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In this work we propose a study to determine the structure of Fe and Co doped TiO2 by using the Fe K-edge, Co K-edge and Ti K- edge X-ray absorption near edge spectroscopy. The detailed analysis of Fe and Co-doped TiO2 before and after Methylene blue (MB) treatment was examined under the irradiation of 35 W xenon arc lamp for 3 h. The materials treated with MB were studied by X-ray absorption spectroscopy, EPR and FT-IR which revealed that the oxidation state of Co2+ was photo-oxidized to Co3+ and Fe3+ was photo-reduced to Fe2+ or less. Thermodynamic, kinetic properties were studied at different reaction temperature and the activation energy (Ea), enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) of activation were calculated for the reaction. The activation energy has been found for TiO2, Fe TiO2 and Co TiO2 as 24.771, 11.413 and 15.801 kJ mol−1 respectively. The structure, morphology and optical properties were studied by XRD, UV-diffuse reflectance spectra, FESEM, TEM and PL. Moreover, electrochemical studies were carried out to demonstrate the oxygen evolution reaction (OER) activity on TiO2, Fe TiO2 and Co TiO2 in 1 M of H2SO4 electrolyte, with a scan rate of 50 mV s−1 and the as-prepared photocatalysts could act as the promising electrode materials for water splitting.

The core-shell structure of Ce-doped TiO2@SiO2@(Ni-Cu-Zn) ferrite noted of CTSF as composite nanoparticles (NPs) was synthesized using a modified sol-gel method. The physicochemical properties of as-prepared products were characterized completely by X-ray diffraction (XRD), Brunauer-Emmit-Teller (BET), X-ray photoelectron spectroscopy (XPS) and superconducting quantum interference device (SQUID), serially. Meanwhile, assessment of the photocatalystic activity of catalyst was performed by ultraviolet-visible spectrometry (UV-vis). The results of the study show that the anatase phase related to the TiO2structure was constructed on the outer shell coating of composite NPs. However, the second phase associated with the Ce structure was not easy to be detected on the XRD pattern, confirming that the doping Ce had been incorporated into the TiO2crystal structure. The mesoporous structure of Ce-doped TiO2layers was demonstrated by the type IV isotherm and H3 type hysteresis loop. The homogenous pore size was generated with the specific surface area up to 111.916m2/g and 0.241cc/g of pore volume. The stoichiometry of the chemical composition formed with fewer defects on the surface of TiO2layers was exhibited by the symmetry curve of Ti 2p3/2and Ti 2p1/2peaks of XPS spectra. Meanwhile, the redox couple corresponding to Ce3+/Ce4+was incorporated inside the thin TiO2coating. Furthermore, the catalyst magnetic NPs can be also separated by using an external magnetic field from the reaction system. The product performance associated with the degradation efficiency was achieved to be 50% in the aqueous solution of methylene blue (MB)

Leukemia stem cells (LSCs) in individuals with chronic myelogenous leukemia (CML) (hereafter referred to as CML LSCs) are responsible for initiating and maintaining clonal hematopoiesis. These cells persist in the bone marrow (BM) despite effective inhibition of BCR–ABL kinase activity by tyrosine kinase inhibitors (TKIs). Here we show that although the microRNA (miRNA) miR-126 supported the quiescence, self-renewal and engraftment capacity of CML LSCs, miR-126 levels were lower in CML LSCs than in long-term hematopoietic stem cells (LT-HSCs) from healthy individuals. Downregulation of miR-126 levels in CML LSCs was due to phosphorylation of Sprouty-related EVH1-domain-containing 1 (SPRED1) by BCR–ABL, which led to inhibition of the RAN–exportin-5–RCC1 complex that mediates miRNA maturation. Endothelial cells (ECs) in the BM supply miR-126 to CML LSCs to support quiescence and leukemia growth, as shown using mouse models of CML in which Mir126a (encoding miR-126) was conditionally knocked out in ECs and/or LSCs. Inhibition of BCR–ABL by TKI treatment caused an undesired increase in endogenous miR-126 levels, which enhanced LSC quiescence and persistence. Mir126a knockout in LSCs and/or ECs, or treatment with a miR-126 inhibitor that targets miR-126 expression in both LSCs and ECs, enhanced the in vivo anti-leukemic effects of TKI treatment and strongly diminished LSC leukemia-initiating capacity, providing a new strategy for the elimination of LSCs in individuals with CML.

Graphene oxide-based composites have been developed as cheap and effective photocatalysts for dye degradation and water splitting applications. Herein, we report reduced graphene oxide (rGO)/Ag/Fe-doped TiO2 that has been successfully prepared using a simple process. The resulting composites were characterized by a wide range of physicochemical techniques. The photocatalytic activities of the composite materials were studied under visible light supplied by a 35 W Xe arc lamp. The rGO/Ag/Fe-doped TiO2 composite demonstrated excellent degradation of methylene blue (MB) in 150 min and 4-nitrophenol (4-NP) in 210 min under visible light irradiation, and trapping experiments were carried out to explain the mechanism of photocatalytic activity. Moreover, electrochemical studies were carried out to demonstrate the oxygen evolution reaction (OER) activity on rGO/Ag/Fe-doped TiO2 in 1 M of H2SO4 electrolyte, with a scan rate of 50 mV s−1. The reductions in overpotential are due to the d-orbital splitting in Fe-doped TiO2 and rGO as an electron collector and transporter.

Little is known about how leukemia cells alter the bone marrow (BM) niche to facilitate their own growth and evade chemotherapy. Here, we provide evidence that acute myeloid leukemia (AML) blasts remodel the BM niche into a leukemia growth-permissive and normal hematopoiesis-suppressive microenvironment through exosome secretion. Either engrafted AML cells or AML-derived exosomes increased mesenchymal stromal progenitors and blocked osteolineage development and bone formation in vivo. Preconditioning with AML-derived exosomes 'primed' the animals for accelerated AML growth. Conversely, disruption of exosome secretion in AML cells through targeting Rab27a, an important regulator involved in exosome release, significantly delayed leukemia development. In BM stromal cells, AML-derived exosomes induced the expression of DKK1, a suppressor of normal hematopoiesis and osteogenesis, thereby contributing to osteoblast loss. Conversely, treatment with a DKK1 inhibitor delayed AML progression and prolonged survival in AML-engrafted mice. In addition, AML-derived exosomes induced a broad downregulation of hematopoietic stem cell-supporting factors (for example, CXCL12, KITL and IGF1) in BM stromal cells and reduced their ability to support normal hematopoiesis. Altogether, this study uncovers novel features of AML pathogenesis and unveils how AML cells create a self-strengthening leukemic niche that promotes leukemic cell proliferation and survival, while suppressing normal hematopoiesis through exosome secretion.

