Semisolid Culture Research Articles

Comparison of different androgenesis protocols for doubled haploid plant production in ornamental pepper (Capsicum annuum L.)

Doubled haploid (DH) plant production plays an important role in pepper (Capsicum annuum L.) breeding for development of new varieties. However, information is lacking for DH plant production in pepper genotypes carrying ornamental value. The major goal of this study was to produce DH ornamental pepper lines having the mentioned value. Androgenic responses of 48 genotypes at the F2 or F3 generation were compared in each of three androgenesis protocols to determine the most effective method. Of the three protocols tested, anthers were placed on two different semisolid culture media and on a double-layer medium also called shed-microspore culture medium. The results revealed that the shed-microspore culture protocol was superior to both semisolid anther culture protocols. The average numbers of total and normal-looking embryos per bud of the most responsive genotype were 102.90 and 34.11, respectively. We report here, a total of 122 regenerated ornamental pepper plants with ornamental value; 63 DH, 52 haploid, and 7 mixoploid plants were produced in the present study. The spontaneous diploidization rate was 51.6% based on flow cytometry analyses. The results showed that the shed-microspore culture protocol could be used effectively for the development of DH lines in ornamental pepper breeding programs.

Aerobic bacteria and fungi from skin lesions of fish in Khartoum state

Objective: This cross-sectional study was conducted from April to July 2014 in Khartoum state, the Sudan, to investigate aerobic bacteria and fungi of skin lesions of fish in 3 different areas in Khartoum. Material and methods: A total of 50 samples were collected from the skin lesions of different types of fish including Synodontis species (n=17), Tilapia niloticus (n=15), Labeo niloticus (n=10), Hydrocynus species (n=4), and Clarias species (n=4). Liquid, semi-solid, and solid culture media like nutrient broth, blood agar, MacConkey agar, sabouraud dextrose agar (SDA), and Simmon's citrate medium were used for the isolation and identification of bacteria and fungi. Besides, Gram staining and biochemical characterization were also conducted. Results: Culturing of the collected samples revealed growth of bacteria from all (100%), and growth of fungi could be found from 32% samples. A number of 188 bacteria were isolated, mainly Staphylococcus species, Bacillus species, Aeromonas species, Pseudomonas species, and Vibrio species. Besides, 16 fungi could be identified containing Aspergillus niger , A. flavus , A. fumigatus , and Phycomycete. Conclusion: Fishes with skin lesions are harboring many pathogenic bacteria and fungi and may act as a source of zoonotic infections and can transmit several pathogens to workers in fish industry and consumers. Therefore, thorough and strict routine inspection of fish is recommended to ensure safety and that there are no serious risks to consumers. http://doi.org/10.5455/javar.2016.c176

Phenolic Compositions of Litchi Shoot Tips and Zygotic Embryos Collected in Different Months and Their Effects on the Explant Browning and Its Control

In the research it was examined the changes in total phenolic contents and seven major phenolic compounds (gallic acids, +(–) catechin, catechol, Chlorogenic acid, o-coumaric acid, rutin and quercetin) of two litchi cultivars (Purbi and Bedana) shoot tips and fruits (for zygotic embryos) collected in different months, in order to determine their effects on the explants browning during establishment stage of shoot tip culture. The concentrations of phenolic compounds varied depending on the cultivars and the months. Phenolic compounds showed various correlation coefficients with the explants browning. Total phenolic content and some individual phenolic compounds including +(–) catechin, catechol, gallic acid, chlorogenic acid and rutin quantified in this study showed significant positive correlations with the explants browning, while o-coumaric acid and quercetin did not exhibit any significant one. According to our results, explants browning are affected by the phenolic compounds at different ranges. In both litchi cultivars, shoot tips and fruits (for zygotic embryos) collected in March exhibited the lowest explants browning during the establishment stage as compared to those collected in the other months. So it may be possible to increase the success of shoot tip and zygotic embryo culture with the selection of the most suitable terms of explants collection. Browning of explants could be controlled by the use of antioxidants both in semi-solid and liquid culture.

