Later Stages Of Maturation Research Articles

The Maximum Burial Depth in Hydrocarbon Source Rocks on Kongquehe Slope in the Tarim Basin

The strata of the Kongquehe slope in the Tarim basin have suffered from long-term denudation, causing the determination of maximum ancient buried depth in mainly hydrocarbon source rocks to be very difficult. The denudation restricts the conduct of research into the hydrocarbon generation history of the source rocks. Through the use of drilling, seismic, logging, and geochemical data, calculation of the amount of denudation of the main unconformable surface, consideration of the present residual thicknesses of major hydrocarbon source rocks and other data, the authors determine maximum ancient buried depth in four major hydrocarbon source rocks and determines the time required to reach the maximum ancient buried depth. The authors have ascertained four main results through comprehensive analysis. (a) A majority of the Jurassic hydrocarbon source rock passed the hydrocarbon-generating threshold during the Neogene period and are now in the early-mature to mature stage. (b) Carboniferous hydrocarbon source rock is only distributed in the Caohu depression, having crossed the hydrocarbon-generating threshold in the Cretaceous period, and is now in the mature to late-mature stage. (c) Most of the middle-upper Ordovician hydrocarbon source rock crossed the hydrocarbon-generating threshold in the late Ordovician period and is now in the late-mature to over-mature stage. There are two blocks in the east research area that appear to contain the maximum ancient buried depth which may have experienced secondary hydrocarbon generation in the late Silurian. (d) Cambrian-Lower Ordovician hydrocarbon source rock is in the late-mature to over-mature stage and may have experienced secondary hydrocarbon generation in the Cenozoic period in most of Kongquehe area.

Enteric methane emission, diet digestibility, and nitrogen excretion from beef heifers fed sainfoin or alfalfa1

Effects of plant-bound condensed tannin (CT)-containing sainfoin vs. CT-free alfalfa (or low-CT alfalfa-sainfoin mixture), plant stage of maturity, and their interaction on enteric methane (CH4) emissions, diet digestibility, and N excretion were studied, using 8 ruminally cannulated beef heifers in 2 sequential short-term experiments (Exp. 1 and 2). In Exp. 1, first growth legumes were harvested daily and offered fresh to heifers. Heifers were assigned to 100% sainfoin or 80% alfalfa:20% sainfoin (as-fed basis). Responses were measured at early (late vegetative to early bud; stage 2 to 3) and late (early flower; stage 5) stage of maturity. In Exp. 2, the same legumes were harvested from second growth (late bud; stage 4) and offered to heifers as hay; 100% sainfoin or 100% alfalfa. In both experiments, heifers were fed once daily at 1× maintenance. When fed as fresh forage (Exp. 1), sainfoin, compared with the alfalfa-sainfoin blend, had greater digestibility of OM (74.7 vs. 70.9%; P = 0.02), yet tended to have lower CP digestibility (73.2 vs. 77.1%; P = 0.059). There was no difference between fresh legumes for CH4 emissions [25.9 g/kg DMI ± 4.02 SE; 8.5% of gross energy intake (GEI) ± 1.26 SE; or 36.8 g/kg digested OM ± 1.75 SE]. The fresh legumes were more digestible at early, rather than at late, maturity and, consequently, enteric CH4 (27.4 vs. 24.4 g/kg DMI; P < 0.004; 8.9 vs. 8.1% GEI; P < 0.008) was greater at early, rather than at later, growth. When fed as hay (Exp. 2), sainfoin, compared with alfalfa, had greater digestibility of OM (60.5 vs. 50.3%; P = 0.007), lower digestibility of CP (64.2 vs. 68.8%; P = 0.004), yet there was no difference between the legume hays for CH4 emissions (22.4 g/kg DMI ± 1.29 SD and 7.1% GEI ± 0.40 SD). However, on the basis of OM digested, CH4 emissions were lower for sainfoin than alfalfa hay (44.3 vs. 59.0 g/kg; P = 0.008). Percentage of total N excretion in urine was less for sainfoin compared with alfalfa, both for fresh legumes in Exp. 1 (74 vs. 78%; P = 0.017) or hay in Exp. 2 (64 vs. 72%; P < 0.001), and increasing maturity lowered urinary N excretion. In conclusion, feeding CT-containing sainfoin partially shifted N excretion from urine to feces, but it had little impact on enteric CH4 emissions from beef cattle fed at maintenance as compared with feeding either 80% alfalfa:20% sainfoin (fresh forages) or 100% alfalfa (hay). Feeding fresh legumes harvested between the late vegetative to early bud stage, compared with harvested at the early flower stage, increased N excreted in urine as well as enteric CH4 emissions from beef cattle fed at maintenance.

