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Background The development of new treatment strategies to improve peripheral nerve repair after injury, especially those that accelerate axonal nerve regeneration, is very important. The aim of this study is to elucidate the molecular mechanisms of how bone marrow stromal cell (BMSC)-derived exosomes (EXOs) participate in peripheral nerve regeneration and whether the regenerative effect of EXOs is correlated with dose. Method BMSCs were transfected with or without an siRNA targeting Ago2 (SiAgo2). EXOs extracted from the BMSCs were administered to dorsal root ganglion (DRG) neurons in vitro. After 48 h of culture, the neurite length was measured. Moreover, EXOs at four different doses were injected into the gastrocnemius muscles of rats with sciatic nerve crush injury. The sciatic nerve functional index (SFI) and latency of thermal pain (LTP) of the hind leg sciatic nerve were measured before the operation and at 7, 14, 21, and 28 days after the operation. Then, the number and diameter of the regenerated fibers in the injured distal sciatic nerve were quantified. Seven genes associated with nerve regeneration were investigated by qRT-PCR in DRG neurons extracted from rats 7 days after the sciatic nerve crush. Results We showed that after 48 h of culture, the mean number of neurites and the length of cultured DRG neurons in the SiAgo2-BMSC-EXO and SiAgo2-BMSC groups were smaller than that in the untreated and siRNA control groups. The average number and diameter of regenerated axons, LTP, and SFI in the group with 0.9 × 1010 particles/ml EXOs were better than those in other groups, while the group that received a minimum EXO dose (0.4 × 1010 particles/ml) was not significantly different from the PBS group. The expression of PMP22, VEGFA, NGFr, and S100b in DRGs from the EXO-treated group was significantly higher than that in the PBS control group. No significant difference was observed in the expression of HGF and Akt1 among the groups. Conclusions These results showed that BMSC-derived EXOs can promote the regeneration of peripheral nerves and that the mechanism may involve miRNA-mediated regulation of regeneration-related genes, such as VEGFA. Finally, a dose-effect relationship between EXO treatment and nerve regeneration was shown.

We investigated the occurrence of mesenchymal stem cell (MSC)-derived exosome uptake and retrograde transport at peripheral nerve endings using bone marrow MSCs (bMSCs) transduced with recombinant CD63-green fluorescent protein (GFP) lentiviral plasmid. GFP was used to track the release of bMSC-derived exosomes and the uptake and transport at peripheral nerve terminals, the dorsal root ganglion (DRG), and the spinal cord. In vitro cell culture and injection of a CD63-GFP exosome suspension into the right gastrocnemius muscle of an in vivo rat model were also performed. Fluorescence microscopy of co-cultured CD63-GFP exosomes and SH-SY5Y or BV2 cell lines and primary cultured DRG cells in a separate experiment demonstrated exosome uptake into DRG neurons and glia. Moreover, we observed both retrograde axoplasmic transport and hematogenous transport of exosomes injected into rat models at the DRG and the ipsilateral side of the anterior horn of the spinal cord using fluorescence microscopy, immunohistochemistry, and Western blot analyses. In conclusion, we showed that exosome uptake at peripheral nerve endings and retrograde transport of exosomes to DRG neurons and spinal cord motor neurons in the anterior horn can occur. In addition, our findings propose a novel drug delivery approach for treating neuronal diseases.

