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The remarkable regeneration capability of plant tissues or organs under culture conditions has underlain an extensive practice for decades. The initial step in plant in vitro regeneration often involves the induction of a pluripotent cell mass termed callus, which is driven by the phytohormone auxin and occurs via a root development pathway. However, the key molecules governing callus formation remain unknown. Here we demonstrate that Arabidopsis LATERAL ORGAN BOUNDARIES DOMAIN （LBD）/ASYMMETRIC LEAVES2-LIKE （ASL） transcription factors are involved in the control of callus formation program. The four LBD genes downstream of AUXIN RESPONSE FACTORs （ARFs）, LBD16, LBD17, LBD18 and LBD29, are rapidly and dramatically induced by callus-inducing medium （CIM） in multiple organs. Ectopic expression of each of the four LBD genes in Arabidopsis is sufficient to trigger spontaneous callus formation without exogenous phytohormones, whereas suppression of LBD function inhibits the callus formation induced by CIM. Moreover, the callus triggered by LBD resembles that induced by CIM by characteristics of ectopically activated root meristem genes and efficient regeneration capacity. These findings define LBD transcription factors as key regulators in the callus induction process, thereby establishing a molecular link between auxin signaling and the plant regeneration program.

Aim： To examine the effects of quercetin, a natural antioxidant, on high glucose （HG）-induced apoptosis of cultured dorsal root ganglion （DRG） neurons of rats. Methods： DRG neurons exposed to HG （45 mmol/L） for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species （ROS） level and apoptosis were determined. The expression of NF-кB, IкBα, phosphorylated IкBα and Nrf2 was examined using RT PCR and Western blot assay. The expression of hemeoxygenase-1 （HO-1）, IL-6, TNF-α, iNOS, COX-2, and caspase-3 were also examined. Results： HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF-κB signaling pathway. Co-treatment with quercetin （2.5, 5, and 10 mmol/L） dose-dependently decreased HG-induced caspase-3 activation and apoptosis. Quercetin could directly scavenge ROS and significantly increased the expression of Nrf-2 and HO-1 in DRG neurons. Quercetin also dose-dependently inhibited the NF-κB signaling pathway and suppressed the expression of iNOS, COX-2, and proinflammatory cytokines IL-6 and TNF-α. Conclusion： Quercetin protects rat DRG neurons against HG-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition, thus may be beneficial for the treatment of diabetic neuropathy.

Aim： To investigate the influences of breviscapine, a flavonoid extracted from Erigeron breviscapus, on the proliferation and migration of vascular smooth muscle cells （VSMCs） cultured in a high glucose medium and the underlying mechanisms. Methods： VSMCs were isolated from thoracic aortas of male Sprague-Dawley rats and cultured in vitro. Cell proliferation was evaluated using Counting Kit-8 cell viability assay. Cell migration was evaluated using transwell migration assay and in vitro scratch assay. The expression and activity of protein kinase C-132 （PKC-132）, extracellular signal-regulated kinase 1/2 （ERK1/2）, p38 mitogen-activated protein kinase （p38）, and JNK mitogen-activated protein kinase （JNK） were measured with Western blotting. Results： Exposure of VSMCs to a high glucose （25 mmol/L） medium significantly increased the proliferation and migration potential as compared to the control group. Pretreatment with breviscapine （65 pmol/L and 108 pmol/L） attenuated high glucose-enhanced pro- liferation and migration of VSMCs. Exposure of VSMCs to the high glucose medium activated both the PKC-132 and ERK1/2 MAPK, but not the p38 and JNK MAPK. Pretreatment with breviscapine （65 pmol/L and 108 pmol/L） blocked high glucose-induced increase of the ERK1/2 activity, but not that of the PKC-132 activity. Conclusion： Our study demonstrated that breviscapine ameliorates high glucose-induced proliferation and migration of VSMCs via inhibiting ERK1/2 MAPK signaling.