Ag nanoparticles with different weight percentage loaded on Ni-Cu-Zn ferrite@SiO 2 @TiO 2 composite were successfully prepared via sol-gel method. The effect of deposited amount of Ag nanoparticles on structural, magnetic and photocatalytic properties of the composite was investigated. XPS characterization confirmed that Ag nanoparticles deposited on the surface of Ni-Cu-Zn ferrite@SiO 2 @TiO 2 composite. Particle size and shape of magnetic recyclable photocatalysts were observed using TEM, showing a core-shell structure with an average particle size of 20 nm of Ag. Based on room temperature magnetic hysteresis curves, as 0.4 wt% Ag immobilized on Ni-Cu-Zn ferrite@SiO 2 @TiO 2 composite revealed the maximum saturation magnetization of 39.9 emu/g. Photocatalytic degradation of Ni-Cu-Zn ferrite@SiO 2 @TiO 2 @Ag was carried out in methylene blue (MB) solutions illuminated under a 35-W Xe arc lamp and color temperature of 6000 K. The results revealed that 83.9% of MB was photodegraded for 0.4 wt% of Ag-loaded Ni-Cu-Zn ferrite@SiO 2 @TiO 2 . Moreover, it can be easily separated and recycled without significant loss of photocatalytic activity after being used five times. Compared to the conventional photocatalysts, the magnetic core–shell photocatalyst is suitable for large scale applications in industrial wastewater system.

Oxygen transportation properties of SmBa0.5Sr0.5Co2O5+δ (SBSC55) layered perovskite has been investigated as a potential cathode for intermediate-temperature solid oxide fuel cells (IT-SOFCs). This research includes the following items: (1) structural characteristics, thermogravimetric behavior, oxygen permeation, chemical bulk diffusion coefficient (Dchem), and (2) the electrochemical performance of long-term testing carried out to evaluate its electrochemical stability. The Dchem values for SBSC55 cathode are 2.6 × 10−6, 9.1 × 10−6 and 1.8 × 10−5 cm2 s−1 at 500 °C, 600 °C, and 700 °C, respectively. The oxygen permeation flux for SBSC55 membrane with 1.0 mm thickness increased from 0.143 mL min−1 cm−2 at 500 °C to 0.406 mL min−1 cm−2 at 800 °C under synthetic air at a flow rate of 50 mL min−1, helium at a rate of 25 mL min−1. The activation energies of oxygen permeation for the high temperature region (700–800 °C) and low temperature region (500–650 °C) are 23.67 and 6.96 kJ mol−1, respectively. It suggested that oxygen diffusion for high temperature range is surface exchange process, low temperature range is bulk diffusion process. The long-term test of cathodic polarization resistance for SBSC55∣SDC∣SSBSC55 half-cell at 600 °C during 96 h has been carried out with an increase rate of 0.30% h−1. Based on the impedance spectra with various oxygen partial pressure results, the rate-limiting process of ORR is determined that the oxygen ion transfer from triple-phase boundary (TPB) and/or two-phase boundary (2PB) sites of cathode is major with the minority of charger transfer reaction.

Acute myeloid leukemia (AML) arises through multistep clonal evolution characterized by stepwise accumulation of successive alterations affecting the homeostasis of differentiation, proliferation, self-renewal, and survival programs. The persistence and dynamic clonal evolution of leukemia-initiating cells and preleukemic stem cells during disease progression and treatment are thought to contribute to disease relapse and poor outcome. Inv(16)(p13q22) or t(16;16)(p13.1;q22), one of the most common cytogenetic abnormalities in AML, leads to expression of a fusion protein CBFβ-SMMHC (CM) known to disrupt myeloid and lymphoid differentiation. Anemia is often observed in AML but is presumed to be a secondary consequence of leukemic clonal expansion. Here, we show that CM expression induces marked deficiencies in erythroid lineage differentiation and early preleukemic expansion of a phenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitor population. Using dual-fluorescence reporter mice in lineage tracking and repopulation assays, we show that CM expression cell autonomously causes expansion of abnormal Pre-Meg/E progenitors with compromised erythroid specification and differentiation capacity. The preleukemic Pre-Meg/Es display dysregulated erythroid and megakaryocytic fate-determining factors including increased Spi-1, Gata2, and Gfi1b and reduced Zfpm1, Pf4, Vwf, and Mpl expression. Furthermore, these abnormal preleukemic Pre-Meg/Es have enhanced stress resistance and are prone to leukemia initiation upon acquiring cooperative signals. This study reveals that the leukemogenic CM fusion protein disrupts adult erythropoiesis and creates stress-resistant preleukemic Pre-Meg/E progenitors predisposed to malignant transformation. Abnormality in Meg/E or erythroid progenitors could potentially be considered an early predictive risk factor for leukemia evolution.

The effects of Sr doping on the Ba-site of SmBaCo2O5+δ in term of structure characteristics, thermal expansion coefficients (TECs), electrical properties and electrochemical performance have been investigated as cathode material for intermediate-temperature solid oxide fuel cells (IT-SOFCs). The TECs of SBSC-based cathodes are calculated from 19.8 − 20.5 × 10−6 K−1 in the temperature range of 100–800 °C, and the TEC values decrease with increasing Sr content. The oxygen content and the average oxidation state of cobalt increase with increasing Sr content determined by the X-ray photoelectron spectroscopy (XPS) and Thermogravimetry analysis (TGA) results. At a given temperature, the electrical conductivity values are in the order as follows: SBSC55 > SBSC73 > SBSC91. This behavior might be due to the increase in electronic hole. The electrical conductivities of SBSC55 at 600 °C are distributed in the range of 660 S/cm of p(O2) = 0.01 atm to 1168 S/cm of p(O2) = 0.21 atm, indicating that the cathode can endure reducing atmosphere. SBSC55 with high electrical conductivity in p(O2) = 0.01 atm is ascribed to SBSC55 with stable double-perovskite structure at such low oxygen partial pressure. The SBSC55 cathode showed the highest power density of 304 mW/cm2 at operating temperature of 700 °C. Based on the electrochemical properties, SBSC55 is a potential cathode for IT-SOFCs.