Objective To investigate whether Hainan papayas has protective effects in an Aβ40-induced primary neuron injury model and elucidate the underlying molecular mechanism. Methods Cultured primary neurons from the dorsal root ganglia (DRG) of Sprague–Dawley (SD) rats were treated with 20 μM Aβ40 peptide, 100 μg/L Hainan papaya water extract, peptide plus extract, or culture medium for 24 h. Cell viability was measured by MTT assay, and neuronal apoptosis was evaluated by DAPI staining. ERK signaling pathway-associated molecule activation and changes in Bax expression were analyzed by Western blotting and immunofluorescence. Results A cell viability rate of (44.11 ± 6.59)% in the Aβ40 group was rescued to (79.13 ± 6.64)% by adding different concentrations of the extract. DAPI showed pyknotic nuclei in 39.5% of Aβ40-treated cells; the fraction dropped to 17.4% in the 100 μg/L extract group. ERK phosphorylation was observed in the Aβ40 group but was ameliorated by pretreatment with 100 μg/L extract. Hainan papaya water extract also prevented Aβ40-induced phosphorylation of MEK, RSK1 and CREB associated with ERK signaling and downregulated Bax expression in the neurons. Conclusion The results suggest that Hainan papaya water extract has protective effects on neurons; the mechanism may be related to suppression of ERK signaling activation.

Crystallisation of proteins is usually achieved with the help of chemical agents. Because there are few general guidelines in determining what agents will help to crystallise a specific protein, suitable crystallisation agents are often found via exhaustive trial-and-error tests by mixing many chemical agents (the collection of which is called a crystallisation screening kit) one-by-one with the protein. Currently, many commercially available crystallisation screening kits have been developed and utilised in practical crystallisation screen experiments. However, information regarding the design of new screening kits has yet to be expanded using a large amount of experimental data. Here, we show the step-by-step design processes of a polyethylene glycol-based screening kit. It was found that the screening performance could be improved by modifying the crystallisation screening kits according to the accumulated data (such as those in the Biological Macromolecule Crystallisation Database (BMCD)), the screening test results and existing knowledge. The screening kit designed in this paper can be used for practical protein crystallisation screen experiments and the method can be used in the design of other crystallisation screening kits.

MicroRNAs (miRNAs) play an important role in the development, differentiation, proliferation, survival, and oncogenesis of cells and organisms including nervous system. However, the role of miRNAs in primary neurons of dorsal root ganglion (DRG) after injury was not clear. In this study, a miRNA microarray analysis was performed, and a total of 21 miRNAs were found to be down-regulated following unilateral sciatic nerve transection. The miR-144, miR-145, and miR-214 were further validated using quantitative reverse transcriptase PCR (qRT-PCR). Moreover, in situ hybridization (ISH) experiments using locked nucleic acid (LNA)-modified DNA oligonucleotide probes verified that miR-144, miR-145, and miR-214 were expressed in primary neurons of DRG and down-regulated following sciatic nerve transection. Predictions of potential miRNA targets involved were identified by performing a bioinformatics analysis. These predictions were tested using miRNA luciferase reporter vectors, with Robo2 and srGAP2 evaluated as the potential targets of miR-145 and miR-214, respectively. The role of miR-145 in cultured primary neurons was also investigated, and the result found that miR-145 miR-145 inhibited neurite growth and down-regulated Robo2 expression. Finding from this study suggested that miRNAs of DRG can mediated the course of regeneration including through Slit-Robo-srGAP signaling pathway after injury.

This paper presents a modification to the conventional vapor diffusion (hanging- or sitting-drop) technique for protein crystallization screening. In this modified method, the reservoir solution is replaced with a desiccant to allow for a larger range of protein solution concentrations, thereby providing more opportunities for crystal formation. This method was tested in both reproducibility and screening studies, and the results showed that it significantly improves the efficiency and reduces the cost of protein crystallization screens.