Influence of enamel matrix derivative on cells at different maturation stages of differentiation.

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2–5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.

Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts.

Primitive erythroid cells, the first red blood cells produced in the mammalian embryo, are necessary for embryonic survival. Erythropoietin and its receptor EpoR, are absolutely required for survival of late-stage definitive erythroid progenitors in the fetal liver and adult bone marrow. Epo- and Epor-null mice die at E13.5 with a lack of definitive erythrocytes. However, the persistence of circulating primitive erythroblasts raises questions about the role of erythropoietin/EpoR in primitive erythropoiesis. Using Epor-null mice and a novel primitive erythroid 2-step culture we found that erythropoietin is not necessary for specification of primitive erythroid progenitors. However, Epor-null embryos develop a progressive, profound anemia by E12.5 as primitive erythroblasts mature as a synchronous cohort. This anemia results from reduced primitive erythroblast proliferation associated with increased p27 expression, from advanced cellular maturation, and from markedly elevated rates of apoptosis associated with an imbalance in pro- and anti-apoptotic gene expression. Both mouse and human primitive erythroblasts cultured without erythropoietin also undergo accelerated maturation and apoptosis at later stages of maturation. We conclude that erythropoietin plays an evolutionarily conserved role in promoting the proliferation, survival, and appropriate timing of terminal maturation of primitive erythroid precursors.

Applying terahertz time-domain spectroscopy to probe the evolution of kerogen in close pyrolysis systems

Terahertz time-domain spectroscopy (THz-TDS) has been used to probe the evolutionary paths of kerogen in selected black mudstone. The evolutionary regime of kerogens (for instance, the immaturity, early maturity, middle maturity, late maturity, and catagenesis stages) can be indicated by the absorption coefficient in the THz region. The present study of identification based on THz-TDS was in good agreement with programmed pyrolysis experiments and suggests that THz technology can act as a nondestructive, contact-free tool for probing the ability to generate hydrocarbons from kerogens.

Pre- and Postharvest Muskmelon Fruit Cracking: Causes and Potential Remedies

Fruit cracking is an important disorder that can cause severe loss of marketable yield and revenue in the muskmelon (Cucumis melo) fruit industry. The physiological and environmental factors causing cracking are poorly understood. Although generally considered a physiological disorder caused by fluctuating environmental conditions, current evidence indicates that this disorder also has a genetic as well as a genotype × environment component. Certain cultivars are more susceptible than others, but wide fluctuations in irrigation, temperature, and nutrition during late fruit maturation stages appear to predispose fruit to cracking. This article summarizes the current state of our understanding of the causes of fruit splitting in muskmelons.

Resolvin E1 regulates osteoclast fusion via DC‐STAMP and NFATc1

Interactions between the immune and skeletal systems in inflammatory bone diseases are well appreciated, but the underlying molecular mechanisms that coordinate the resolution phase of inflammation and bone turnover have not been unveiled. Here we investigated the direct actions of the proresolution mediator resolvin E1 (RvE1) on bone-marrow-cell-derived osteoclasts in an in vitro murine model of osteoclast maturation and inflammatory bone resorption. Investigation of the actions of RvE1 treatment on the specific stages of osteoclast maturation revealed that RvE1 targeted late stages of osteoclast maturation to decrease osteoclast formation by 32.8%. Time-lapse vital microscopy and migration assays confirmed that membrane fusion of osteoclast precursors was inhibited. The osteoclast fusion protein DC-STAMP was specifically targeted by RvE1 receptor binding and was down-regulated by 65.4%. RvE1 did not affect the induction of the essential osteoclast transcription factor nuclear factor of activated T cells c1 (NFATc1) or its nuclear translocation; however, NFATc1 binding to the DC-STAMP promoter was significantly inhibited by 60.9% with RvE1 treatment as shown in electrophoresis mobility shift assay. Our findings suggest that proresolution mediators act directly on osteoclasts, in addition to down-regulation of inflammation, providing a novel mechanism for modulating osteoclast signaling in osteolytic inflammatory disease.