The hematopoietic stem cell (HSC) niche is composed of a complex set of stromal support cells that maintain HSCs and promote normal hematopoiesis. We now know that molecular changes within the hematopoietic niche contribute to leukemia development. Leukemia cells often reorganize the hematopoietic niche to promote and support their own survival and growth. Here we will summarize recent works that decipher the normal hematopoietic niche cellular components and describe how the leukemia-transformed niche contributes to hematological malignances. Finally, we will discuss recent publications that highlight a possible role for exosomes in the leukemia-induced niche reorganization. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.

We previously reported that long-term administration of a low dose of gastrin and epidermal growth factor (GE) augments β-cell neogenesis in late-stage diabetic autoimmune mice after eliminating insulitis by induction of mixed chimerism. However, the source of β-cell neogenesis is still unknown. SRY (sex-determining region Y)-box 9(+) (Sox9(+)) ductal cells in the adult pancreas are clonogenic and can give rise to insulin-producing β cells in an in vitro culture. Whether Sox9(+) ductal cells in the adult pancreas can give rise to β cells in vivo remains controversial. Here, using lineage-tracing with genetic labeling of Insulin- or Sox9-expressing cells, we show that hyperglycemia (>300 mg/dL) is required for inducing Sox9(+) ductal cell differentiation into insulin-producing β cells, and medium hyperglycemia (300-450 mg/dL) in combination with long-term administration of low-dose GE synergistically augments differentiation and is associated with normalization of blood glucose in nonautoimmune diabetic C57BL/6 mice. Short-term administration of high-dose GE cannot augment differentiation, although it can augment preexisting β-cell replication. These results indicate that medium hyperglycemia combined with long-term administration of low-dose GE represents one way to induce Sox9(+) ductal cell differentiation into β cells in adult mice.

The purpose of the research was to determine the photocatalytic degradation efficiency of acid orange 7 (AO7) using 2 mol% Fe-doped TiO2 as photocatalysts under UV irradiation. Fe-doped TiO2 powder synthesized by solid-reaction technique reveals the average particle size of 0.91 μm, BET surface area of 11.6 m2/g, a wide pore-size distribution with the average pore diameter of 3.2 nm and pore volume of 0.035 cm3/g. Within the experiment, AO7 was prepared as a synthetic textile wastewater at the initial concentration of 34 mg/L along with using various doses of Fe-doped TiO2 photocatalysts. The results indicated that Fe-doped TiO2 with dose of 0.100 g in 100 mL of AO7 reached the highest removal efficiency about 88.7%.

Mesenchymal stromal cells are an important component of the adult hematopoietic stem cell niche. They are a diverse population of cells that include a hierarchy of primitive, intermediate, and mature osteoprogenitors that support HSCs and supply the bone with matrix producing osteoblast. To understand the different roles played by individual types of progenitors, it is necessary to separate individual populations and analyze them in a controlled environment. Here we describe two transplantation models, an ectopic bone forming assay and an intravenous injection assay, in which niche components can be isolated and manipulated to dissect their individual properties.

Mesenchymal stromal cells are an important component of the adult hematopoietic stem cell niche. They are a diverse population of cells that include a hierarchy of primitive, intermediate, and mature osteoprogenitors that support HSCs and supply the bone with matrix producing osteoblast. To understand the different roles played by individual types of progenitors, it is necessary to separate individual populations and analyze them in a controlled environment. Here we describe two transplantation models, an ectopic bone forming assay and an intravenous injection assay, in which niche components can be isolated and manipulated to dissect their individual properties.

Graphene oxide-based composites have been developed as cheap and effective photocatalysts for dye degradation and water splitting applications. Herein, we report reduced graphene oxide (rGO)/Ag/Fe-doped TiO

MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, whereas the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142(-/-) animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142(-/-) B cells express elevated levels of B-cell-activating factor (BAFF) receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142(-/-) mice rescues the B-cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B-cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.

The aim of the work is to study the effects of oxygen vacancy and surface hydroxyl group density on the photocatalytic activity of Fe3+-doped TiO2, and to investigate how the titanium dioxide doped with different levels of the Fe3+ influenced their physical and chemical characterizations. The photocatalysts were characterized by X-ray diffraction (XRD), thermogravimetric analysis (TGA), electron spin resonance (ESR), X-ray photoelectron spectroscope (XPS) and Fourier transform infrared (FTIR) spectra. The results revealed that adsorbed hydroxyl group density significantly influenced the photocatalytic activity, and a small amount of Fe3+ can act as a photo-generated hole and a photo-generated electron trap and inhibit the electron–hole recombination. The 0.10%-Fe3+-TiO2 with the highest surface hydroxyl group density revealed the maximum rate constant of 0.716 and the optimal photocatalytic degradation of MB. As Fe3+ doping levels are larger than 0.10%, the cluster of Fe Ti ′ – V ¨ O – Fe Ti ′ generated gradually. This implied that an excessive amount of Fe3+ doped into TiO2 is detrimental to the photocatalytic activity due to the formation of Fe Ti ′ – V ¨ O – Fe Ti ′ clusters and enhances the recombination of photogenerated electrons and holes.

Hematopoietic stem cells (HSCs) reside in regulatory niches in the bone marrow (BM). Although HSC niches have been extensively characterized, the role of endosteal osteoblasts (OBs) in HSC regulation requires further clarification, and the role of OBs in regulating leukemic stem cells (LSCs) is not well studied. We used an OB visualization and ablation mouse model to study the role of OBs in regulating normal HSCs and chronic myelogenous leukemia (CML) LSCs. OB ablation resulted in increase in cells with a LSK Flt3(-)CD150(+)CD48(-) long-term HSC (LTHSC) phenotype but reduction of a more highly selected LSK Flt3(-)CD34(-)CD49b(-)CD229(-) LTHSC subpopulation. LTHSCs from OB-ablated mice demonstrated loss of quiescence and reduced long-term engraftment and self-renewal capacity. Ablation of OB in a transgenic CML mouse model resulted in accelerated leukemia development with reduced survival compared with control mice. The notch ligand Jagged-1 was overexpressed on CML OBs. Normal and CML LTHSCs cultured with Jagged-1 demonstrated reduced cell cycling, consistent with a possible role for loss of Jagged-1 signals in altered HSC and LSC function after OB ablation. These studies support an important role for OBs in regulating quiescence and self-renewal of LTHSCs and a previously unrecognized role in modulating leukemia development in CML.