Aim： To investigate the effect of farrerol, a major active component isolated from a traditional Chinese herb ＂Man-shan-hong＂ （the dried leaves of Rhododendron dauricum L） on fetal bovine serum （FBS）-induced proliferation of cultured vascular smooth muscle cells （VSMCs） of rat thoracic aorta. Methods： VSMCs proliferation, DNA synthesis and cell cycle progression were studied using the MTT assay, bromodeoxyuridine （BrdU） incorporation and flow cytometry, respectively. The mRNA levels of cell cycle proteins were quantified using real-time RT-PCR, and the phosphorylation of ERK1/2 was determined using Western blotting. Reporter gene and receptor binding assays were employed to study the interaction between farrerol and estrogen receptors （ERs）. Results： Farrerol （0.3-10 pmol/L） inhibited VSMC proliferation and DNA synthesis induced by 5% FBS in a concentration-dependent manner. The effects were associated with G1 cell cycle arrest, down-regulation of cell cycle proteins and reduction in FBS-induced ERK1/2 phosphorylation. Using a reporter gene, it was found that farrerol （3 μmol/L） induced 2.1-fold transcription of ER. In receptor binding assays, farrerol inhibited the binding of [3H]estradiol for ERα and ERβ with ICsovalues of 57 pmol/L and 2.7 μmol/L, respectively, implying that farrerol had a higher affinity for ERβ. Finally, the inhibition of VSMC proliferation by farrerol （3 μmol/L） was abolished by the specific ERβ antagonist PHTPP （5 μmol/L）. Conclusion： Farrerol acts as a functional phytoestrogen to inhibit FBS-induced VSMC proliferation, mainly via interaction with ERβ, which may be helpful in the treatment of cardiovascular diseases related to abnormal VSMCs proliferation.

In order to investigate the expression of glycerol-3 -phosphate dehydrogenase by GCY1 gene in recombinant Saccharomyces cerevisiae, induction culture of the S. cerevisiaestrain was performed with SD-URA 2% galactose, 3 × YP ＋ 6% glucose, SC-URA 2% galactose, and SC-URA 2% galactose ＋ 5% NaCI glyeerol-3-phosphate dehydregenase, the cultured S. cerevisiaewas comminuted followed by full-automatic high-speed purification, and SDS-PAGE gel electrophoresis was performed for molecular weight of the GST fusion protein. The results showed that after shaking culture of the S. cerevisiae containing GCY1 at 25 ℃, the OD values of its 3 × YP ＋ 6% glucose culture and SC-URA 2% galaetose ＋ 5% NaC1 culture were 8.75 and 7.35, respectively. It was shown by purification with a Profinia low-pressure liquid chromatograph that only the S. cerevisiae cultured in SC-URA 2% galactose ＋ 5% NaC1 medium expressed glycerel-3-phosphate de- hydrogenase, the molecular weight of which was detected as 65 ku by SDS-PAGE gel electrophoresis.

The objective of this study was to investigate the effects of mineralized bone nodules, formed in vitro by bone marrow stromal cells （BMSCs）, on the new bone formation in bone defect and on implant surface. The mineralized bone nodules were generated by culture of Lewis rats BMSCs on titanium disks in osteogenic induction medium. The gap-healing animal model was used to create the bone defect facing the disk. The titanium disks in the presence orB group or in the absence of NB group bone nodules were randomly placed into one of the rat distal femurs. This self-control design was used to compare the bone formation in defects and on titanium surface, by Micro-CT, fluorescence staining, histological and histomorphometric analysis. The new bone formation parameters in bone defect area of B group were significantly higher than those of NB group at 2 weeks, including bone volume fraction, trabecular thickness and bone area ratio. The bone nodules pre-stained with Alizarin red disappeared mostly at 2 weeks, while the red fluorescence reappeared in the newly formed bone away from the disk surface. For the bone-implant contact, B group showed lower values than NB group at 2 weeks, but no significant difference was found at 4 weeks. Our results indicate that the mineralized bone nodules can be resorbed in vivo and promote the early osteogenesis in the bone defects, and bone nodules may be applicable for new bone generation in bone defect or modification of tissue engineering scaffold.

[ Objective ] This study aimed to explore immature embryo culture of Lagerstroemia indica and investigate the appropriate conditions for growth and differentiation. [ Method] Immature embryos of L. indica were employed as the explants for germination induction to establish aseptic lines. Based on that, the effects of different hormone levels and culture conditions on immature embryo culture of L. indica were analyzed. [ Result ] Peeled immature embryos of L. indica were germinated easily, leading to a germination rate of 100%. The optimal initial medium was MS ＋ BA0.5 ＋ NAA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ; the optimal shoot induction medium was MS ＋ BA0.5 ＋ NAA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ＋ coconut milk 10% ; the optimal rooting medium was MS ＋ BA0.5 ＋ IBA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ＋ coconut milk 10%. [ Conclusion] This study provided a technical reference for subsequent optimized breeding of L. indica.