Subcomponents of Vitamine B Complex Regulate the Growth and Development of Human Brain Derived Cells

The work is focused on the role of single components of Vitamin B complex: pyridoxine, riboflavin, thiamine, and nicotinamide in the processes of growth and development of the human brain derived cells. We, also, have assayed the activity of Xanthine Oxidase in the presence of above mentioned subcomponents to delineate the possible mechanism of their action. Results indicate that the all components of Vitamin B complex might be responsible for cells’ growth, maturation, proliferation. During early period of the cells’ growth the most important components were thiamine and Pyridoxine, initiating cells’ proliferation (number of the cells in one field: 2556, 17±355, 68, 2179,0±223,55, resp) vs control (1562,94±146,45), whereas during the late stages of maturation the most important components responsible for differentiation, were riboflavin and nicotinamide (3774.77± 188.41, 3558.82±152.90 resp. vs control 2905±263.75; p<0.035). In comparison with the all other subcomponents of Vitamin B complex only in pyridoxine containing samples, XO activity was specifically inhibited by allopurinol (percentile of inhibition 160,00±60,00 vs control 31,03±6,92, p<0,05). We have concluded that Pyridoxine might interact with XO and regulate its activity. All components of Vitamin B complex are able to initiate cells development and growth; however, they are supposed to be selectively utilized in time dependent manner to guarantee the highest efficiency of the cells development, maturation and proliferation

Modification of Carotenoid Levels by Abscission Agents and Expression of Carotenoid Biosynthetic Genes in ‘Valencia’ Sweet Orange

The effect of 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) and ethephon on peel color, flavedo carotenoid gene expression, and carotenoid accumulation was investigated in mature 'Valencia' orange ( Citrus sinensis L. Osbeck) fruit flavedo at three maturation stages. Abscission agent application altered peel color. CMNP was more effective than ethephon in promoting green-to-red (a) and blue-to-yellow (b) color at the middle and late maturation stages and total carotenoid changes at all maturation stages. Altered flow of carotenoid precursors during maturation due to abscission agents was suggested by changes in phytoene desaturase (Pds) and ζ-carotene desaturase (Zds) gene expression. However, each abscission agent affected downstream expression differentially. Ethephon application increased β-carotene hydroxilase (β-Chx) transcript accumulation 12-fold as maturation advanced from the early to middle and late stages. CMNP markedly increased β- and ε-lycopene cyclase (Lcy) transcript accumulation 45- and 15-fold, respectively, at midmaturation. Patterns of carotenoid accumulation in flavedo were supported in part by gene expression changes. CMNP caused greater accumulation of total flavedo carotenoids at all maturation stages when compared with ethephon or controls. In general, CMNP treatment increased total red carotenoids more than ethephon or the control but decreased total yellow carotenoids at each maturation stage. In control fruit flavedo, total red carotenoids increased and yellow carotenoids decreased as maturation progressed. Trends in total red carotenoids during maturation were consistent with measured a values. Changes in carotenoid accumulation and expression patterns in flavedo suggest that regulation of carotenoid accumulation is under transcriptional, translational, and post-translational control.