Design of optimal beamformers that withstand system perturbations such as channel mismatch, sensor position error, and pointing error has been a key issue in real-world applications of arrays. This paper aims to characterize the array performance in relation to the random perturbations from a statistical perspective. In the synthesis stage, directivity index and front-to-back ratio are employed as the performance measures for beamformer optimization. Filter coefficients of the arrays are determined using the least-squares and convex optimization approaches using the preceding performance measures. Next, Monte Carlo sampling are conducted to simulate the stochastic system perturbations following either uniform distribution or normal distribution. Statistics including the sample mean, maximum, minimum, and the maximum likelihood (ML) of the preceding performance measures are calculated. Three regularization criteria based on max-mean, max-min, and max-ML of performance measures are proposed for choosing regularization parameters used in beamformer optimization. The max-mean criterion was found most useful to determine either a simple constant or a frequency-dependent regularization parameter. To validate the proposed methods, experiments of beam patterns and automatic speech recognition test were conducted for directional and diffuse noise suppression problems, where optimal beamformers designed with the regularization parameter selected by the preceding procedures were utilized.

We successfully prepared the TiO 2 @Mn-Zn ferrite composite powder for magnetic photocatalyst. The core Mn-Zn ferrite powder was synthesized by using steel pickling liquor and used alkaline batteries as the starting materials. The shell TiO 2 nanocrystal was prepared by sol–gel hydrolysis precipitation of titanium isopropoxide (Ti(OC 3 H 7 ) 4 ) on the Mn-Zn ferrite powder. The thickness of the titania shell was found to be approximately 2 nm. The core of Mn-Zn ferrite is of spherical or elliptical shape and the particle size of the core is in the range of 20–40 nm. The magnetic Mn-Zn ferrite nanopowder is uniformly encapsulated in a titania layer forming the core–shell structure of TiO 2 @Mn-Zn ferrite powder. The magnetic photocatalyst can be successfully used to treat dye waste waters.

In this study, we identified a CD105+CD90.1−CD133−CD55− (CD133−CD55−) population in the fetal skeletal element that can generate bone and bone marrow. Besides osteoblasts and chondrocytes, the CD133−CD55− common progenitors can give rise to marrow reticular stromal cells and perivascular mesenchymal progenitors suggesting they function as the fetal common skeletal progenitor. Suppression of CXCL12 and Kitl expression in CD133−CD55− common progenitors severely disrupted the BM niche formation but not bone generation. Thus, CD133−CD55− common progenitors are the main source of CXCL12 and Kitl producing cells in the developing marrow.

The artimisinins are a class of antimalarial compounds whose antiparasitic activity is mediated by induction of reactive oxygen species (ROS). Herein, we report that among the artimisinins, artesunate (ARTS), an orally bioavailable compound has the most potent antileukemic activity in AML models and primary patients' blasts. ARTS was most cytotoxic to the FLT3-ITD+ AML MV4-11 and MOLM-13 cells (IC50 values of 1.1 and 0.82μM respectively), inhibited colony formation in primary AML and MDS cells and augmented cytotoxicity of chemotherapeutics. ARTS lowered cellular BCL-2 level via ROS induction and increased the cytotoxicity of the BCL-2 inhibitor venetoclax (ABT-199). ARTS treatment led to cellular and mitochondrial ROS accumulation, double stranded DNA damage, loss of mitochondrial membrane potential and induction of the intrinsic mitochondrial apoptotic cascade in AML cell lines. The antileukemic activity of ARTS was further confirmed in MV4-11 and FLT3-ITD+ primary AML cell xenografts as well as MLL-AF9 syngeneic murine AML model where ARTS treatment resulted in significant survival prolongation of treated mice compared to control. Our results demonstrate the potent preclinical antileukemic activity of ARTS as well as its potential for a rapid transition to a clinical trial either alone or in combination with conventional chemotherapy or BCL-2 inhibitor, for treatment of AML.

Microenvironment cues received by haematopoietic stem cells (HSC) are important in regulating the choice between self-renewal and differentiation. On the basis of the differential expression of cell-surface markers, here we identify a mesenchymal stromal progenitor hierarchy, where CD45−Ter119−CD31−CD166−CD146−Sca1+(Sca1+) progenitors give rise to CD45−Ter119−CD31−CD166−CD146+(CD146+) intermediate and CD45−Ter119−CD31−CD166+CD146−(CD166+) mature osteo-progenitors. All three progenitors preserve HSC long-term multi-lineage reconstitution capability in vitro; however, their in vivo fates are different. Post-transplantation, CD146+ and CD166+ progenitors form bone only. While Sca1+ progenitors produce CD146+, CD166+ progenitors, osteocytes and CXCL12-producing stromal cells. Only Sca1+ progenitors are capable of homing back to the marrow post-intravenous infusion. Ablation of Sca1+ progenitors results in a decrease of all three progenitor populations as well as haematopoietic stem/progenitor cells. Moreover, suppressing production of KIT-ligand in Sca1+ progenitors inhibits their ability to support HSCs. Our results indicate that Sca1+ progenitors, through the generation of both osteogenic and stromal cells, provide a supportive environment for hematopoiesis., How the environment of the niche regulates haematopoietic stem cells (HSC) is unclear. Here, the authors identify a mesenchymal stromal progenitor hierarchy and identify Sca1+ cells as common progenitors for mesenchymal stromal cells in the adult niche that provide a supportive environment for hematopoiesis.

