but cannot operate continuously.

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) play a vital role in replenishment of blood cells. In addition to growth factors, energy metabolism plays an important role in cellular proliferation. Oxidative phosphorylation that occurs in the mitochondria is the major source of ATP. In this study, we have investigated the role of peroxisome proliferator-activated-γ coactivator-1α (PGC-1α), a major regulator of mitochondrial biogenesis, in hematopoiesis. PGC-1α is expressed in HSC/HPCs. Loss of PGC-1α minimally affects basal hematopoiesis; however, it significantly impairs stress hematopoiesis. Recovery of hematopoiesis poststress involves rapid proliferation of HSC/HPCs. Growth factors stimulate HSC/HPC proliferation in a dose-dependent manner and this response is modulated by oxygen tension. Although severe hypoxic conditions inhibit HSC/HPC proliferation, mild hypoxia enhances the clonogenic potential; however, the mechanism underlying this phenomenon remains largely unknown. Our studies demonstrate that PGC-1α-mediated mitochondrial biogenesis is critical for the increased clonogenic potential of progenitors under mild hypoxia. Metabolic programming and increased glucose uptake can drive rapid progenitor cell proliferation under relatively low oxygen tension only if the HPC has the capacity to increase PGC-1α expression and mitochondrial biogenesis. Loss of PGC-1α also impairs the long-term repopulating potential of HSCs. Our findings may have therapeutic applications for rapid recovery of blood cells following myeloablation.

In vitro stimulation of HSCs with growth factors generally leads to their depletion. Understanding the molecular mechanisms underlying expansion of HSCs in vivo following myeloablation could lead to successful expansion of HSCs ex vivo for therapeutic purposes. Current findings show that mTORC1 is activated in HSPCs following 5-fluorouracil treatment and that mTORC1 activation is dependent on mitochondrial ETC capacity of HSPCs. Moreover, expression of PGC-1 family members, proteins that regulate mitochondrial biogenesis, in HSPCs following 5-fluorouracil treatment changes; also, these proteins play a stage specific role in hematopoietic recovery. While PRC regulates HSCs' expansion during early recovery phase, PGC-1α regulates progenitor cell proliferation and recovery of hematopoiesis during later phase. During early recovery phase, PRC expression, mitochondrial activity and mTORC1 activation are relatively higher in PGC-1α−/− HSCs compared to WT HSCs, and PGC-1α−/− HSCs show greater expansion. Administration of rapamycin, but not NAC, during early recovery phase improves WT HSC numbers but decreases PGC-1α−/− HSC numbers. The current findings demonstrate that mTOR activation can increase HSC numbers provided that the energy demand created by mTOR activation is successfully met. Thus, critical tuning between mTORC1 activation and mitochondrial ETC capacity is crucial for HSC maintenance/expansion in response to mitogenic stimulation.

: This project is to investigate how the solar wind/interplanetary magnetic field (IMF) controls the equatorial ionospheric dynamics during intense geomagnetic storms.

Cell motility is a very critical phenomenon that plays an important role in the development of eukaryotic organisms. One of the well studied cell motility phenomena is chemotaxis, which is described as a directional movement of cell in response to changes in external chemotactic gradient. Numerous studies conducted both in unicellular organism and in mammalian cells have demonstrated the importance of phosphatidylionositol-3 kinase (PI3K) in this process. In addition, it is now well established that although PI3K plays an activation role in chemotaxis, the role of phosphatases is also critical to maintain this dynamic cyclical process. Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that is a key player in regulating PI3K signaling. PP2A is abundantly and ubiquitously expressed and has been highly conserved during the evolution of eukaryotes. PP2A is composed of three protein subunits, A, B, and C. Subunit 'A' is a 60-65 kDa structural component, 'C' is a 36-38 kDa catalytic subunit, and 'B' is a 54-130 kDa regulatory subunit. The core complex of PP2A is comprised of the A and C subunits, which are tightly associated and this dimeric core complexes with the regulatory B subunit. The B subunit determines the substrate specificity as well as the spatial and temporal functions of PP2A. PP2A plays an important role in regulating multiple signal transduction pathways, including cell-cycle regulation, cell-growth and development, cytoskeleton dynamics, and cell motility. This review focuses on the role of PP2A in regulating motility of normal and transformed cells.