Ca2+influx and the store-operated Ca2+entry pathway undergo regulation during mouse oocyte maturation

In preparation for fertilization, mammalian oocytes undergo optimization of the mechanisms that regulate calcium homeostasis. Among these changes is the increase in the content of the Ca(2+) stores ([Ca(2+)]ER), a process that requires Ca(2+) influx. Nevertheless, the mechanism(s) that mediates this influx remains obscure, although is known that [Ca(2+)]ER can regulate Ca(2+) influx via store-operated Ca(2+) entry (SOCE). We find that during maturation, as [Ca(2+)]ER increases, Ca(2+) influx decreases. We demonstrate that mouse oocytes/eggs express the two molecular components of SOCE--stromal interaction molecule 1 (Stim1) and Orai1--and expression of human (h) Stim1 increases Ca(2+) influx in a manner that recapitulates endogenous SOCE. We observe that the cellular distribution of hStim1 and hOrai1 during maturation undergoes sweeping changes that curtail their colocalization during the later stages of maturation. Coexpression of hStim1 and hOrai1 enhances influx throughout maturation but increases basal Ca(2+) levels only in GV oocytes. Further, expression of a constitutive active form of hStim1 plus Orai1, which increases basal Ca(2+) throughout maturation, disturbs resumption of meiosis. Taken together, our results demonstrate that Ca(2+) influx and SOCE are regulated during maturation and that alteration of Ca(2+) homeostasis undermines maturation in mouse oocytes.

Investigation of tocotrienol biosynthesis in rice (Oryza sativa L.)

Rice tocotrienol (T3) has gained attention due to its physiological activities (e.g., antiangiogenesis). However, the biosynthetic pathway for T3 production in rice grain has not been well studied. We hypothesized that T3 biosynthesis enzymes and/or precursors play an important role in T3 production in whole grain. This proposal was evaluated in rice (Oryza sativa L.) by PCR and HPLC techniques. Grain tocopherol as well as flag leaf vitamin E levels were also investigated for comparison. For rice samples 14days after flowering, grain was abundant in T3, but not in flag leaf. Expression of a gene encoding homogentisate geranylgeranyltransferase (HGGT, which has long been believed to be important for T3 production) differed significantly between grain and flag leaf. We then investigated rice samples during the grain maturation period, and found that grain T3 and HGGT levels increased in the early stage and then reached a plateau. T3 precursors such as homogentisate and geranylgeranyl pyrophosphate decreased during maturation. No increase in grain T3 from the middle to late stages of maturation and a decrease in T3 precursors during maturation suggest that HGGT would be an essential, but not limiting factor for T3 biosynthesis, and T3 precursors could regulate the T3 level in grain. The results of this study would be useful for nutraceutical purposes (e.g., development of T3-overproducing rice for the prevention of angiogenic disorders).

New Betulinic Acid Derivatives for Bevirimat-Resistant Human Immunodeficiency Virus Type-1

Bevirimat (1, BVM) is an anti-HIV agent that blocks HIV-1 replication by interfering with HIV-1 Gag-SP1 processing at a late stage of viral maturation. However, clinical trials of 1 have revealed a high baseline drug resistance that is attributed to naturally occurring polymorphisms in HIV-1 Gag. To overcome the drug resistance, 28 new derivatives of 1 were synthesized and tested against compound 1-resistant (BVM-R) HIV-1 variants. Among them, compound 6 exhibited much improved activity against several HIV-1 strains carrying BVM-R polymorphisms. Compound 6 was at least 20-fold more potent than 1 against the replication of NL4-3/V370A, which carries the most prevalent clinical BVM-R polymorphism in HIV-1 Gag-SP1. Thus, compound 6 merits further development as a potential anti-AIDS clinical trial candidate.

The impact of fruit maturation on bioactive microconstituents, inhibition of serum oxidation and inflammatory markers in stimulated PBMCs and sensory characteristics of Koroneiki virgin olive oils from Messenia, Greece

Olive fruits from the Koroneiki cultivar (Olea europaea L.) grown in Messenia, Greece, were hand-picked from the same trees in progressive maturity stages, covering three months, and processed identically with a commercial olive mill and a three-phase decanter. Data on quality parameters, and antioxidant activity of the obtained oils were collected by employing the conventional analytical methods set by European Union Commission Regulation no. 61/2011. Additionally, the potential of oils' polar extract to inhibit total serum lipid oxidation and inflammatory markers in stimulated human mononuclear cells was assayed. The results showed that ripening caused an increase in monounsaturated and decrease in polyunsaturated fatty acids, as well as an increase in phenolic compounds - mainly hydroxytyrosol - and in squalene. The extracts' ferric reducing power was in line with the increase of phenolic compounds. In later stages of maturation, lipoprotein oxidation was less potent and the decrease of inflammatory markers in stimulated human mononuclear cells was more powerful. Sensory evaluation detected differences in oils' "bitter" attributes, while the analysis of oils' volatiles revealed quantitative differences.