Effect of phloroglucinol on rooting and in vitro acclimatization of papaya (Carica papaya L. var. Maradol Roja)

The development of somatic embryogenesis in papaya (Carica papaya L.) on semisolid culture medium provides an alternative for propagation and breeding of this species. However, a critical factor in the protocol is the low survival of plants in ex vitro conditions. The aim of this study was to determine the effect of the product phloroglucinol on rooting and in vitro acclimatization of somatic embryo-derived papaya shoots using sterile zeolite. In vitro shoots were placed in culture vessels with phloroglucinol at 0, 79, 118.5, or 158 μM with 9.8 μM of indole-3-butyric acid and zeolite as a support on half-strength Murashige and Skoog semi-solid medium. Different morphological and physiological variables were evaluated after 27 d of culture. In the treatments with phloroglucinol, very good rooting development in shoots from somatic embryogenesis was achieved. The phloroglucinol stimulated the root elongation and formation of new roots with respect to the agar control. The best results for in vitro shoots were obtained with 79 μM phloroglucinol plus 9.8-μM indole-3-butyric acid, where 100% rooting, with 2.40-cm root length, 1.76 root number and the highest values of photosynthesis (6.497 μmol CO2 m−2 s−1) was achieved in 27 d of culture compared to the control. This facilitated a better survival rate state of plants (96.5%) in ex vitro acclimatization conditions.

Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations

Development of FF-10101, a Novel Irreversible FLT3 Inhibitor, Which Overcomes Drug Resistance Mutations

Micropropagation of breadfruit (A. altilis) enhanced using a bioreactor system

Breadfruit (Artocarpus altilis) is an important starchy food crop with much potential to support livelihoods in the Pacific region. The region has the greatest diversity of breadfruit, with three known species and hybrids. Seeded varieties are largely found in Melanesia and Micronesia, while seedless varieties are found in Polynesia. The seedless varieties are propagated using marcotts, root suckers and root cuttings, and the seeded varieties use seeds. To support increased demands for food for the growing population and to supply market demands, improved methods of propagation are needed. The use of conventional in vitro technology, combined with a temporary immersion bioreactor system, reduced the field-planting readiness time from 44 to 30 weeks. Plantlets produced using the bioreactor were more vigorous, sturdier and taller than those cultured in the conventional semi-solid static tissue culture system. Breadfruit seedlings generated via the bioreactor system are undergoing field evaluation in three breadfruit orchards in Nadi, Fiji, alongside marcotts and root suckers. The analysis is based on the best source of planting material in terms of stem girth, plant height, early fruiting, fruiting patterns, and nutrition and production aspects of varieties. Bioreactor-generated plantlets have also been distributed to Pacific Island countries for evaluation. The research by the Centre for Pacific Crops and Trees (CePaCT) of the Secretariat of the Pacific Community (SPC) based in Suva, Fiji is part of the Pacific breadfruit project funded by the Australian Centre for International Agricultural Research (ACIAR).

Effect of Decitabine on Megakaryocyte Culture of Steroid-resistant ITP Patients

To investigate the effect of decitabine and plasma of ITP patients on in vitro cultrue of megakaryocytes in bone marrow of steroid-resistant ITP patients. Bone marrow mononuclear cells were isolated from 20 steroid-resistant ITP patients, both methyl cellulose semisolid culture system (to observe and count the number of megakaryocytes colony-forming unit) and liquid culture system (to analysis the expression rate of CD41a(+) cells) were used for megakaryocyte cultrue. The experiments were divided into 4 groups according to the different components of the culture system, group A was control, group B was added with decitabine, group C with ITP plasma, group D with both decitabine and ITP plasma, and the rest of the culture components were the same in the 4 groups except the above-mentioned materials. Morphology of megakaryocytes was observed by inverted and light microscopy. The expression rate of CD41a⁺ cells in culture was analysed by flow cytometric. Different concentration of decitabine showed different effect on megakaryocyte growth of steroid-resistant ITP patients and the optimal concentration to differentiate into megakaryocyte for bone marrow mononuclear cells is 3.0 µmol/L. Compared with group A, both megakaryocyte colony forming units (CFU) and expression rate of CD41a⁺ cells in group B were statistically significantly higher (P < 0.05). As compared with group A, the megakaryocyte colony-forming units in group C decreased with statistically significant difference, while compared with group C, the megakaryocyte colony-forming units in group D obviously increased with statistically significant difference. Decitabine is able to induce bone marrow mononuclear cells of steroid-resistant ITP patients to differentiate into megakaryocyte and the optimal concentration is 3.0 µmol/L; ITP plasma is able to inhibit the megakaryocyte growth of steroid-resistant ITP patients.