This paper demonstrates that optimum weighting coefficients and inverse filters for microphone arrays can be accomplished, with the aid of a systematic methodology of mathematical programming. Both far-field and near-field array problems are formulated in terms of convex optimization formalism. Three application examples, including data-independent far-field array design, nearfield array design, and pressure field interpolation, are presented. In far-field array design, array coefficients are optimized to tradeoff Directivity Index for White Noise Gain or the coefficient norm, while in nearfield array convex optimization is applied to design Equivalent Source Method-based Nearfield Acoustical Holography. Numerical examples are given for designing a far-field two-dimensional random array comprised of thirty microphones. For far-field arrays, five design approaches, including a Delay-And-Sum beamformer, a Super Directivity Array, three optimal arrays designed using l1,l2,andl∞-norms, are compared. Numerical and experimental results have shown that sufficiently high White Noise Gain was crucial to robust performance of array against sensor mismatch and noise. For nearfield arrays, inverse filters were designed in light of Equivalent Source Method and convex optimization to reconstruct the velocity field on a baffled spherical piston source. The proposed nearfield design is benchmarked by those designed using Truncated Singular Value Decomposition and Tikhonov Regularization. Compressive Sampling and convex optimization is applied to pressure field reconstruction, source separation and modal analysis with satisfactory performance in both near-field and far-field microphone arrays.

Introduction: Acute myeloid leukemia (AML) is a highly aggressive form of leukemia that results a poor survival outcome. Currently, diagnosis and prognosis are based on invasive single-point bone marrow biopsies (iliac crest). Although non-invasive positron emission tomography (PET) imaging has been developed for almost all solid tumors and some hematological malignancies, there is currently no non-invasive imaging specific to AML available, representing an unmet clinical need. About 85% of AML cells express CD33, and expression levels of CD33 has been correlated with poor survival outcomes (Pollard et al., Blood (2012)), making it an ideal candidate for immuno-PET. Therefore, our primary goal is to develop anti-CD33 immuno-PET for detecting CD33+ AML. The secondary goal is to assess the feasibility of CD33 PET image-guided external beam targeted radiation delivery in combination with chemotherapy (AraC). Methods: Murine anti-human CD33 monoclonal antibody clone p67.6 was conjugated to DOTA and radiolabeled with Cu-64 for imaging studies. In vivo PET-CT imaging and bio distribution of 64Cu-DOTA-anti-CD33 in vivo was carried out in NSG mice bearing CD33+ (MV4-11, HL60) AML cells. CD33-negative MM1s cells (multiple myeloma) were used as negative control. The AML and MM bearing mice were injected with 64Cu-DOTA-anti-CD33 (100 µCi/10 µg) and serial PET imaged at 24-48 h. We developed a functional total marrow irradiation (fTMI) treatment, where mice received total marrow irradiation (TMI) (2 Gy) and boost radiation (2 Gy) to regions with increased CD33 activity, followed by 2 days of AraC (40 mg/kg) and 24h later a bone marrow transplant. Furthermore, a humanized anti-human CD33 monoclonal antibody was generated and tested for immunogenicity against CD33 in AML cell lines and patient samples for future clinical studies. Results: PET-CT imaging and biodistribution studies of 64Cu-DOTA-anti-CD33 clearly indicates a CD33+ PET signal in the femur, tibia, humerus joints, L spine and spleen in AML-bearing mice, but not in multiple myeloma-bearing mice or in cold anti-CD33-DOTA treated leukemic mice (Figure 1A and B). Our new imaging method was able to detect CD33+ AML with a favorable sensitivity (71.4%) and specificity (100%). Based on detailed whole-body 3D imaging and validated with biodistribution studies, we discovered preferential regions in the skeletal system with differential CD33 activity, indicating the spatial heterogeneity of AML. CD33+ PET intensity was observed in the following descending order: femur≥lspine>humerus>tibia (Figure 1C). Next, using the PET-CT images, we targeted these CD33-active regions using the fTMI treatment plan. We have developed a unique irradiation system which allows targeting only skeletal tissues while sparing major organs like gut, lung and liver, unlike conventional total body irradiation (TBI). This approach will also provide an opportunity to escalate doses to specific regions in the body without damaging other unintended targets. The fTMI (2 Gy TMI and 2 Gy boost) treatment plan increased the medial survival of the mice to 43 days versus that of untreated control mice (26 days) or AraC treated mice (33 days) Figure(1D). Since the preclinical study was carried out using murine anti-CD33 antibody clone p67.6, we further tested the newly generated humanized anti-human-CD33 monoclonal antibody. This humanized antibody detects CD33 in both AML cell lines and human patient sample (Fig 1E). Conclusion: This study is the first to use an anti-CD33 monoclonal antibody for non-invasive immuno-PET-based imaging for AML detection, showing high sensitivity and specificity. This whole body imaging may be useful for AML diagnosis not only in the entire skeletal system, but also in the extramedullary organs, and for longitudinal monitoring of treatment response. Unlike conventional TBI, Image-guided fTMI along with conventional chemotherapy may result in an improved prognosis, as unintended major organs are spared from radiation. Disclosures Vallera: GT Biopharma: Consultancy, Research Funding. Stein:Celgene: Speakers Bureau; Amgen Inc.: Speakers Bureau.

AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values Taken together, these results support 8-Cl-Ado as a promising novel agent via a unique mechanism of action for FLT3-ITD AML, mediated by miR-155 degradation. 8-Cl-Ado also synergizes with TKIs both in vitro and in vivo thereby representing a potentially novel clinical approach for FLT3-ITD AML. A single-agent phase I/II clinical trial in relapsed/refractory AML is already ongoing at our institution. Figure. Figure. Disclosures Wennerberg: Novartis: Research Funding. Gandhi:Pharmacyclics: Research Funding.