The difference between Noggin and basic fibroblast growth factor for the neural precursor differen- tiation from human embryonic stem cells has not been studied. In this study, 100 tJg/L Noggin or 20 IJg/L basic fibroblast growth factor in serum-free neural induction medium was used to differen- tiate human embryonic stem cells H14 into neural precursors using monolayer differentiation. Two weeks after induction, significantly higher numbers of neural rosettes formed in the Noggin-induced group than the basic fibroblast growth factor-induced group, as detected by phase contrast micro- scope. Immunofluorescence staining revealed expression levels of Nestin, [3-111 Tubulin and Sox-1 were higher in the induced cells and reverse-transcription PCR showed induced cells expressed Nestin, Sox-1 and Neurofilament mRNA. Protein and mRNA expression in the Noggin-induced group was increased compared with the basic fibroblast growth factor-induced group. Noggin has a greater effect than basic fibroblast growth factor on the induction of human embryonic stem cell differentiation into neural precursors by monolayer differentiation, as Noggin accelerates and in- creases the differentiation of neural precursors.

The present study analyzed changes in Wnt3a expression during differentiation of adipose-derived stern cells into cholinergic neurons. Immunocytochemistry and immunofluorescence revealed significantly increased nestin, neuron-specific enolase, microtubule-associated protein 2, and choline acetyltransferase expression in adipose-derived stem cells isolated from Sprague-Dawley rats and cultured in vitro in neural-induced medium. These expressions increased with prolonged induction time. Real-time reverse transcription-PCR and western blot assay results demonstrated significantly increased choline acetyltransferase and Wnt3a protein and mRNA expressions, respectively, in adipose-derived stem cells following induction. Choline acetyltransferase expression positively correlated with Wnt3a protein and mRNA expressions. These results demonstrated that neural-induced medium induced differentiation of adipose-derived stem cells into cholinergic neuronal-like cells, with subsequent increased Wnt3a expression.

[ Objective] Peanut in vitro regeneration system was optimized to provide technical support for its genetic transformation and mutagenesis. [ Method ] The effect of gibberellin, light and genotype on somatic embryogenesis and plantiet regeneration of peanut was studied using Guihua peanut cuhivar as materials, leallets as explants, MS ＋ 10 mg/L 2, 4-D as somatic embryo induction medium, and MS ＋3 mg/L 6-BA ＋0. 8 mg/L NAA ＋ （0 - 15 mg/L） GA3 as plantlet induction me- dium. [Result]The somatic embryo induction rate of five peanut varieties under light condition of light： dark = 14 h： 10 h was significantly higher compared with that under dark condition by 7, 5 - 37.5 percent points. Among different peanut varieties, Guihua 26 represented the highest somatic embryo induction rate of 62. 8%, while Guihua 833 represented the lowest somatic embryo induction rate of 21.7%. Adding 5 - 15 mg/LGA3 in plantlet induction medium was conducive to improving the induction rate of plandets derived from somatic embryos. With addition of 5 mg/L GA3, Guihua 26 and Guihna 771 represented the highest plantlet induction rate of 42.8% and 35.3%, respectively; with addition of 15 mg/L GA3, Guihua 836, Guihua 1026 and Guihna 833 represented the highest plantlet in- duction rate of 38.7%, 33.3% and 26.4L%, respectively. Among different combinations of peanut variety and GA3 concentration, plantlet induction rate reached the highest in the combination Guihua 26 ＋ 5 mg/L CA.3 arid reached the lowest in the combination Guihua 836 ＋ 15 mg/L GA3. [ Conclusion] Appropriate light conditions are conducive to peanut somatic embryogenesis in vitro. Adding GA3 in plantlet induction medium is conducive to promoting plantlet regeneration. Guihua 26 and Guihua 836 are the best peanut varieties among the experimental genotypes for somatic embryogenesis and plant regeneration.