Efficient microtuber production of potato in modified nutrient spray bioreactor system

A laboratory scale bioreactor system has been developed using nutrient spray technology for in vitro mass production of potato microtubers. Its effectiveness on the production of microtubers was investigated and compared with conventional liquid and semi-solid culture systems through bioreactor. Optimal culture conditions such as spray intervals, varying concentrations of 6-benzylaminopurine (BAP) and explants density were determined for the NSB. In order to determine optimal spray intervals, liquid medium was sprayed inside the NSB at different intervals (½, 1–4h) of which the 1h interval resulted in the highest number of shoots (3.47) and length (8.99cm). Number of microtubers produced (5.13) was highest with 1h intervals and fresh weight of microtubers (0.90g) was highest for ½h interval. Different concentrations of BAP (0.5, 1.0 and 1.5mg/L) were used to evaluate its effect on microtuberization. It was observed that number and diameter of microtubers were increased (5.31 and 0.96cm) when 0.5mg/L BAP was supplemented in MS medium. We found fresh weight of microtubers (0.97g) was increased when 1.0mg/L BAP were added to the medium. In order to determine suitable explants density, single nodes grouped into five categories e.g 30, 45, 60, 75, and 90 and placed in the NSB system. A density of 60 explants resulted in increases in shoot length (17.5cm) number of internodes (12.5) and with highest amount of chlorophyll (40.2mg/g) as well as with highest number and fresh weight of microtubers (4.43 and 0.89g, respectively). Out of the three culture systems, the NSB performed best where 1.5–2.0 fold increases in shoot growth and microtuberization without hyperhydration. The NSB also produced the highest number (4.67), fresh weight (0.86g) and diameter (0.78cm) of microtubers. From this study we may conclude that the NSB system has good potential for commercial mass production of potato micro-tuber.

Real-time Recording of Cytosolic Calcium Levels in Arabidopsis thaliana Cell Cultures during Parabolic Flights

In plants, like in other organisms, calcium (Ca2+) is an important second messenger which participates in the conversion of environmental signals into molecular responses. There is increasing evidence, that sensing of changes in gravitation or reorientation of tissues is an example for such signaling cascades in which Ca2+ is involved. In order to determine g-dependent changes in the cytosolic calcium (Ca $^{2+}_{\text {cyt}})$ concentration of plant cells, semisolid transgenic callus cell cultures of Arabidopsis thaliana (A.t.), expressing the calcium sensor YC3.6 (cameleon), were exposed to g-forces between 1.8g and μg during parabolic flights. Using such cells, intracellular calcium transients can be monitored by FRET in vivo and in real-time. Interestingly we observed a slight decrease of the Ca $^{2+}_{\text {cyt}}$ level during the hypergravity phases of a parabola but a significant increase of the Ca $^{2+}_{\text {cyt}}$ concentration during microgravity. Application of known Ca2+ inhibitors and antagonists yielded the following effects: nifedipine (Ca2+ channel blocker) showed no effect, whereas LaCl3, GdCl3 (both inhibitors of uptake at the plasma membrane), DPI (inhibitor of NADP oxidase), and DMSO (solvent) diminished the gravity-alteration-related Ca $^{2+}_{\text {cyt}}$ response. EGTA (binding of Ca2+) and eosin yellow (inhibitor of a plasma membrane-located Ca2+ pump) suppressed the respective Ca $^{2+}_{\text {cyt}}$ changes entirely. We thus conclude that the significant increase in Ca $^{2+}_{\text {cyt}}$ under microgravity is largely due to extracellular Ca2+ sources.

Antibacterial activity of endophytic fungi from leaves of Indigofera suffruticosa Miller (Fabaceae).