Altered B cell development and fuctions in Adenosine Deaminase deficient patients

Event Abstract Back to Event Altered B cell development and fuctions in Adenosine Deaminase deficient patients Immacolata Brigida1*, Aisha V. Sauer1, Francesca Ferrua2, Stefania Giannelli1, Samantha Scaramuzza1, Maria Pia Cicalese2, Miriam Casiraghi2, Elisabetta Traggiai3, Mirjam Van Der Burg4 and Alessandro Aiuti1, 5 1 San Raffaele Ospital, HSR-TIGET, department of regenerative medicine stem cells and gene therapy, Italy 2 San Raffaele Scientific Institute, Milan, Pediatric Immunohematology and Bone Marrow Transplantation Unit, Italy 3 Novartis Institute for Research in Biomedicine, Switzerland 4 Erasmus MC, University Medical Center, Department of Immunology, Netherlands 5 Tor Vergata University, Department of Systems Medicine, Italy Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune deficiency and dysregulation due to metabolic toxicity. Alterations in B cell development and function have been poorly studied in these patients. Enzyme replacement therapy (ERT) and hematopoietic stem cell gene therapy (GT) are therapeutic options in patients lacking a suitable bone marrow transplant donor. We studied bone marrow (BM) of ADA-deficient patients and investigate the ability of ERT and GT to restore normal B cell differentiation and functions in BM and PB. We found that BM B cells from untreated ADA-deficient patients show an increased proportion of pre-B1 B-cells and a progressive decrease in later stage of maturation. This is in agreement with observation that the strongest selective advantage for ADA-transduced cells is observed at the transition from immature to naïve cells. BM alterations were overcome in patients treated with GT or ERT, but in the latter group immature B cells were expanded. In the periphery, transitional B cells accumulate under ERT and persist long-term, while after GT, transitional B cell frequency progressively normalized. Down regulation of BAFF-R and higher BAFF plasma levels were observed both in ERT and GT patients. B cell proliferative responses after BCR/TLR triggering were severely impaired in ERT patients but improved after GT. Our findings confirm that transfer of ADA gene into autologous hematopoietic stem cells restores B-cell development and functions. Keywords: Gene Therapy, ADA-SCID, B cells, B cell development, Autoimmunity Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Translational immunology and immune intervention Citation: Brigida I, Sauer AV, Ferrua F, Giannelli S, Scaramuzza S, Cicalese M, Casiraghi M, Traggiai E, Van Der Burg M and Aiuti A (2013). Altered B cell development and fuctions in Adenosine Deaminase deficient patients. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00413 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Immacolata Brigida, San Raffaele Ospital, HSR-TIGET, department of regenerative medicine stem cells and gene therapy, Milan, Italy, brigida.immacolata@hsr.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Immacolata Brigida Aisha V Sauer Francesca Ferrua Stefania Giannelli Samantha Scaramuzza Maria Pia Cicalese Miriam Casiraghi Elisabetta Traggiai Mirjam Van Der Burg Alessandro Aiuti Google Immacolata Brigida Aisha V Sauer Francesca Ferrua Stefania Giannelli Samantha Scaramuzza Maria Pia Cicalese Miriam Casiraghi Elisabetta Traggiai Mirjam Van Der Burg Alessandro Aiuti Google Scholar Immacolata Brigida Aisha V Sauer Francesca Ferrua Stefania Giannelli Samantha Scaramuzza Maria Pia Cicalese Miriam Casiraghi Elisabetta Traggiai Mirjam Van Der Burg Alessandro Aiuti PubMed Immacolata Brigida Aisha V Sauer Francesca Ferrua Stefania Giannelli Samantha Scaramuzza Maria Pia Cicalese Miriam Casiraghi Elisabetta Traggiai Mirjam Van Der Burg Alessandro Aiuti Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. 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Polyphenol content and antioxidant activity of maqui (Aristotelia chilensis Molina Stuntz) during fruit development and maturation in Central Chile