To evaluate the effects of five different surface treatments on the bond strength between fiber posts and resin cement.Fifty fiber posts were randomly divided into 5 groups for different surface treatments. Group A was treated with silane coupling agent (Clearfil Porcelain Bond Activator,Kuraray); Group B was treated with silane coupling agent and then coated with dentin bonding agent (Clearfil SE Bond,Kuraray); Group C was immersed in 30% H(2)O(2); Group D was immersed in 30 % H(2)O(2) and then treated with silane coupling agent; Group E received no surface treatment (control group). After bonding to resin cement, each group was then divided into 2 subgroups equally, while one group was stored in sodium chloride for 24 h at 37 degrees centigrade, and the other group was stored in sodium chloride for 24 h at 37 degrees centigrade and then subjected to thermal cycling for 10000 times. Microtensile bond strengths were tested and the data was statistically analyzed using SPSS17.0 software package.The microtensile bond strength before thermal cycling was (6.7±0.7) MPa for group A,(14.4±1.1) MPa for group B,(10.7±0.9) MPa for group C,(16.0±1.0) MPa for group D and (6.7±1.0) MPa for group E. After thermal cycling, the microtensile bond strength was (6.7±0.7) MPa for group A, (13.1±0.7) MPa for group B, (9.0±0.7) MPa for group C, (15.0±0.9) MPa for group D and (5.6±0.7) MPa for group E. The results showed that surface treatments had significant impact on the bond strength (P0.05). There was no significant difference between group A and group E. Group D had the highest bond strength compared with the other groups. Microtensile bond strengths were significantly different before and after thermalcycling treatment in each group (P0.05).Thermocycling treatment decreases the bond strength of fiber posts to resin cement with these 5 surface treatments. Coupled with 30 % H(2)O(2) solution and silanization, the bonding strength of fiber posts to resin cement can increase significantly.

To evaluate the effect of different bonding procedure and different root region on the bond strength of fiber posts to root canal.Forty-two extracted intact human maxillary central incisors were endodontically treated with post spaces be prepared and divided into 6 groups randomly. The fiber posts were then luted with the following different bonding procedures. Group I: total etch dentin bonding agent Luxabond + self-adhesive system Rely XTM Unicem; Group II: total etch dentin bonding agent Luxabond + dual-cure rein cement Luxacore; Group III: self etch dentin bonding agent Contax + self -adhesive system Rely X TM Unicem; Group IV :self etch dentin bonding agent Contax + dual -cure rein cement Luxacore; Group V:self-adhesive system Rely X TM Unicem; Group VI:dual-cure rein cement Luxacore. After dowel cementation, thin-slice push-out test was performed. The bonding interface and dentin surface were examined under scanning electron microscope. The data was statistically analyzed using SPSS13.0 software package.The results showed that different bonding procedures had significant different impacts on the bond strength (P0.05).Group I and Group III had significantly higher bond strength compared with other groups. Resin tags of dentin bonding interface were observed in Group I and Group II and intimate adaptations of resin cement with the substrate were revealed.Coupled with total etch or self etch dentin bonding agent, self- adhesive system Rely X TM Unicem can improve the bonding strength of fiber posts obviously.

The Slit–Robo GTPase-activating proteins (srGAPs) play an important role in neurite outgrowth and axon guidance; however, little is known about its role in nerve regeneration after injury. Here, we studied the expression of srGAPs in mouse dorsal root ganglia (DRG) following sciatic nerve transection (SNT) using morphometric and immunohistochemical techniques. Reverse transcriptase polymerase chain reaction and Western blot analysis indicated that srGAP1 and srGAP3, but not srGAP2, were expressed in normal adult DRG. Following unilateral SNT, elevated mRNA and protein levels of srGAP1 and srGAP3 were detected in the ipsilateral relative to contralateral L3–4 DRGs from day 3 to day 14. Immunohistochemical results showed that srGAP1 and srGAP3 were largely expressed in subpopulations of DRG neurons in naive DRGs. However, after SNT, srGAP3 in neurons was significantly increased in the ipsilateral relative to contralateral DRGs, which peaked at day 7 to day 14. Interestingly, DRG neurons with strong srGAP3 labeling also coexpressed Robo2 after peripheral nerve injury. These results suggest that srGAPs are differentially expressed in murine DRG and srGAP3 are the predominant form. Moreover, srGAP3 may participate in Slit–Robo signaling in response to peripheral nerve injury or the course of nerve regeneration. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

To study The protective effect of puerarin on Abeta(25-35)-induced PC12 cell injury.PC12 cells were treated with puerarin for 0.5 h, then incubated with Abeta(25-35) (50 micromol/L) for 24 h to investigate the production of reactive oxygen species (ROS), mitochondrial membrane potential levels and Caspase-3 activation; The expressions of Bax, bcl-2 were measured by Western Blotting.Preincubation of the cell with puerarin could inhibit the ROS and increase mitochondrial membrane potential levels. Puerarin was also found to increase the Bcl-2/Bax ratio and reduce Caspase-3 activation.Puerarin may act as an intracellular ROS scavenger, and its antioxidant properties may protect against Abeta(25-35)-induced cell injury.