Endophytic fungi were isolated from healthy leaves of Indigofera suffruticosa Miller, a medicinal plant found in Brazil which is used in folk medicine to treat various diseases. Among 65 endophytic fungi isolated, 18 fungi showed activity against at least one tested microorganism in preliminary screening, and the best results were obtained with Nigrospora sphaerica (URM-6060) and Pestalotiopsis maculans (URM-6061). After fermentation in liquid media and in semisolid media, only N. sphaerica demonstrated antibacterial activity (in Potato Dextrose Broth-PDB and in semisolid rice culture medium). In the next step, a methanolic extract from rice culture medium (NsME) and an ethyl acetate extract (NsEAE) from the supernatant of PDB were prepared and both exhibited antimicrobial activity against Gram-negative and Gram-positive bacteria. The best result was observed against Staphylococcus aureus, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 1.56 mg/mL and 6.25 mg/mL, respectively, for NsME and MIC and MBC values of 0.39 mg/mL and 3.12 mg/mL, respectively, for NsEAE. This study is the first report about the antimicrobial activity of endophytic fungi residing in I. suffruticosa leaves, in which the fungus N. sphaerica demonstrated the ability to produce bioactive agents with pharmaceutical potential, and may provide a new lead in the pursuit of new biological sources of drug candidates.

248. A Bona Fide Mammalian Replicator Enhances All Aspects of Episomal Gene Transfer Into Human Hematopoietic Progenitor Cells

Episomal gene transfer for gene therapy applications is limited at present mainly because of luck of long term persistence of episomes in the nucleus of cells. However, episomal vectors may offer a valid alternative to viral vectors as well as to gene editing strategies. To expand the potential of episomal vectors, we used the Initiation of Replication region, (IR), from β-globin gene locus, which is capable to promote DNA replication ectopically (Wang L et al 2004) and can rescue DNA replication within the context of Scaffold /Matrix Attachment Region (S/MAR) based episomal vector (Giannakoppulos A. et al 2009). We constructed two episomal vectors, derived from the protopype S/MAR based episomal vector pEPI-eGFP (6.7Kb), namely, pEP-IR and its control pEPI-SFFV, by replacing CMV promoter of eGFP by the Spleen Focus forming Virus (SFFV) promoter, while vector pEP-IR carries the IR element just upstream the SFFV promoter. Stress Induced Duplex Destabilization (SIDD) analysis of the novel plasmids indicated that these changes would not disrupt their function as episomal vectors and could be beneficial. All three vectors (pEP-IR and control vectors pEPI-SFFV and pEPI-eGFP) showed excellent episomal properties upon transfer into K562 cells. Additionally, they showed transfection efficiency around 30% upon transfer into CD34+ cells from peripheral blood, from adult, mobilised, healthy donors. CD34+/eGFP+ cells sorted by FACS were placed in semisolid cultures and produced differentiated cell-colonies within 14 days. Non-fluorescent colonies do not contain plasmids, as documented previously too (Papapetrou E. et al 2006). Vector pEP-IR shows an enhancement of all aspects of vector performance: 100% fluorescent colonies against (0%) for pEPI-eGFP as found previously (Papapetrou E. et al 2006) and 56% for pEPI-SFFV, in Fluorescent microscopy; increase of plasmid copy number per fluorescent cell, which was 1.78 for pEP-IR against 1.12 for pEPI-SFFV, as estimated by Real Time PCR. The ratio of the number of fluorescent, stably transfected colonies to the number of transfected, seeded cells presenting the rate of establishment in semisolid cultures of transfected CD34+ cells, was 5.5% for pEPI-SFFV and 15% for pEP-IR. It is thus determined that the relative capacity for establishment for pEP-IR is 3-fold higher than that of control vector pEPI-SFFV. Finally, estimation of eGFP mRNA showed that pEP-IR supports 3 times higher eGFP expression per plasmid copy, compared to pEPI-SFFV. Vector pEPI-IR in a series of over five transfections of CD34+ cells, routinely produced fluorescent cells in the area of 99.8%. Practically, vector pEP-IR is expected to be established in all cells that give rise to the differentiated cell-progeny of CD34+ cells, that is in 10% to 20% of the total sorted CD34+/eGFP+ cells. These estimates for vector pEPI-IR can be of clinical importance.

