ALBERT

Description: Differences in soil P among silvopasture, grassland, and arable lands have been well established. Nevertheless, most of the reports compare soil properties under long-term sites. Thus, there exists little information on the effect of the conversion of silvopasture to arable or grassland use on soil P pools. The objective of the study was to determine the impact of converting silvopasture system (SP) into arable cropping and grassland system on the distribution of P pools and potential P bioavailability. We compared the following systems: SP system, SP converted to arable cropland (SP-AL), SP converted to grassland (SP-GL), and for comparative purposes, a long-term arable cropland (AL). The P fractionation was performed by a sequential extraction scheme, using acid and alkaline extractants on samples collected from the 0–10 and 10–20 cm soil layers. It was assumed that the large variations in soil-P fractionations are caused by the different management practices associated with land conversion. The results of P fractionation showed a dominance of calcium-bound P, HCl-extractable Pi constituted up to 36% of the soil total P (TP). However, the type of land use did not affect this P fraction. On the other hand, the reduction in labile-P i and NaOH-P i fractions observed at the SP-AL site may have led to the decline in readily available P. The soil total organic P (TP o ) content was 8% and 17% lower at SP-AL compared to SP and SP-GL site, respectively. Labile organic-P (labile-P o ) content was markedly higher at SP site compared to arable soils, and was ≈ 10% of TP o . The NaOH-P o constituted the highest fraction of the organic-P pool (55%–79% of TP o ) across all the study systems, and was positively correlated with TP o ( p 〈 0.01). The study indicates that conversion of SP system in temperate regions to arable cropping with conventional tillage seems to result in the reduction of P availability compared to SP, indicating SP as an important land-use practice.

Description: Fluidized beds have been commercially utilized in gas-solid contacting processes such as FCC process. Attempts have been made for bubbling fluidized beds to enhance gas exchange between the bubble phase and the emulsion phase as well as to increase bubble hold up. In this study, low-density superfine particles 5070S was selected whose average diameter is about 10 μm. Unlike agglomerate particulate fluidization of superfine particles on which other researchers have reported, the smaller bubbles whose diameter is less than 10 mm uniformly dispersed in the whole bed. Additionally, the gas exchange rate with 5070S became larger than FCC particle. We concluded that such a unique gas-solid behavior is probably due to the proper density difference between particle aggregate and fluidizing gas.

Description: Various kinds of functional cells differentiated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have recently been developed and expected for use in human regenerative medicine. However, the safety and efficacy of ESC/iPSC-based therapies must be carefully evaluated prior to clinical application, by using reliable animal models. The common marmoset (CM, Callithrix jacchus) is known to be a suitable preclinical model for clinical translation studies, and CM ESCs have already been established by us. Hematopoietic stem/progenitor cells (HSCs/HPCs) are one of very useful cells for transplantation therapy to treat various diseases including leukemia. However the shortage of their donors becomes a huge social problem and the expansion of HSCs/HPCs in vitro is known to be very difficult. We have previously demonstrated that CM ESCs showing indefinite self-renewal can be differentiated into hematopoietic lineages by the forced expression of hematopoietic transcription factor (TAL1/SCL). However the efficiency of their hematopoietic differentiation was quite low (less than 5%). Therefore the development of new method to promote hematopoietic differentiation of CM ESCs more efficiently is needed. To promote hematopoietic differentiation of CM ESCs, we focused on self-renewal pathway of CM ESCs and oxygen levels during EB formation. We have reported that self-renewal of CM ESCs is regulated by phosphoinositide 3-kinases (PI3Ks)-protein kinase B (AKT) pathway that is known to regulate cell cycle and cell proliferation as well as cell survival (Nii et al., 2014). On the other hand, the differentiation of mouse ESCs to hematopoietic precursors such as hemangioblasts, bipotential progenitors of endothelial and hematopoietic cells, can be enhanced by hypoxic condition (Ramírez-Bergeron et al., 2004). In addition, expansion of HSCs/HPCs can be increased by hypoxic condition in vitro (Danet et al., 2003). Thus, we hypothesized that the suppression of ESC self-renewal by the inhibition of PI3K-AKT pathway under hypoxic condition would improve hematopoietic differentiation of CM ESCs. To test our hypothesis that the inhibition of self-renewal pathway of CM ESCs could promote their hematopoietic differentiation, we treated CM ESCs with PI3K inhibitor (LY: LY294002) for the first 4 days of EB formation and examined the proportion of CD34+ cells by flow cytometric analysis, and found that the populations of CD34+ cells were significantly increased in the presence of LY. Moreover, the day8-EBs treated with LY gave rise to significantly more hematopoietic colonies than controls in colony forming unit (CFU) assay. These results indicated that hematopoietic differentiation was significantly enhanced by the inhibition of PI3K-AKT pathway in the process of EB formation. To further promote hematopoietic differentiation of CM ESCs, we conducted EB formation assay of CM ESCs and induced their differentiation into HPCs under hypoxic condition. We found that the hypoxic condition (5% O2) significantly increased the proportion of both CD34+ and CD34+/CD117+ cells in day8-EBs especially when PI3K-AKT pathway was inhibited by the LY treatment. These results were also obtained from human ESCs. In the present study, we demonstrated that transient treatment of PI3K inhibitor during EB formation under hypoxia condition promoted hematopoietic differentiation of human and CM ESCs, which might contribute to the development of the valuable experimental system using CM ESCs in order to test new strategies of human regenerative medicine. Disclosures No relevant conflicts of interest to declare.