Abstract 3616 Poster Board III-552 Despite being an excellent alternative to bone marrow or mobilized peripheral blood as a source of hematopoietic stem progenitor cells (HSPCs), the limiting factor to wider use of cord blood (CB) in transplantation is the 10-fold lower cell dose in a typical CB unit compared to harvested bone marrow or peripheral blood stem cells. Successful ex-vivo expansion of HSPCs as well as increasing transplantation efficiency by adopting protocols that enhance homing and engraftment of transplanted HSPC provides hope of making the applicability of a single unit of CB wider in the adult population. Interaction and adhesion of HSPCs with extracellular matrix (ECM) is an important event that regulates cell differentiation, proliferation, survival/ apoptosis as well as migration of HSPCs. Based on evidence present in the literature it appears that in addition to cooperative action between adhesion and growth factors, integrin mediated cell adhesion also provides distinct growth regulatory cues. Although it is known that in hematopoietic system, β1 integrin plays an important role in the interaction of HSPCs with integrin ligands, the underlying molecular mechanisms regulating βl integrin activity in hematopoietic cells remains largely unknown. Based on studies in other systems- Integrin linked kinase (ILK) appears to be an important molecule. ILK not only modulates β1 integrin activity, but also functions as an adapter protein, physically coupling downstream signals from both integrins and growth factors. ILK is preferentially expressed in murine stem / early progenitor cells (based on I. Lemiscka's Stem cell database) and we have found that ILK is expressed in both primitive (CD38-/loCD34+) and committed progenitor (CD38+ CD34+) cells from CB by western blot. Moreover, using a co-immunoprecipitation approach, we found that in freshly isolated CD34+CB cells ILK interacts with β1 integrin. To investigate the role of ILK in adhesion-dependent and -independent growth of CB CD34+ cells both in the presence and absence of growth factors we expressed constitutive active ILK (CAILK) or dominant negative ILK (DNILK) in CD34+CB cells. In addition, we have also evaluated the effect of modulating ILK activity on chemotaxis of CD34+ CB cells towards stromal derived factor-1 (SDF-1)/CXCL12. We found that expression of CAILK enhances expansion of total CD34+ cells as well as colony forming cells stimulated ex-vivo by growth factors (stem cell factor- SCF; Flt-3 ligand and thrombopoietin-TPO), compared to cells transfected with vector alone. In contrast, expression of DNILK inhibited expansion of CD34+CB cells; this effect was more pronounced when cells were cultured in the absence of fibronectin, the ECM. Expression of CAILK increases cell-cycling since a greater proportion of cells were in ‘S’-phase compared to cells expressing DNILK or vector alone, both when the cells were expanded in the presence or absence of fibronectin for 20h. Expression of CAILK also leads to improved survival of CD34+ cells in the absence of serum and growth factors. Interestingly, β-catenin could be detected in CD34+ cells expressing CAILK, but not in cells transfected with DNILK or vector alone. Furthermore, in response to growth factor stimulation, Akt is phosphorylated in cells expressing either CAILK or DNILK; however, the amount of Akt phosphorylation was lower in cells expressing DNILK. Expression of CAILK improved modestly but not significantly the chemotaxis of CD34+ cells towards SDF-1 compared to cells expressing vector alone. However, DNILK significantly impaired chemotaxis of CD34+CB cells towards SDF-1. This impairment of chemotaxis is associated with defective actin polymerization in response to SDF-1, both at the ‘leading-edge’ and ‘tail’ of a polarized DNILK expressing CD34+ cell. Our findings implicate a role for ILK in both growth factor stimulated ex-vivo expansion of HSPCs as well as SDF-1 mediated chemotaxis. This may have potential implications in the therapeutic use of CB cells. Disclosures: No relevant conflicts of interest to declare.

CXCL12 and its receptor CXCR4 play an important role in hematopoietic stem/progenitor cell (HSPC) migration from and retention within the bone marrow. HSPCs are very selective in their chemotactic response and undergo chemotaxis only in response to CXCL12. In addition to CXCR4, HSPCs express receptors for various other chemokines; however, the role of these receptors is not well understood. Freshly isolated CD34+ cells (highly enriched for HSPCs) from cord blood (CB) express low levels of CCR5; however, if the cells were washed with acidic buffer before Ab staining to remove any ligand bound to CCR5, then nearly 80% of CD34+ CB cells were found to express CCR5 on the cell surface. Although none of the CCR5 ligands investigated in this study (CCL3, CCL4, and CCL5) induced chemotaxis, at relatively high concentrations they transiently enhanced CXCL12-mediated chemotaxis of CD34+ CB cells. In contrast, CXCL12-mediated adhesion of cells to VCAM-1-coated surfaces was reduced if CD34+ CB cells were pretreated with these CCR5 ligands for 15 min. The effect of these chemokines on CXCL12-mediated responses was not at the level of CXCR4 expression, but on downstream signaling pathways elicited by CXCL12. Pretreatment with CCR5 chemokines enhanced CXCL12-mediated Akt phosphorylation, but down-modulated calcium flux in CD34+ CB cells. Modulation of CXCL12-mediated responses of CD34+ cells by CCR5 chemokines provides a possible mechanism that underlies movement of HSPCs during inflammation.