IN VITRO CULTURE OF SPORES FROM WOODWARDIA FIMBRIATA SMITH

Woodwardia fimbriata Smith is a fern species belonging to the family Blechnaceae. It is native from western North America and known by the common name of giant chain fern. The present study reports Woodwardia fimbriata in vitro spore germination, gametophyte and sporophyte development and subsequent acclimatization of young plants to ex vitro conditions. Spores of W. fimbriata were obtained by exchange with other botanical gardens (via Index Seminum). Spores were successfully surface sterilized with commercial bleach and germination took place in liquid culture medium, consisting in half-strength Murashige and Skoog mineral salts and 2% sucrose. For sporophyte induction and development, gametophytes were transferred to different semi-solid (0.25% gelrite) culture media, consisting on half-strength Murashige and Skoog mineral salts and 2% sucrose, supplemented with five concentrations of NaH2PO4 (0, 50, 100, 150 and 200 mg/L) or to peat:perlite (1:1) sterile substrate. Two different photon flux density levels were also compared during the process. Best results concerning sporophyte number were observed on peat:perlite substrate (3.92 sporophytes/gametophytic cluster) and 75 µmol/m2/s. Well-developed sporophytes were potted in peat:perlite (1:1) compost and transferred to acclimatization conditions. Survival rates ranged from 90 to 95%.

α-Galactosylceramide suppresses murine eosinophil production through interferon-γ-dependent induction of NO synthase and CD95.

α-Galactosylceramide (α-GalCer), a pleiotropic immunomodulator with therapeutic potential in neoplastic, autoimmune and allergic diseases, activates invariant natural killer T-cells throughCD1-restricted receptors for α-GalCer on antigen-presenting cells, inducing cytokine secretion. However the haemopoietic effects of α-GalCer remain little explored. α-GalCer-induced modulation of eosinophil production in IL-5-stimulated bone marrow cultures was examined in wild-type (BALB/c, C57BL/6) mice and their mutants lacking CD1, inducible NOS (iNOS), CD95 and IFN-γ, along with the effects of lymphocytes; IFN-γ; caspase and iNOS inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs) and LTD4 ; and dexamethasone. α-GalCer (10(-6) -10(-8) M) suppressed IL-5-stimulated eosinopoiesis by inducing apoptosis. α-GalCer pretreatment in vivo (100 μg·kg(-1) , i.v.) suppressed colony formation by GM-CSF-stimulated bone marrow progenitors in semi-solid cultures. α-GalCer and dexamethasone synergistically promoted eosinophil maturation. Suppression of eosinophil production by α-GalCer was prevented by aminoguanidine and was undetectable in bone marrow lacking iNOS, CD95, CD28; or CD1d. Separation on Percoll gradients and depletion of CD3+ cells made bone marrow precursors unresponsive to α-GalCer. Responsiveness was restored with splenic lymphocytes. Experiments with (i) IFN-γ-deficient bone marrow, alone or co-cultured with spleen T-cells from wild-type, but not from CD1d-deficient, donors; (ii) IFN-γ neutralization; and (iii) recombinant IFN-γ, showed that these effects of α-GalCer were mediated by IFN-γ. Effects of α-GalCer on eosinophil production were blocked by LTD4 and NSAIDs. α-GalCer activation of IFN-γ-secreting, CD1d-restricted lymphocytes induced iNOS-CD95-dependent apoptosis in developing eosinophils. This pathway is initiated by endogenous regulatory lymphocytes, antagonised by LTD4 , NSAIDs and aminoguanidine, and modified by dexamethasone.

Umbilical cord blood for autologous transfusion in the early postnatal ontogeny: analysis of cell composition and viability during long-term culturing.

Changes in cell composition and viability as well as the content and functional activity of hemopoietic progenitor cells were analyzed during long-term (up to 1 month at 4°C) storage of human umbilical cord blood cells. No significant quantitative changes in erythrocytes were found during this period. The total content and viability of leukocytes changed, which resulted in the prevalence of mononuclear cells (lymphocytes and monocytes). Analysis of functional activity of hemopoietic stem cells in semisolid culture revealed a decrease in the relative content of CFU during the first week of storage [corrected] and inability of cells to colony formation after 2 weeks.

Interleukin-10 inhibits autonomous myelopoiesis in patients with myelofibrosis.

The spontaneous formation of colony-forming units granulocyte/macrophage (CFU-GM) in semisolid cultures has been shown to be due to the endogenous release of cytokines and/or to the hypersensitivity of cells against growth factors. We have reported that increased autonomous CFU-GM growth is an in vitro characteristic of myelofibrosis (MF) which may reflect aberrant hematopoiesis in vivo. Because of its cytokine synthesis-inhibiting action, we speculated that interleukin-10 (IL-10) may inhibit pathological overproduction of myeloid cells in MF by suppression of autonomous myelopoiesis. In this study, IL-10 significantly inhibited autonomous CFU-GM formation in vitro from peripheral blood mononuclear cells (PB MNC) in 10 of 11 patients with MF tested. In all patients, there was a mean inhibition of 69% ranging from 35% to 100%. Suppression of autonomous CFU-GM formation by IL-10 was dose dependent and reversible by the addition of anti-IL-10 antibodies. Our results indicate that IL-10 is a potentially useful molecule to affect aberrant myelopoiesis in patients with MF.