Description: Introduction There is still a need for a general test easily implementable and widely available that may be used to screen all patients on DOACs. A recent study has suggested that the dilute Russell Viper Venom Time (DRVV-T) could be used for the monitoring of DOACs but these results have been generated in vitro. The primary objective of this study is to analyse and compare the results obtained with the DRVV Screen and Confirm tests to the plasma drug levels measured by LC-MS/MS. We also aimed at proposing specific cut-off associated with supratherapeutic levels at Ctrough. Finally, a comparison of our results with those obtained with PT for rivaroxaban and aPTT for dabigatran is also provided. Methods Thirty-two rivaroxaban and 31 dabigatran platelet poor plasma samples from real-life patients were included in the study. Dilute Russell's Viper Venom time was measured using STA®-Staclot®DRVV Screen and Confirm reagents. Prothrombin Time and aPTT have been performed with Triniclot PT Excel S® and RecombiPlastin 2G® and with STA®C.K. Prest and SynthasIL®, respectively. The Hemoclot Thrombin Inhibitor® and the Biophen DiXaI® have been performed to estimate plasma concentration of dabigatran and rivaroxaban, respectively. All methodologies were performed on a STA-R Evolution® analyser according with the recommendation of the manufacturer, except for RecombiPlastin 2G® which were performed on an ACL-TOP®. All of these tests have been performed according to the recommendations of the manufacturer. The reference LC-MS/MS measurement of plasma drug concentrations were validated according to FDA Guidelines for Industry for Bioanalytical Method (for rivaroxaban) and to the Validation European Medicines Agency guidelines (for dabigatran). Results The plasma concentrations range from 6 to 426ng/mL for rivaroxaban and from 0 to 386ng/mL for dabigatran as determined by LC-MS/MS. Tables 1 and 2 summarize Spearman correlations and Bland-Atlman analyses for rivaroxaban and dabigatran, respectively. Figures 1 and 2 provide the results of STA®-Staclot®DRVV Screen and Confirm versus LC-MS/MS measurements. Bland-Altman graphs are also provided. Discussion STA®-Staclot®DRVV-Screen and Confirm shows a better correlation than PT or aPTT. Bland-Altman analyses reveal an overestimation of approximately 40ng/mL and large 5th-95th limits of agreement with both STA®-Staclot®DRVV-Screen and Confirm. Specific cut-offs associated with supratherapeutic level (〉200ng/mL) at Ctrough have been defined. For STA®-Staclot®DRVV-Screen, results below 125 seconds or below a ratio of 3 could exclude plasma concentrations 〉200ng/mL for rivaroxaban and dabigatran (Figure 1 - A and B). For STA®-Staclot®DRVV-Confirm, cut-offs must be adapted independently (Figure 2 - A and B). Results below 75 seconds or below a ratio of 2, could exclude rivaroxaban plasma concentrations 〉200ng/mL. For dabigatran, the threshold could be defined at 90 seconds or at a ratio of 2.5. Conclusion Thanks to its good correlation with plasma drug level, DRVVT can be more informative than PT and aPTT, to exclude supra-therapeutic level of rivaroxaban and dabigatran at Ctrough. However, due to overestimations in plasma drug level, it cannot be recommended to accurately estimate plasma drug concentrations which require more specific coagulation assays or LC-MS/MS measurements. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2311 The human regenerative medicine by the transplantation of the functional cells differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) have great potential of contributing to the treatments for various diseases, and thus have attracted huge public attention. However, the risk of unwelcome tumor formation originated from transplanted cells in recipients remains to be solved. Therefore the safety and efficacy of ESC/iPSC-based therapies should be carefully evaluated using reliable animal disease models before their clinical application. Among experimental animal models, common marmoset (CM, Callithrix jacchus), one of NEW WORLD monkeys, has recently been recognized as a useful non-human primate because they are small, easy to handle, highly reproductive and genetically very similar to humans. We have continuously investigated the characteristics of ESCs and iPSCs derived from CM. Understanding the self-renewal pathways in ESCs/iPSCs is crucial for the development of improved technology to culture and differentiate them into functional cells of potential therapeutic use. It has been reported that the maintenance of self-renewal in human or mouse ESCs/iPSCs require basic fibroblast growth factor (bFGF) or leukemia Inhibitory factor (LIF) respectively, however the growth factors required for the culture of CM ESCs/iPSCs have not been clearly determined. To clarify whether LIF or bFGF is more appropriate to maintain self-renewal of CM ESCs in culture, we examined the proliferation rate of CM40, a CM ESC line, maintained in the presence or absence of LIF or bFGF. CM ESCs were passaged at a ratio of 1:3 every 3 to 4 days. We found that the number of OCT3/4+cells was significantly increased by the addition of bFGF but not of LIF compared to control (w/o cytokines). Similar results were obtained when Cj11, another CM ESC line, was used. These results indicate that bFGF is essential for culturing CM ESCs, but LIF is dispensable. It has been reported that bFGF and its downstream PI3K-AKT and MEK-ERK pathways are important for maintenance of ESCs in human. Thus we examined whether PI3K-AKT and MEK-ERK pathway play crucial roles in the maintenance of self-renewal in CM ESCs. CM40 was cultured in the medium containing bFGF in the presence of PI3K inhibitor (LY294002) or MEK inhibitor (PD0325901). We found that the percentage and number of OCT3/4+ cells were gradually decreased in the presence of LY294002 (10 μM or 20 μM), suggesting that PI3K-AKT pathway is essential for the self-renewal of CM ESCs. Furthermore, the percentage and number of OCT3/4+cells were gradually decreased by addition of PD0325901 (1 μM or 5 μM) in the course of 4 passages, indicating that MEK-ERK pathway also plays a role in the self-renewal of CM ESCs. Next we examined if inhibition of self-renewal pathway such as PI3K-AKT or MEK-ERK promote hematopoietic differentiation in CM ESCs. One of methods for inducing hematopoietic cells from ESCs is embryoid body (EB) formation which is a conventional technique frequently used for in vitro differentiation of ESCs. Thus to induce hematopoietic differentiation, we performed EB formation assay by plating single-cell suspension of CM ESCs (3 × 105 cells) in StemLine II supplemented with 50 ng/ml BMP4 and 50 ng/ml VEGF with or without 10 μM LY294002 or 5 μM PD0325901 for 2 days. Then we removed half the medium and added fresh medium with the same final concentrations of BMP4, VEGF, LY294002 and PD0325901, plus 25 ng/ml SCF, 25 ng/ml TPO and 25 ng/ml FLT3L to expand the hematopoietic progenitors. We found that addition of LY294002 or PD0325901 increased the population of cells positive for CD34, a marker for hematopoietic stem/progenitor and endothelial cells, in day4-EBs. These CD34+cells showed hematopoietic differentiation potential proved by colony forming unit (CFU) assay Taken together, inhibition of self-renewal pathway such as PI3K-AKT or MEK-ERK in CM ESCs is thought to promote their hematopoietic differentiation by EB formation. Our findings might be useful to develop a better technology of the culture and hematopoietic differentiation of CM ESCs as well as to test efficacy and safety of ESC-derived hematopoietic cells using CM disease models for the future ESC/iPSC-based human regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