Maqui ( Aristotelia chilensis [Molina] Stuntz, Elaeocarpaceae) is a Chilean native species which produces small berries that are mainly collected from the wild. The health benefits of maqui fruit are attributed to their high polyphenol content as well as their wide variety of anthocyanins and flavonols. One of the main factors that affect the polyphenol content in fruit is the maturity stage at harvest. The objective of this study was to determine total phenol and total anthocyanin content and antioxidant activity (by ferric reducing ability of plasma [FRAP] assay) of maqui fruits harvested at different fruit maturity stages from two wild populations located in Central Chile. Each maturity stage was determined by days from fruit set, berry size, and soluble solids. Total phenol content declined while total anthocyanin content increased from the green to light red stage. Nevertheless, both total phenol and anthocyanin content increased from the light red to dark purple stage. The highest anthocyanin content and antioxidant activity was found in the late maturity stage (dark purple). The results show that ripening in maqui fruit can be expected with 1100 growing degree-days (91 d after fruit set) in Central Chile. At this moment of harvest, fruits with 18-19° Brix have the highest anthocyanin content and antioxidant activity (FRAP). This study constitutes the first advances in the understanding of maqui fruit ripening and corresponding antioxidant activity.

Heat Shock Protein 70 Cytosolic Sequestration by Excess of Free Alpha-Globin Chains Is a Key Mechanism of the Ineffective Erythropoiesis in β-Thalassemia Major Patients

AbstractAbstract 823 Background:Despite extensive knowledge of the molecular defects causing β-thalassemia major (TM), less is known about the mechanisms responsible of the ineffective erythropoiesis. This latter is characterized by accelerated erythroid differentiation, apoptosis and maturation arrest at the polychromatophilic stage. It explains, at least in part, the profound anemia observed in this disease. Although it has been proposed that both the precipitation of unmatched globin chains as well as the accumulation of unbound iron could lead to oxidative stress and subsequent hemolysis, the mechanism by which apoptosis and maturation arrest are induced remains unclear. Hypothesis:We have previously demonstrated that normal human erythroid cell maturation requires the transcription factor GATA-1 and an transient activation of caspase-3 at the basophilic stage (Zermati Y, J Exp Med 2001). Although GATA-1 is a capsase-3 target, we have shown that it was protected during erythroid maturation by the nucleus translocation of heat shock protein 70 (Hsp70) at the onset of caspase-3 activation (Ribeil JA, Nature 2007). Hsp70 is an universally highly conserved protein chaperone involved in the regulation of protein folding and in the prevention of damaging aggregates formation (Hart HU, Nature 2011). We hypothesized that, in addition to AHSP the cognate chaperone of α-globin chains, Hsp70 could also serve as a secondary chaperone in case of excess of free α-globin chains or aggregates to reduce their toxicity. As a result, this interaction could be detrimental by preventing the nuclear localization of Hsp70 leading to the degradation of GATA-1, maturation arrest and apoptosis in TM erythroid mature cells. Results:During erythroid maturation of CD34+ derived from TM peripheral blood (n=8) or bone marrow (n=3), we have first shown by confocal microscopy that, in contrast to CD34+ from healthy donor (n=10), Hsp70 nuclear translocation did not occur at the late stage of maturation. As a result, GATA-1 was poorly expressed, end stage erythroid maturation was arrested and apoptosis occurred. Hsp70/α-globin complexes were evidenced by confocal analysis in cytosol (specific co-localization), by immunoprecipitation of lysed differentiated erythroid TM cells in liquid culture, and by yeast two-hydrid experiments. In order to better characterize this interaction, we have demonstrated by molecular modeling, docking and molecular dynamic simulation (3D models of the human full-length Hsp701-701generated by homology modeling from crystallographic structures) that α-globin bounds to a highly electronegative cavity formed by NBD, SBD and LID (3 major domains of Hsp70). The Hsp70/α-globin complex is characterized by extensive protein-protein interactions stabilized by multiple hydrogen bonds that we have accurately characterized.Then, to demonstrate the pathological role of the Hsp70 cytosolic sequestration, by lentivirus transfection of TM CD34+ cells, we have expressed a nucleus-targeted mutant of Hsp70 and a wtHsp70 (overexpressing Hsp70). As expected, both lentivirus increased nuclear Hsp70 expression and rescued GATA-1 expression. They also increased TM erythroid cell maturation (more mature cells -acidophils and reticulocytes- than in control transfected cells -21.5% vs 11,8%, p<0,05) and decreased apoptosis (50% decreased). Transfection with a retrovirus mutant GATA-1 (μGATA-1, uncleavable by actived caspase-3) had similar positive effects on erythroid maturation and cell death. Finally, by flow cytometry analysis we could observe that concomitantly to GATA-1 protection in these experiments the percentage of F cells was significantly increased (+20%) at end stages of maturation. To ensure the specificity of our findings, β-globin gene lentivirus transfection of TM CD34+ cells led to disappearance of Hsp70/α-globin interactions in the cytosol, Hsp70 nuclear localization, GATA-1 protection and normal erythroid maturation. Conclusion:Our data indicate that, cytosolic Hsp70 sequestration by α-globin chains prevents its nuclear localization and is a key mechanism of the ineffective erythropoiesis observed in β-TM patients. In order to increase nuclear Hsp70 translocation, developing drugs or small molecules that could increase Hsp70 expression or better disrupt the Hsp70/α-globin complex could be a novel approach of targeted therapies to improve erythropoiesis in TM. Disclosures:Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Disruption of MDM2-p53 Interactions by a Small Molecule Inhibitor RG7112 Increases Apoptosis and Impairs Polyploidization of Human Megakaryocytes