Protein Enrichment of Opuntia Spp. Using Different Biotechnological Treatments

Chihuahua desert possesses 60% of the Opuntia species (nopal) naturally grown in Mexico. These species are considered a reserve material due to its tolerance to adverse weather conditions, and some have been used as cattle forage regardless their low protein content. One of the traditional ways for protein enrichment is through yeast fermentation using S. cerevisae. In the present work, several routes have been assessed to augment cellulose bio availability during fermentation. The protein enrichment process, coupled with an additional thermal, enzymatic or fungal treatment in a semi-solid culture is evaluated. The yeast concentration was the constant parameter in the culture at 15% wet basis. Six different treatments were proposed considering :a. Thermal treatment; b. Fermentation using Phanerochaete chrysosporium A594; c. Novozyme 188 and Celluloclast thydrolysis; d. Fermentation with S. cerevisiae at 37° C for 48 h, 180 rpm. For each sample, lignin, cellulose and protein were measured. The results are as follows. Protein at original sample was of 0.042%, and its content (%) in the different treatment combinations was: a and d: 16.5; a,b and d:22.8; a,c and d:10.6; d:14.3; b and d: 25.9, c and d:8.75. Therefore, thermal processing did not contribute to the increase in protein content in the final product, neither was the enzymatic pre-treatment. Fungal solid state fermentation contributed significantly to the increment in protein content due, principally, to the lignin depletion in the fermentation media.

Effects of CD3McAb and rhIL-2 activated bone marrow on the killing and purging of leukemia cells.

We investigated the roles of CD3McAb and rhIL-2 activated bone marrow in the killing and purging of leukemia cells. Cytotoxicity of activated bone marrow was detected with MTT assay. CFU-GM level in activated bone marrow and the destruction of leukemia cells were measured using the semi-solid cell culture. Immune activation markers in activated bone marrow were detected by indirect immunofluorescence assay. Bone marrow activated by CD3McAb and rhIL-2 displayed significantly upregulated the killing and purging abilities on the leukemia cell line K562 and HL-60. Such effects were superior to that of bone marrow activated by rhIL-2 or CD3McAb alone (P < 0.05, P < 0.01). Activation by rhIL-2 and (or) CD3McAb exerted no obvious influence on CFU-GM level in bone marrow. Compared with bone marrow activated by rhIL-2 or CD3McAb alone, the synergistic effect of both CD3McAb+ and hIL-2 caused significant increase of CD3(+), CD8(+), CD19(+), CD25(+), CD38(+), and CD56(+) levels. Our study indicates that CD3McAb enhanced the killing and purging effects of rhIL-2 activated bone marrow on leukemia cells.

A whole-genome microarray study of Arabidopsis thaliana semisolid callus cultures exposed to microgravity and nonmicrogravity related spaceflight conditions for 5 days on board of Shenzhou 8.

The Simbox mission was the first joint space project between Germany and China in November 2011. Eleven-day-old Arabidopsis thaliana wild type semisolid callus cultures were integrated into fully automated plant cultivation containers and exposed to spaceflight conditions within the Simbox hardware on board of the spacecraft Shenzhou 8. The related ground experiment was conducted under similar conditions. The use of an in-flight centrifuge provided a 1 g gravitational field in space. The cells were metabolically quenched after 5 days via RNAlater injection. The impact on the Arabidopsis transcriptome was investigated by means of whole-genome gene expression analysis. The results show a major impact of nonmicrogravity related spaceflight conditions. Genes that were significantly altered in transcript abundance are mainly involved in protein phosphorylation and MAPK cascade-related signaling processes, as well as in the cellular defense and stress responses. In contrast to short-term effects of microgravity (seconds, minutes), this mission identified only minor changes after 5 days of microgravity. These concerned genes coding for proteins involved in the plastid-associated translation machinery, mitochondrial electron transport, and energy production.