Recounts the life and career of Dr. Jules Aarons, a pioneer on using satellites to study Earth's ionosphere beginning with the launch of Sputnik. Dr. Jules passed away on November 21, 2008, in Newton, Massachusetts, at the age of 87.

CXCL12 and its receptor CXCR4 play an important role in hematopoietic stem/progenitor cell (HSPC) migration from and retention within the bone marrow. HSPCs are very selective in their chemotactic response and undergo chemotaxis only in response to CXCL12. In addition to CXCR4, HSPCs express receptors for various other chemokines; however, the role of these receptors is not well understood. Freshly isolated CD34+ cells (highly enriched for HSPCs) from cord blood (CB) express low levels of CCR5; however, if the cells were washed with acidic buffer before Ab staining to remove any ligand bound to CCR5, then nearly 80% of CD34+ CB cells were found to express CCR5 on the cell surface. Although none of the CCR5 ligands investigated in this study (CCL3, CCL4, and CCL5) induced chemotaxis, at relatively high concentrations they transiently enhanced CXCL12-mediated chemotaxis of CD34+ CB cells. In contrast, CXCL12-mediated adhesion of cells to VCAM-1-coated surfaces was reduced if CD34+ CB cells were pretreated with these CCR5 ligands for 15 min. The effect of these chemokines on CXCL12-mediated responses was not at the level of CXCR4 expression, but on downstream signaling pathways elicited by CXCL12. Pretreatment with CCR5 chemokines enhanced CXCL12-mediated Akt phosphorylation, but down-modulated calcium flux in CD34+ CB cells. Modulation of CXCL12-mediated responses of CD34+ cells by CCR5 chemokines provides a possible mechanism that underlies movement of HSPCs during inflammation.

Molecular mechanisms preserving hematopoietic stem cell (HSC) self-renewal by maintaining a balance between proliferation, differentiation, and other processes are not fully understood. Hyperactivation of the mammalian target of rapamycin (mTOR) pathway, causing sustained proliferative signals, can lead to exhaustion of HSC repopulating ability. We examined the role of the novel ras gene Rheb2, an activator of the mTOR kinase, in colony-forming ability, survival, and repopulation of immature mouse hematopoietic cells. In a cell line model of mouse hematopoietic progenitor cells (HPCs), we found enhanced proliferation and mTOR signaling in cells overexpressing Rheb2. In addition, overexpression of Rheb2 enhanced colony-forming ability and survival of primary mouse bone marrow HPCs. Expansion of phenotypic HSCs in vitro was enhanced by Rheb2 overexpression. Consistent with these findings, Rheb2 overexpression transiently expanded phenotypically defined immature hematopoietic cells after in vivo transplantation; however, these Rheb2-transduced cells were significantly impaired in overall repopulation of primary and secondary congenic transplantation recipients. Our findings suggest that HPCs and HSCs behave differently in response to growth-promoting signals stimulated by Rheb2. These results may have value in elucidating mechanisms controlling the balance between proliferation and repopulating ability, a finding of importance in clinical uses of HPCs/HSCs.

Neutrophils play an important role in the host's defense against infection with various pathogenic organisms. Granulocyte colony stimulating factor (G-CSF) is regarded as a major regulator of neutrophil production and function. Mice lacking G-CSF or its receptor are neutropenic. IL-6 is another cytokine that has been shown to promote neutrophil production and modulate the function of many types of immune cells. We have analyzed G-CSF/IL-6 double deficient (G-CSF(- / - )/IL-6(- / - )) mice to gain an insight into the possible contribution of IL-6 to the residual granulopoiesis in G-CSF-deficient (G-CSF(- / - )) mice. Furthermore, we have evaluated the ability of G-CSF(- / - )/IL-6(- / - ) mice to combat an experimental infection with Candida albicans. Our data shows that IL-6 plays a role in granulopoiesis during early post natal period but it is dispensable for steady-state granulopoiesis in adult mice. However, adult G-CSF(- / - )/IL-6(- / - ) mice are more susceptible to Candida infection than similarly infected G-CSF(- / - ) mice. Although, the candidacidal function of neutrophils of G-CSF(- / - )/IL-6(- / - ) mice is deficient, the ability to produce IFN-gamma and TNF-alpha in response to Candida infection is not compromised. Similarly, nitric oxide production by peritoneal macrophages from G-CSF(- / - )/IL-6(- / - ) mice in response to Candida is comparable to G-CSF(- / - ) mice.