Increasing evidence suggests that the cancer cells take shelter in different osteoblastic and adipocytic niches, where they hide from chemotherapy and continue to survive. As yet, how leukemia cells alter the bone marrow (BM) adipocytic niches to facilitate their expansion and assist them in evading chemotherapy is unclear. We have previously shown that the acute myeloid leukemia (AML) cells directly or through their exosomes, reprogram BM osteoblastic niche which facilitates their expansion and suppress normal hematopoiesis(Kumar B et al, Leukemia 2018,32(3):575-587). In this study , we provide further evidences that AML and Acute lymphoid leukemia (ALL) transformed the BM adipocytic niche to facilitate their expansion and suppress normal hematopoiesis. Using MLL-AF9 (AML) knock-in mouse, MLL-AF9 or BCR-ABL(p190, ALL) HSC transduction transplantation leukemia mice models, we performed flow cytometry analysis to show that the leukemia cells stimulate expansion of BM derived CD45-Ter119-CD31-CD166-Sca1+CD140a+(PaS)MSCs population compared to normal mice (p=0.04, p=0.001 and p=0.002 respectively).Further, the BM osteoblasts specific Osteocalcin mRNA expression in sorted stroma cells and flow cytometry based osteoblasts population (CD45-Ter119-CD31- CD166+Sca1-) numbers were also significantly reduced in AML (p=0.04) and ALL (p=0.02) mice models suggesting bone loss with the leukemia development. Similar to osteoblasts loss, mature adipocytes (Perilipin, PPARg mRNA) were also significantly reduced in the ALL/AML mice compared to control. The triglyceride content and white adipose tissue(WAT) mass was diminished in leukemic mice , suggesting leukemia may have utilized adipocyte for survival. Adipocyte loss in the leukemia mice was accompanied by long term hematopoietic stem cells(LT-HSC ) and erythroid megakaryocyte progenitor (MEP) populations reduction in the leukemic mice (p=0.01 and p=0.02 respectively). To dissect the mechanism of adipocytes reduction is either due to adipocyte loss or adipocytes maturation defect in leukemic mice, we analyzed different stromal progenitors in normal and leukemic mice and identified that the leukemia cells stimulate the growth of BM derived adipocytic committed progenitors (CD45-Ter119-CD31-CD166-Sca1+CD140a+CD29+CD24-) and blocked the chondrocyte/osteoblastic/adipocytic multipotent progenitors (CD45-Ter119-CD31- CD166-Sca1+CD140a+CD29+CD24+) population (p=0.02,p=0.01 respectively).Despite the increase in number of MSCs and adipocytic progenitors, the in-vitro adipocytic differentiation potential of sorted adipocyte committed progenitors was severely compromised in ALL and AML compared to control. The WAT western blot analysis showed significantly increased expression of ATGL and pHSL(Ser-563) expression involved in triglyceride lipolysis in the leukemic mice .The ALL leukemic adipocytic stroma had increased expression of IL-1β and IL-6 cytokine levels compared to normal stroma and provided more survival advantage to leukemia cells in in-vitro co-culture experiments in nutrient deprived conditions and during chemo-radiotherapy treatment. Further, the ATGL and HSL pharmacological inhibitors rescued leukemia induced lipolysis, reduced leukemia proliferation and increased chemotherapy induced apoptosis in leukemia cells. Overall, this data strongly suggests the notion of progressive decline in functional LT-HSCs & normal hematopoiesis, adipocytes and osteoblasts numbers with leukemia progression due to activation of lipolytic enzymes resulting in increased availability of fatty acids for leukemia expansion and is a common feature in both lymphoid and myeloid leukemias. Disclosures No relevant conflicts of interest to declare.

Summary Bone marrow vascular niches sustain hematopoietic stem cells (HSCs) and are drastically remodeled in leukemia to support pathological functions. Acute myeloid leukemia (AML) cells produce angiogenic factors, which likely contribute to this remodeling, but anti-angiogenic therapies do not improve AML patient outcomes. Using intravital microscopy, we found that AML progression leads to differential remodeling of vasculature in central and endosteal bone marrow regions. Endosteal AML cells produce pro-inflammatory and anti-angiogenic cytokines and gradually degrade endosteal endothelium, stromal cells, and osteoblastic cells, whereas central marrow remains vascularized and splenic vascular niches expand. Remodeled endosteal regions have reduced capacity to support non-leukemic HSCs, correlating with loss of normal hematopoiesis. Preserving endosteal endothelium with the small molecule deferoxamine or a genetic approach rescues HSCs loss, promotes chemotherapeutic efficacy, and enhances survival. These findings suggest that preventing degradation of the endosteal vasculature may improve current paradigms for treating AML., Graphical Abstract, Highlights • AML leads to progressive remodeling of endosteal stroma • HSC loss is spatiotemporally correlated with endosteal remodeling • In vivo imaging reveals transendothelial migration of healthy hematopoietic cells • Rescue of endosteal vessels preserves HSCs and enhances the efficacy of chemotherapy, Multi-modal microscopy of acute myeloid leukemia progression within the bone marrow reveals focal and progressive remodeling of endosteal blood vessels coupled to loss of osteoblasts, hematopoietic stem cells (HSCs), and HSC niches. Preserving endosteal vessels increases the number of surviving HSCs and improves the efficacy of chemotherapy.

In this paper, we examine the hypothesis that world oil market is “one great pool” by investigating the integration between China's and four major crude oil markets. Using a nonlinear correlation measure, we find that the price co-movement between China's and international oil prices is stronger in the long-term than in the short-term. Employing a threshold error correction model, we find that long-term equilibrium relationships display significant asymmetric effects and exist in a regime only. Moreover, international oil prices can drive China's oil prices to run towards long-term equilibrium level, but not vice versa. Finally, we also investigate volatility transmission using BEKK–GARCH models. Both in-sample and out-of-sample evidences indicate that only unidirectional volatility spillover running from benchmark markets to China's oil market can be found. Benchmark markets dominate China's oil market. Overall, our results do not support the “one great pool” hypothesis.

This paper demonstrates that inverse source reconstruction can be performed using a methodology of particle filters that relies primarily on the Bayesian approach of parameter estimation. In particular, the proposed approach is applied in the context of nearfield acoustic holography based on the equivalent source method (ESM). A state-space model is formulated in light of the ESM. The parameters to estimate are amplitudes and locations of the equivalent sources. The parameters constitute the state vector which follows a first-order Markov process with the transition matrix being the identity for every frequency-domain data frame. Filtered estimates of the state vector obtained are assigned weights adaptively. The implementation of recursive Bayesian filters involves a sequential Monte Carlo sampling procedure that treats the estimates as point masses with a discrete probability mass function (PMF) which evolves with iteration. The weight update equation governs the evolution of this PMF and depends primarily on the likelihood function and the prior distribution. It is evident from the simulation results that the inclusion of the appropriate prior distribution is crucial in the parameter estimation. (C) 2013 Acoustical Society of America.

Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in mouse embryos and are critical regulators of lymphoid lineage fate determination. However, it remains unclear how Notch regulates the balance between HSC self-renewal and differentiation in the adult bone marrow (BM). Here we report a novel mechanism that prevents HSCs from undergoing premature lymphoid differentiation in BM. Using a series of in vivo mouse models and functional HSC assays, we show that leukemia/lymphoma related factor (LRF) is necessary for HSC maintenance by functioning as an erythroid-specific repressor of Delta-like 4 (Dll4) expression. Lrf deletion in erythroblasts promoted up-regulation of Dll4 in erythroblasts, sensitizing HSCs to T-cell instructive signals in the BM. Our study reveals novel cross-talk between HSCs and erythroblasts, and sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and differentiation. (Blood. 2013;121(6):918-929)

Mouse mast cell protease 4 (mMCP-4), the mouse counterpart of human mast cell chymase, is thought to have proinflammatory effects in innate or adaptive immune responses associated with mast cell activation. However, human chymase can degrade the proinflammatory cytokine TNF, a mediator that can be produced by mast cells and many other cell types. We found that mMCP-4 can reduce levels of mouse mast cell-derived TNF in vitro through degradation of transmembrane and soluble TNF. We assessed the effects of interactions between mMCP-4 and TNF in vivo by analyzing the features of a classic model of polymicrobial sepsis, cecal ligation and puncture (CLP), in C57BL/6J-mMCP-4-deficient mice versus C57BL/6J wild-type mice, and in C57BL/6J-Kit(W-sh/W-sh) mice containing adoptively transferred mast cells that were either wild type or lacked mMCP-4, TNF, or both mediators. The mMCP-4-deficient mice exhibited increased levels of intraperitoneal TNF, higher numbers of peritoneal neutrophils, and increased acute kidney injury after CLP, and also had significantly higher mortality after this procedure. Our findings support the conclusion that mMCP-4 can enhance survival after CLP at least in part by limiting detrimental effects of TNF, and suggest that mast cell chymase may represent an important negative regulator of TNF in vivo.

α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis is controversial. We identified a subset of hematopoietic stem cells (HSCs) in the bone marrow (BM) that expressed β7 integrin. These β7(+) HSCs were capable of multilineage, long-term reconstitution and had an inherent competitive advantage over β7(-) HSCs. On the other hand, HSCs that lacked β7 integrin (β7KO) had reduced engraftment potential. Interestingly, quantitative RT-PCR and flow cytometry revealed that β7KO HSCs expressed lower levels of the chemokine receptor CXCR4. Accordingly, β7KO HSCs exhibited impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on BM endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with its endothelial ligand MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment.

The lineage restriction of prospectively isolated hematopoietic progenitors has been traditionally assessed by bulk in vitro culture and transplantation of large number of cells in vivo. These methods, however, cannot distinguish between homogenous multipotent or heterogeneous lineage-restricted populations. Using clonal assays of 1 or 5 cells in vitro, single-cell quantitative gene expression analyses, and transplantation of mice with low numbers of cells, we show that a common myeloid progenitor (CMP) is Sca-1(lo)lin(-)c-Kit(+)CD27(+)Flk-2(-) (SL-CMP; Sca-1(lo) CMP) and a granulocyte/macrophage progenitor (GMP) is Sca-1(lo)lin(-)c-Kit(+)CD27(+)Flk-2(+)CD150(-/lo) (SL-GMP; Sca-1(lo) GMP). We found that mast cell progenitor potential is present in the SL-CMP fraction, but not in the more differentiated SL-GMP population, and is more closely related to megakaryocyte/erythrocyte specification. Our data provide criteria for the prospective isolation of SL-CMP and SL-GMP and support the conclusion that mast cells are specified during hematopoiesis earlier than and independently from granulocytes.

We used mast cell-engrafted genetically mast cell-deficient C57BL/6-Kit(W-sh/W-sh) mice to investigate the roles of mast cells and mast cell-derived tumor necrosis factor in two models of severe bacterial infection. In these mice, we confirmed findings derived from studies of mast cell-deficient WBB6F(1)-Kit(W/W-v) mice indicating that mast cells can promote survival in cecal ligation and puncture (CLP) of moderate severity. However, we found that the beneficial role of mast cells in this setting can occur independently of mast cell-derived tumor necrosis factor. By contrast, using mast cell-engrafted C57BL/6-Kit(W-sh/W-sh) mice, we found that mast cell-derived tumor necrosis factor can increase mortality during severe CLP and can also enhance bacterial growth and hasten death after intraperitoneal inoculation of Salmonella typhimurium. In WBB6F(1)-Kit(W-sh/W-sh) mice, mast cells enhanced survival during moderately severe CLP but did not significantly change the survival observed in severe CLP. Our findings in three types of genetically mast cell-deficient mice thus support the hypothesis that, depending on the circumstances (including mouse strain background, the nature of the mutation resulting in a mast cell deficiency, and type and severity of infection), mast cells can have either no detectable effect or opposite effects on survival during bacterial infections, eg, promoting survival during moderately severe CLP associated with low mortality but, in C57BL/6-Kit(W-sh/W-sh) mice, increasing mortality during severe CLP or infection with S. typhimurium. Refereed/Peer-reviewed

Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.

Sepsis is a complex, incompletely understood and often fatal disorder,1 typically accompanied by hypotension,2 that is considered to represent a dysregulated host response to infection.3,4,5 Neurotensin (NT) is a 13-amino-acid peptide that, among its multiple effects, induces hypotension.6 We find that intraperitoneal and plasma concentrations of NT are increased in mice after severe caecal ligation and puncture (CLP), a model of sepsis, and that mice treated with a pharmacological antagonist of NT, or NT-deficient mice, exhibit reduced mortality during severe CLP. In mice, mast cells can degrade NT and reduce NT-induced hypotension and CLP-associated mortality, and optimal expression of these effects requires mast cell expression of neurotensin receptor 1 and neurolysin. These findings show that NT contributes to sepsis-related mortality in mice during severe CLP and that mast cells can lower NT concentrations, and suggest that mast cell-dependent reduction in NT levels contributes to the ability of mast cells to enhance survival after CLP.

RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (−/−) mast cells and examined FcϵRI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in −/− mast cells, including decreased basal FcϵRI expression, slowed FcϵRI internalization, elevated IgE + Ag–induced degranulation and IL-6 production, and the decreased ability of −/− cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface FcϵRI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen–activated −/− mast cells. By contrast, correction of these −/− phenotypes required a functional Vps9 domain. Thus, FcϵRI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.