The Effects of Lipocalin (LCN2) on Hematopoiesis in Primary Myelofibrosis

Myelofibrosis (MF) is characterized by anemia, thrombocytopenia and marrow fibrosis. Malignant rather than normal hematopoietic progenitor cells (HPC) preferentially proliferate. A form of MF can also occur in patients with a prior history of PV or ET which is referred to as PV or ET related MF (PV-MF and ET-MF). In MF, excessive proliferation of marrow stromal cells occurs in response to cytokines elaborated by the malignant clone. Previously, we found that lipocalin-2 (LCN2) levels were elevated in patient plasmas with MPN, and a greatest degree of elevation was observed in the PMF plasma and PMF conditioned medium. We also found that LCN2 increased PMF HPC proliferation and increased numbers of JAK2V617F homozygous HPCs, but depressed normal BM HPC (Lu. et al, 2013 ASH meeting).In this study, a series of experiments were performed to further explore the effects of LCN2 on MF pathogenesis. ELISA data showed that plasma LCN2 levels not only were higher in all MF patients, plasma LCN2 levels in PV-MF as well as ET- MF were significantly greater than that in PV or ET plasma (p=0.0002 and p=0.0175, respectively). Importantly, in MPN patients, a non-parametric spearman correlation coefficient analysis suggested an inverse relationship between the patients’ hemoglobin levels and LCN2 levels (coefficient=-0.35, p<0.0001) and between platelet counts and LNC2 levels (coefficient=-0.26, p=0.0044). Immunofluorescence staining showed a greater proportion of PMF MNCs expressed LCN2. And immunohistochemical results revealed that LCN2 expressing cells were present in the PMF bone marrow and spleens samples and the LCN2 expression was restricted to cells belonging to the myeloid lineage rather than erythroid precursors or megakaryocytes. These data strongly confirmed that a higher level of LCN2 was expressed in MF patients. We then found that LCN2 significantly increased the generation of ROS (reactive oxygen species) in normal BM CD34+ cells but not PMF CD34+ cells (p<0.05). Furthermore, exposure of normal marrow to the LCN2 dramatically increased the CD34+/annexin V+ cell population in a dose dependent fashion. These apoptotic effects were blocked by addition of N-Acetyl-Cysteine (NAC), a ROS scavenger. By contrast, LCN2 did not increase the degree of apoptosis of PMF CD34+ cells when added at similar doses. These data indicate that LCN2 selectively promotes the survival of PMF CD34+ cells but leads to the apoptosis of normal BM CD34+ cells by increasing ROS. With 6 different individual BM samples, we demonstrated that PMF MNC cells that produce high levels of LCN2 as well as a recombinant form of LCN2 strongly promoted BM marrow adherent cells (MAC) proliferation. Similarly, LCN2 added to semisolid cultures of normal marrow cells resulted in the appearance of increased numbers of fibroblast-like cells. In addition, the ability of LCN2 to promote MAC proliferation was blocked by addition of NAC. These results suggest that LCN2 related generation of ROS induces MAC proliferation. The MAC in both control and LCN2 containing conditions expressed vimentin, Von Willebrand factor (VWF) and α-smooth muscle actin (a-SMA), while in LCN2 cultures expressed higher levels of CD105 (Endoglin) and CD106 (VCAM-1). These MAC therefore expressed features characteristic of mesenchymal stem cells, fibroblasts and endothelial cells. The MAC under control conditions expressed CD90+ on their cell surface, while incubation with LCN2 resulted in diminished cell surface CD90 expression, and its localization to the nucleus.We propose that LCN2 acts in an autocrine fashion to promote malignant PMF hematopoiesis as documented by its ability to preferentially favor the appearance of JAK2V617F homozygous hematopoietic colonies in vitro, but also acts in a paracrine fashion to suppress the proliferation of normal HPC and to affect the marrow microenvironment by promoting stromal cell proliferation. Our findings indicate that LCN2 affects these numerous biological effects by promoting the generation of ROS. We also propose that therapeutic strategies that can impair the actions of LCN2 or inactivate ROS are likely to be beneficial to MPN patients since they improve disease related cytopenias as well as impede the development of progressive marrow fibrosis. Such agents affecting this alternative therapeutic target might potentially be used alone or in combination with other presently available therapeutic modalities. DisclosuresNo relevant conflicts of interest to declare.

