Overexpression of OsAGO1b Induces Adaxially Rolled Leaves by Affecting Leaf Abaxial Sclerenchymatous Cell Development in Rice.

BACKGROUND: ARGONAUTE 1 (AGO1) proteins can recruit small RNAs to regulate gene expression, involving several growth and development processes in Arabidopsis. Rice genome contains four AGO1 genes, OsAGO1a to OsAGO1d. However, the regulatory functions to rice growth and development of each AGO1 gene are still unknown. RESULTS: We obtained overexpression and RNAi transgenic lines of each OsAGO1 gene. However, only up- and down-regulation of OsAGO1b caused multiple abnormal phenotypic changes in rice, indicating that OsAGO1b is the key player in rice growth and organ development compared with other three OsAGO1s. qRT-PCR assays showed that OsAGO1b was almost unanimously expressed in leaves at different developmental stages, and strongly expressed in spikelets at S1 to S3 stages. OsAGO1b is a typical AGO protein, and co-localized in both the nucleus and cytoplasm simultaneously. Overexpression of OsAGO1b caused adaxially rolled leaves and a series of abnormal phenotypes, such as the reduced tiller number and plant height. Knockdown lines of OsAGO1b showed almost normal leaves, but the seed setting percentage was significantly reduced accompanied by the disturbed anther patterning and reduced pollen fertility. Further anatomical observation revealed that OsAGO1b overexpression plants showed the partially defective development of sclerenchymatous cells on the abaxial side of leaves. In situ hybridization showed OsAGO1b mRNA was uniformly accumulated in P1 to P3 primordia without polarity property, suggesting OsAGO1b did not regulate the adaxial-abaxial polarity development directly. The expression levels of several genes related to leaf polarity development and vascular bundle differentiation were observably changed. Notably, the accumulation of miR166 and TAS3-siRNA was decreased, and their targeted OSHBs and OsARFs were significantly up-regulated. The mRNA distribution patterns of OSHB3 and OsARF4 in leaves remained almost unchanged between ZH11 and OsAGO1b overexpression lines, but their expression levels were enhanced at the regions of vascular bundles and sclerenchymatous cell differentiation. CONCLUSIONS: In summary, we demonstrated OsAGO1b is the leading player among four OsAGO1s in rice growth and development. We propose that OsAGO1b may regulate the abaxial sclerenchymatous cell differentiation by affecting the expression of OSHBs in rice.

PLA/PEG-PPG-PEG/dexamethasone implant prepared by hot-melt extrusion for controlled release of immunosuppressive drug to implantable medical devices, Part 2: in vivo evaluation.

Hot-melt extrusion (HME) plays an important role in preparing implants as local drug delivery systems in pharmaceutical fields. Here, a new PLA/PEG-PPG-PEG/Dexamethasone (PLA/F68/Dex) implant prepared by HME has been developed. Importantly, the implant was successfully achieved to control release of immunosuppressive drug to an implanted device. In particular, this implant has not been reported previously in pharmaceutical fields. FTIR and XRD were adopted to investigate the properties of the samples. The in vivo release study showed that the maximum value of Dex release from the implants was approximately 50% at 1 month. The in vivo degradation behavior was determined by UV spectrophotometer and scanning electron microscopy studies, and the weight loss rate of the implants were up to 25% at 1 month. Furthermore, complete blood count (CBC) test, serum chemistry and major organs were performed, and there is no significant lesion and side effects observed in these results. Therefore, the results elucidated that the new PLA/F68/Dex implant prepared by HME could deliver an immunosuppressive drug to control the inflammatory reaction at the implant site.

Preparation and characterization of poly(pluronic-co-L-lactide) nanofibers for tissue engineering.

In this work, a new kind of biodegradable poly(pluronic-co-L-lactide) (Pluronic-PLLA copolymers) was successfully prepared by melt-polycondensation method from L-lactide, Pluronic and isophorone diisocyanate (IPDI). The obtained copolymers were characterized by (1)H NMR, FT-IR, X-ray, and TGA/DTA. Meanwhile, three-dimensional (3-D) porous scaffolds based on Pluronic-PLLA were prepared by the electrospinning method, the factors of concentration, flow rate and voltage that influence the formation of the Pluronic-PLLA nanofibers were studied and the structure of Pluronic-PLLA nanofibers were investigated by scanning electron microscopy (SEM). MTT results revealed that the Pluronic-PLLA scaffolds had good biocompatibility and nontoxicity. Morphological study using fluorescence micrographs and scanning electron microscopy showed that in vitro osteoblast cell culture demonstrated the electrospun Pluronic-PLLA composite scaffolds could provide a suitable environment for good cell attachment. These results suggested that such Pluronic-PLLA nanofibers membranes might have prospective applications in tissue engineering field.

PLA/F68/dexamethasone implants prepared by hot-melt extrusion for controlled release of anti-inflammatory drug to implantable medical devices: I. Preparation, characterization and hydrolytic degradation study.

Dexamethasone (Dex)-loaded implants were prepared by poly(d,l-lactic acid) (PLA) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) copolymer (PEG-PPG-PEG, Pluronic F68) using hot-melt extrusion method. The purpose of this research was to develop a hot-melt extruded implant PLA/F68/Dex for controlling release of Dex at the implant site. Drug loading and encapsulation efficiency were determined by UV spectrophotometer analysis. The maximum value of the drug loading and encapsulation efficiency for the implants was up to 48.9% and 97.9%, respectively. Differential scanning calorimetry was used to evaluate stability and interaction between the implant and drug. We had studied the water uptake of PLA/F68 implants kept constant at about 12% due to the water was absorbed to a large extent. Dex release profile in vitro was studied, and the results showed that the maximum value of the release rate was approximately 20%. The degradation behavior was confirmed by mass loss and scanning electron microscopy. In addition, the in vivo biocompatibility study indicated that the implants had no negative influence as a foreign material in the body response.