Leukemia stem cells (LSCs) of chronic myeloid leukemia (CML) are refractory to tyrosine kinase inhibitor treatment, persist in the residual disease, and are important source for disease recurrence. Better understanding CML LSCs will help devise new strategies to eradicate these cells. The BALB/c mouse model of CML using retroviral bone marrow transduction and transplantation is a widely used mouse model system for CML, but LSCs in this model are poorly characterized. Here, we show that lineage negative CD150− side population (CD150−SP), but not CD150+SP, are CML LSCs in this model, although both CD150−SP and CD150+SP cells are enriched for long-term hematopoietic stem cells in normal BALB/c mice. We previously showed that BCR-ABL transformation activates protein lysine deacetylase SIRT1 and inhibition of SIRT1 sensitizes CML stem/progenitor cells to tyrosine kinase inhibitors by acetylating and activating p53. In this study, we demonstrate that SIRT1 homozygous knockout substantially reduces CD150−SP CML LSCs, and compromises the maintenance of CML LSCs in the BALB/c model. We identified several molecular alterations in CD150−SP LSCs that included the elevated expression of cyclin-dependent kinase Cdk6 facilitating LSC activation and significantly reduced p53 expression. SIRT1 knockout suppressed Cdk6 expression and likely increases p53 protein functions through deacetylation without increasing its expression. Our results shed novel insight into CML LSCs and support a crucial role of SIRT1 in CML LSCs. Our study also provides a novel means for assessing new agents to eradicate CML LSCs. Stem Cells 2015;33:3437–3451

Snake or honeybee envenomation can cause substantial morbidity and mortality, and it has been proposed that the activation of mast cells by snake or insect venoms can contribute to these effects. We show, in contrast, that mast cells can significantly reduce snake-venom–induced pathology in mice, at least in part by releasing carboxypeptidase A and possibly other proteases, which can degrade venom components. Mast cells also significantly reduced the morbidity and mortality induced by honeybee venom. These findings identify a new biological function for mast cells in enhancing resistance to the morbidity and mortality induced by animal venoms.

T cells are produced in the thymus from progenitors of extrathymic origin. As no specific markers are available, the developmental pathway of progenitors preceding thymic colonization remains unclear. Here we show that progenitors in murine fetal liver and blood, which are capable of giving rise to T cells, NK cells and dendritic cells, but not B cells, can be isolated by their surface expression of paired immunoglobulin-like receptors (PIR). PIR expression is maintained until the earliest intrathymic stage, then downregulated before the onset of CD25 expression. Unlike intrathymic progenitors, generation of prethymic PIR+ progenitors does not require Hes1-mediated Notch signaling. These findings disclose a prethymic stage of T-cell development programmed for immigration of the thymus, which is genetically separable from intrathymic stages.

Five Fc receptor homologs (FcRH1-5) possessing inhibitory and/or activating signaling motifs are differentially expressed during B cell differentiation in humans. In this analysis we describe their three mouse orthologs, moFcRH1, moFcRH2 and moFcRH3. The moFcRH genes are located in a chromosome 3 region that is syntenic with the FcRH locus on human chromosome 1. They encode proteins with 2-5 Ig-like domains that share 20-61% extracellular identity with their human counterparts. One moFcRH1 isoform lacks a transmembrane domain as do both moFcRH2 isoforms. The other moFcRH1 isoform and two moFcRH3 isoforms have transmembrane domains and cytoplasmic ITIM and ITAM-like consensus sequences implying their inhibitory or activating signaling potential. Whereas the moFcRH1 and moFcRH3 orthologs are preferentially expressed at different stages in B cell differentiation, the structurally novel moFcRH2 gene is expressed in non-lymphoid tissues. The highly restricted pattern of moFcRH3 expression suggests this member of the phylogenetically conserved FcRH family may have an important immunoregulatory role in marginal zone B cells.

ADDITIONAL INDEX WORDS. Carica papaya, phloem unloading, fruit sugar uptake, sucrose synthetase, fruit development ABSTRACT. An invertase gene was isolated and its mRNA activity and protein levels were determined during papaya (Carica papaya L.) fruit development. A complete invertase cDNA (AF420223) and a partial sucrose synthase cDNA (AF420224) were isolated from papaya fruit cDNA libraries. The invertase cDNA encoded a predicted polypeptide of 582 residues (MW 65,537 Da), and was 68% and 45% identical with carrot apoplastic and vacuolar invertases, respectively. Key amino acids indicative of an apoplastic invertase were conserved. A full-length gene corresponding to the putative apoplastic invertase cDNA was isolated and was organized into seven exons and six introns. Exon 2 (9 bp long) encoded part of a highly conserved region (NDPNG/A). Invertase mRNA and activity levels increased during fruit maturation and sugar accumulation just before ripening. In contrast, sucrose synthase mRNA levels were high during early fruit growth and low during the fruit sugar accumulation stage. A 73-kDa cell wall extractable protein that cross-reacted with carrot apoplastic invertase antisera substantially increased during papaya fruit maturation and declined in full ripe fruit. The increase in invertase protein levels occurred 2 to 4 weeks before maturity and was markedly higher than the overall increase in enzyme activity at this stage. Subsequently, the increase in enzyme activity was higher than the increase in protein levels between 2 weeks before maturity and fully ripe. The results suggested that mRNA level and invertase activity were related to maturity. The data suggested that the invertase was apoplastic, and that post-translational control of enzyme activity occurred, in which a signifi cant accumulation of invertase occurred before the peak of enzymes activity. Papaya (Carica papaya) sweetness is a major quality factor with a minimum 11.5% soluble solids being required for Hawaiiʼs fruit (Paull et al., 1997). Sugar accumulation occurs during the last phase of fruit development (Chan et al., 1979; Selvaraj et al., 1982). The activity of a NaCl-soluble acid invertase (Zhou and Paull, 2001) parallels sugar accumulation during the last phase of fruit maturation while sucrose synthase (SS) activity is associated with early fruit growth. Fruit removal and defoliation altered both sugar level and invertase activity in papaya fruit (Zhou et al., 2000), suggesting that invertase was involved in phloem unloading during sugar accumulation. Invertases enable the transport of sucrose into the apoplast and vacuole by plasma membrane and tonoplast hexose transporters. Numerous forms of invertases (β-fructosidase; EC 3.2.1.26) are characterized by solubility, pH optima, iso-electric points, and