Description: Because blood cells can be obtained with relatively easy and safe procedure, they have been routinely used for transfusion and transplantation purposes. And they are now considered as attractive cell sources for developing new gene therapies including cancer therapy using various immune cells, and regenerative therapy using hematopoietic stem cells or induced pluripotent stem cells (iPS) cells. For example, chimeric antigen receptor modified autologous T cells have been considered as effective therapy for various cancers. And iPS cells have been easily established from peripheral T cells for the purpose of treating various diseases. However, in spite of these possibilities, the development of the safer and more efficient genetic modification methods of hematopoietic cells is imminent. In this study, we developed the novel measles viral (MV) vector which enables us to transduce multiple genes into immune cells. The wild type measles virus is one of the aerosol-transmitted viruses and has strong infectious capacity to immune cells, and epithelial cells via signaling lymphocyte activation molecule (SLAM) or nectin-4. First, we modified the wild type measles virus genome to non-transmissible and non-lytic, and equipped with the ability of transducing multiple genes, at most six genes, into target cells. Briefly, the intrinsically non-segmented wild type virus genome was divided into two segments and point mutations were introduced into the virus genes encoding hemagglutinin and the matrix protein. Moreover, as the fusion protein gene was removed from the virus genome, the virus could not replicate in neighbor cells. We examined the gene transduction efficiency of the gene modified measles virus (H8-Fd-MV vector) into hematopoietic cells. We first constructed the H8-Fd-MV vector with GFP gene and transduced into hematopoietic progenitor cells and immune cells from human cord blood and peripheral blood. We observed that almost all of HPCs from cord blood (99.7% in floating cells expressed CD34), T cells (99.9% in CD3+ cells), and B cells (98.2% in CD19+ cells) from peripheral blood expressed GFP at two days after the transduction. Especially, to express GFP gene in human peripheral T cells, it was not necessary to pre-stimulate them with CD3/CD28 beads (99.6% in stimulating T cells (72.9% in SLAM+ cells) v.s. 82.6 % in non-stimulating cells (37.4% in SLAM+ cells)). T cells from cord blood showed almost all naïve phenotype (CD4+ cells: 93.2±1.8% in CD45RA+CCR7+ cells, 1.7±1.1% in CD45RA+CCR7- cells; CD8+ cells: 41.6±5.7% in CD45RA+CCR7+ cells, and 41.9±11.0% in CD8+CD45RA+CCR7- cells) and T cells transduced by MV vector expressed GFP more (CD4+ cells: 80.3±13.7%, and CD8+ cells: 82.5±8.5%)　than those transduced by Sendai viral vector (CD4+ cells: 15.5±0.7%, and CD8+ cells: 17.4±5.4%). These data suggested that H8-Fd-MV vector could transduce GFP gene efficiently into various T cell lineages including naïve T cells, which had been difficult to be transduced with classical gene transduction methods. We next generated H8-Fd-MV vector for expressing 6 genes (OCT4, SOX2, KLF4, L-MYC, PIN1, and GFP) and transduced into stimulated T cells. After 3 days, GFP+ T cells expressed all of these 6 genes. We also detected that more than 50% of the stimulated T cells with IL-2 expressed GFP at 14 days after the transduction. After 27 days from transfection, embryonic stem cell (ES cell)-like colonies were picked up and analyzed the character. These cells showed ES cell morphology over 20 times passages and expressed pluripotent marker (NANOG, OCT4, Tra-1-60, Tra-1-81). We also found T cell receptor rearrangements in these cells. Embryoid bodies from these cells expressed three germ line markers in vitro. We next examined the hematopoietic differentiation of these cells using coculture system with murine embryonic aorta-gonad-mesonephros region-derived stromal cell line (AGM-3 cells). The co-cultured cells harvested at day 12 expressed CD34 and CD45. These data suggested that we established iPS cells from terminal differentiated T cells using H8-Fd-MV vector for expressing reprogramming factor. These results indicated that multiple genes were expressed efficiently in immune cells using H8-Fd-MV vector. Highly efficient transduction ability of MV vector for T cells would enable us to develop new gene therapy targeting cancer using gene modified T cells as well as organ regeneration using iPS cells. Disclosures No relevant conflicts of interest to declare.

Description: Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry may serve as a tool to purify zebrafish neutrophils. Disclosures No relevant conflicts of interest to declare.

Description: Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FAgenes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA− cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.