Abstract 1082Megakaryocyte (MK) development is characterized by polyploidization, cytoplasmic maturation and proplatelet formation, which culminates in the release of platelets into the circulation. The tumor suppressor p53 plays a critical role in the regulation of both cell cycle and apoptosis; its function is tightly controlled by the murine double minute (MDM2) protein which facilitates p53 degradation and inhibits p53 transcriptional activity. MK ploidy results from a disruption of normal cell cycle progression termed endomitosis while platelet release is believed to depend on apoptotic processes. The role of p53-MDM2 in MK in these two processes has not been clearly defined. A small molecule RG7112, which disrupts MDM2-p53 interaction, has shown promising anti-tumor effects in phase I clinical trials. This beneficial outcome has, however, been associated with the development of thrombocytopenia. We, therefore, used RG7112 as pharmacological probe to examine the effects of disruption of the MDM2-p53 regulatory loop on MK. We determined the effects of RG7112 on primary human MK by utilizing an in vitro system in which MK were generated from BM-derived CD34+ cells. We first demonstrated that both p53 and MDM2 transcripts are up-regulated as MK differentiation progresses. The ability of CD34+ cells to proliferate in the absence or presence of various concentrations of RG7112 was then evaluated both in liquid cultures and in CFU-MK colony assays. CD34+ cells exposed to 10 μM RG7112 for 7 days generated 70% fewer viable cells as compared to control cells exposed to the inactive form of the drug (p value = 0.0038). Furthermore, CD34+ cells treated with RG7112 formed up to 40% less CFU-MK colonies as compared to untreated cells. An assessment of apoptosis of MK precursors generated in the presence of RG7112 revealed that 69.5+2.1% were Annexin V positive as compared to 31.5+3.5% present in control cultures. These findings are consistent with the previously reported role of RG7112 in inducing p53 activation and apoptosis. Interestingly, phenotypical characterization of the viable cells generated under identical culture conditions, showed that RG7112 treatment did not interfere with the ability of CD34+ cells to acquire markers of MK differentiation during the first 7 days of culture since similar degrees of CD41 and CD42 expression were observed in the absence and in the presence of the drug. Likewise, exposure of MK precursors to the drug for 7 additional days (i.e. later stages of maturation) did not influence CD41 and CD42 expression. By contrast, cells differentiated in the presence of 5 μM RG7112 generated 50% fewer polyploid MK with greater than 4N DNA content as compared to those treated with the inactive form of the drug. Moreover, the negative effects on ploidy were associated with p53 activation, as assessed by the increased levels of p21 protein, a direct target of p53 which is known to limit polyploidization of primary MK. Finally, platelets generated in vitro were analyzed phenotypically and quantitated by dual labeling with anti-CD41 antibodies and thiazole orange (TO). The number of CD41+/TO+platelets derived from MK generated in the presence of RG7112 was reduced by 22% as compared to control. Based on these findings, we conclude that RG7112 impacts megakaryopoiesis by two potential mechanisms: 1) Impairing the ability of CD34+ cells to generate MK precursors due to increased apoptosis; 2) Limiting polyploidization during the late stages of development due to phamacological activation of p53. A combination of these two effects may provide an explanation for thrombocytopenia observed in patients receiving this drug and suggests that p53 plays an important role in normal human thrombocytopoiesis. Disclosures:Iancu-Rubin:Roche: Research Funding. Hoffman:Roche: Research Funding.