Defect in Ribosome Biogenesis Contributes to Impaired Megakaryopoiesis in Primary Myelofibrosis

Primary myelofibrosis (PMF) is a clonal hematologic malignancy resulting from the transformation of a pluripotent hematopoietic progenitor cell leading to increased hematopoiesis and overproduction of abnormal blood cells. PMF is characterized by bone marrow (BM) fibrosis, extramedullary hematopoiesis, increased numbers of circulating CD34+ cells, splenomegaly, and a propensity to evolve to AML. Patients also display anemia and thrombocytopenia and harbor abnormal, immature megakaryocytes (Mks) in their BM and spleen. Numerous mutations are associated with PMF including JAK2, CALR, MPL, TET2, CBL, ASXL1, and IDH.The presence of abnormal Mks in the BM of PMF patients is an important characteristic of the disease, as these cells are believed to contribute to the fibrosis.Here, we performed an analysis of Mk differentiation from individuals with PMF compared to healthy individuals as a way to better understand the mechanism leading to their defects. CD34+ cells obtained from PMF and healthy donors were cultivated in presence of thrombopoietin to induce Mks differentiation. Under these conditions, we observed that the proliferative capacity of CD34+ cells from PMF patients was increased as compared to normal CD34+ cells. At the same time, the percentage of mature Mks obtained was reduced. Semi-solid culture conditions confirmed the proliferative advantage of the PMF CD34+ cells. To investigate the mechanism responsible for this phenotype, we performed genome wide expression analysis of sorted Mks from patients versus controls. Nine PMF patients with different mutations (5 JAK2V617F, 1 MPLW515L, 2 CALR and 1 triple negative) and 4 healthy donors were included in the study. Gene Set Enrichment Analysis (GSEA) analysis revealed that the top scoring pathways included the RPS14 signature, ribosome biogenesis, rRNA metabolic process, and rRNA processing. We first studied the RPS14 geneset, which consists of a group of genes that were up-regulated in CD34+ cells after knock-down of RPS14, a component of the small ribosome subunit. Using shRNA against RPS14, we discovered that its knock down leads to a significant reduction in the development of mature Mks from normal CD34+ cells.The microarray data also revealed changes in the expression of several myeloid genes. CEBPa and GFI-1 were over-expressed in PMF Mks, whereas GFI-1b and PF4, which are associated with Mk differentiation, were down-regulated. Surprisingly the expression of GATA1, a master regulator of megakaryopoiesis, was not changed. However, consistent with previous report, we discovered that GATA1 protein level was reduced in PMF Mks. GATA1 deficient Mks are impaired in their maturation, so we reasoned that the reduction in GATA1 contributes to the defects in PMF. Indeed, we found that overexpression of GATA1 in PMF CD34+ cells rescued Mk differentiation. To understand how GATA1 protein but not mRNA, is reduced, we knocked down RPS14 in the JAK2 mutant SET2 megakaryoblastic cell line. As predicted, we observed a reduction in GATA1 protein, but not mRNA. By ribosomal profiling, we saw that the shRPS14 led to an abnormal ribosomal profile with a reduction of the 40s peak. Finally, by RT-PCR analysis of the ribosomal and polysomal fractions, we found that GATA1 mRNA was less abundant in the polysomal fraction in cells expressing the shRPS14. This finding is consistent with recent data showing that alterations in ribosomal genes cause a decrease in GATA1 protein in Diamond-Blackfan Anemia.Although we identified the RPS14 gene signature, we did not observe changes in RPS14 expression or in any of the ribosomal subunits. Since our GSEA analysis also identified deficiencies in three ribosomal biogenesis pathways, we looked for genes present in all three signatures. NOP58, a ribonucleoprotein required for ribosome biogenesis, was significantly reduced in all samples of PMF Mks. To link the reduction in NOP58 expression with PMF associated mutations, we expressed MPLW515L or the CALR type II mutant in hematopoietic progenitor cells and detected a reduction in NOP58 expression. Our findings suggest that the activation of JAK/STAT signaling leads to a reduction in NOP58, which contributes to defects in ribosome assembly, decreased GATA1 expression and subsequent impairment in Mk differentiation. This work reveals, for the first time, that defective ribosomes contribute to the pathogenesis of PMF and suggests new avenues for therapeutic intervention. DisclosuresStein:Incyte Corporation: Honoraria, Speakers Bureau; Sanofi Oncology: Honoraria.