ABSTRACT CXCL12/stromal cell-derived factor 1 is a member of the CXC family of chemokines that plays an important role in hematopoiesis and signals through CXCR4 and CXCR7. Two splice variants of human CXCL12 (CXCL12α and CXCL12β) induce chemotaxis of CXCR4 + cells and inhibit X4 infection. Recent studies described four other novel splice variants of human CXCL12; however, their antiviral activities were not investigated. We constructed and expressed all of the CXCL12 splice variants in Escherichia coli . Recombinant proteins were purified through a His affinity column, and their biological properties were analyzed. All six CXCL12 variants induced chemotaxis of CXCR4 + and CXCR7 + cell lines. Enhancement of survival and replating capacity of human hematopoietic progenitor cells were observed with CXCL12α, CXCL12β, and CXCL12ε but not with the other variants. CXCL12γ showed the greatest antiviral activity in X4 inhibition assays and the weakest chemotaxis activity through CXCR4. The order of potency in X4 inhibition assays was as follows: CXCL12γ > CXCL12β > CXCL12α > CXCL12θ > CXCL12ε > CXCL12δ. The order of anti-human immunodeficiency virus (HIV) activity was associated with the number of BBXB motifs present in each variant; the most potent inhibitor was CXCL12γ, with five BBXB domains. The results suggest that the different C termini of CXCL12 variants may contain important molecular determinants for the observed differences in antiviral effects and other biological functions. These studies implicate CXCL12γ as a potent HIV-1 entry inhibitor with significantly reduced chemotaxis activity and small or absent effects on progenitor cell survival or replating capacity, providing important insight into the structure-function relationships of CXCL12.

Mitotic arrest deficiency 2 (Mad2) is a component of mitotic spindle checkpoint proteins and is essential for accurate chromosome segregation. We investigated a role for Mad2 in hematopoiesis using Mad2-haploinsufficient (Mad2(+/-)) mice. Mad2(+/-) bone marrow (BM) and spleen manifested decreased absolute numbers and cycling status of immature, but not mature, hematopoietic progenitor cells. Mad2(+/-) BM granulocyte-macrophage colony-forming units (CFU-GMs) did not manifest synergistic proliferation in re- sponse to stem cell factor (SCF) plus GM-CSF. The percentage of annexin V+ cells was higher in Mad2(+/-) than Mad2(+/+) c-Kit(+)lin(-) BM after culture with SCF and GM-CSF. However, no significant difference in phosphorylation of extracellular signal-related kinase (Erk1/2) at Thr202/ Tyr204 and Akt at Ser473 between Mad2(+/-) and Mad2(+/+) BM c-Kit+lin- cells was observed. Immunoprecipitation assays performed in human MO7e cells demonstrated physical association of c-Kit with Mad2. Moreover, stimulation with SCF plus GM-CSF led to dissociation of Mad2 from c-Kit. Confocal microscopy demonstrated that Mad2 colocalized with c-Kit in the cytoplasm of MO7e cells. These results suggest that Mad2 is involved in synergistic growth of immature hematopoietic progenitor cells in response to SCF plus GM-CSF, effects that may be mediated via physical association of Mad2 with c-Kit.

G-CSF and GM-CSF play important roles in regulating neutrophil production, survival, differentiation, and function. However, we have shown previously that G-CSF/GM-CSF double-deficient [knockout (KO)] mice still develop a profound neutrophilia in bone marrow and blood after infection with Candida albicans. This finding suggests the existence of other systems, which can regulate emergency neutrophil production. We have now developed an "in vitro" technique to detect and characterize a neutrophil-promoting activity (NPA) in the media conditioned by mouse embryonic fibroblasts (MEFs) derived from G-CSF(-/-)/GM-CSF(-/-) mice. NPA is produced in vitro by the MEFs after stimulation with LPS or heat-inactivated C. albicans. Although M-CSF added directly to bone marrow cultures does not sustain granulocyte production, our studies indicate that production of NPA requires activation of the M-CSF receptor (c-fms). First, G-CSF(-/-)/GM-CSF(-/-) MEFs produce high levels of NPA after stimulation with LPS or C. albicans, and G-CSF/GM-CSF/M-CSF triple-KO MEFs do not. Second, the production of NPA by the G-CSF(-/-)/GM-CSF(-/-) MEFs is reduced significantly upon incubation with neutralizing antibodies to M-CSF or c-fms. Third, NPA production by G-CSF(-/-)/GM-CSF(-/-)/M-CSF(-/-) fibroblasts is enhanced by supplementing culture medium with M-CSF. Thus, stimulation of c-fms by M-CSF is a prerequisite for the production of NPA.