POSTHARVEST RESPONSE OF PEACH AND NECTARINE CULTIVARS TO 1-METHYLCYCLOPROPENE TREATMENT

With the registration of 1-methylcyclopropene (1-MCP) in the USA and large scale use on apples, the question was posed of how effective the product would be in maintaining the flesh firmness of tree ripened peaches. In NC where peaches are sold primarily through retail outlets immediately after harvest, high quality tree-ripened peaches are expected. However, to meet this expectation peaches are picked at a more advanced stage of maturity and they are softer with a reduced shelf life. Six peach (Prunus persica L. Batsch) cultivars and one nectarine cultivar were harvested at tree-ripe maturity. The cultivars were selected to represent a range of maturity from early to late season. The cultivars included in this study were: 'Candor', 'Goldprince', 'Carolina Red', 'Redhaven', 'Contender', 'China Pearl', and 'Big Red'. After harvest the fruit were cooled overnight to remove field heat and then treated with 1-MCP for 24 h at 5°C. After treatment with one µL/L 1-MCP the fruit were held at 21°C for up to 8 days to simulate retail and consumer conditions after purchase. Treatment with 1-MCP resulted in fruit with higher flesh firmness, particularly during the first 5 days at 21°C, compared to untreated fruit. Treated fruit were significantly firmer than the untreated fruit after approximately seven days, but this was somewhat dependent on harvest maturity. On fruit harvested at a later stage of maturity that had significantly softened, 1-MCP had little effect. One µl/L reduced the respiration rate of the fruit. Effect on soluble solid concentration was not consistent. It was concluded that use of 1-MCP would allow a tree-ripened peach to maintain slightly greater flesh firmness for a longer period of time when held under room temperature conditions. However, the commercial economics of this treatment need to be determined.

Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr.

During the process of subculture of embryogenic cultures, sometimes they may become non-embryogenic, which is not desirable. However, this offers an opportunity to explore the mechanisms underlying cell fate determination and the maintenance of embryogenic potential of explants during the process of somatic embryogenesis. In a previous study, differential expression of microRNAs (miRNAs) has been detected between embryogenic and non-embryogenic cultures as well as during somatic embryo maturation of Larix kaempferi (Lamb.) Carr. However, little is known about the target genes of these miRNAs during these cellular differentiation processes. In this study, full-length cDNA of the MYB homologue from L. kaempferi, LaMYB33, was cloned. Sequence analysis showed that the miR159 target sequence is present in LaMYB33. The isolation of the miRNA-guided cleavage products of LaMYB33 further suggested that this gene is regulated by miRNA. LaMYB33 transcript levels between embryogenic and non-embryogenic cultures and during the late stage of somatic embryo maturation were measured and the results showed opposite patterns in the expression of LaMYB33 and mature miR159. Based on the relationships between the expression patterns of LaMYB33 and mature miR159, we concluded that the post-transcriptional regulation of LaMYB33 by miR159 participates in the maintenance of embryogenic or non-embryogenic potential and somatic embryo maturation, providing new insights into the regulatory mechanisms of somatic embryogenesis.

CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair

AbstractMonocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell–restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2+Ly6C+ macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2-mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.