To investigate the adequate luting cements for zirconia ceramics to dentin.Blocks of sintered zirconia ceramics were randomly divided into 4 groups with 8 slices in each. After saliva immersion,airborne-particle abraded ceramic specimens were cleaned with phosphoric acid gel(containing 35% phosphoric acid) and then bonded to dentin with these four kinds of luting cements. After preserved in 37 degrees centigrade distilled water for 24 hours, the shear bonding strength of these specimens was tested and the data was analyzed with SPSS12.0 software package.The Multilink Automix could attain the highest shear bonding strength and the 3M RelyXTM Unicem AplicapTM could attain higher shear bonding strength, which were both significantly higher than in the Tokuso Ionomer and Shofu Polycarboxylate Cement groups(P0.05).Total etching resin luting cement is an ideal option to the bonding of zirconia ceramics and can provide a strong bonding.

To investigate the effect of silica coating by sol-gel process on bonding strength of zirconia ceramics to dentin.Blocks of sintered zirconia ceramics were cut and randomly divided into 4 groups,16 slices in each group. Each group was subject to one of the 4 kinds of surface treatment (control group, sandblasting, sandblasting +silicone, sandblasting + silica coating + silicone) and then bonded to dentin with resin cement. After preservation in 37 degrees centigrade distilled water for 24 hours, the shear bonding strength of these specimens was tested and the data was analyzed with SAS6.12 software package for analysis of variance. The surface modality of the ceramics was observed under scanning electron microscopy (SEM).The group of sandblasting+ silica coating + silicone attained the highest shear bonding strength, which was significantly different from the other groups(P=0.000);There was no significant difference between the sandblasting and sandblasting + silicone group (P=0.827), which was significantly different from the control group(P=0.001).Silica coating by sol-gel process, coupled with silicone, can significantly increase the bonding strength of zirconia ceramics to dentin.

Secreted Slit proteins have previously been shown to signal through Roundabout (Robo) receptors to negatively regulate axon guidance and cell migration. During vertebrate development, Slit proteins have also been shown to stimulate branching and elongation of sensory axons and cortical dendrites. In this study, Slit1/Robo2 mRNA and protein expressions were detected in adult rat dorsal root ganglion (DRG) and in cultured DRG neurons. Treatment of both models with recombinant, soluble Slit1 protein was found to promote neurite outgrowth and elongation. In contrast, treatment with a recombinant human Robo2/Fc chimera inhibited neurite outgrowth and elongation. When adult DRG and cultured DRG neurons were pretreated with soluble recombinant human Robo2/Fc chimera, neurite outgrowth and elongation was not induced. These findings indicate that Slit1/Robo2 signaling may have a role in regulating peripheral nerve regeneration.

To study the bonding strength of zirconia ceramics with 4 kinds of luting cement materials.Blocks of sintered zirconia ceramics were cut and randomly divided into 4 groups with 16 slices in each group. They were treated with sandblasting and bonded with 4 kinds of luting cements respectively. After preserved in 37 degrees C distilled-water for 24 hours and 30 days, the shear bonding strength of these specimens was tested and the data were analyzed by SAS6.12 software package and bond section were observed by scanning electron microscope.Two-way ANOVA revealed that the group of PanaviaF could attain the highest shear bonding strength: (34.7+/-3.44) MPa (after 24 hours), (31.5+/-3.44) MPa (after 1 month), which was significantly different from other treatment methods (P0.01). The initial shear bonding strength of the groups of resin-reinforced glass ionomer was (15.5+/-2.71) MPa, (16.0+/-1.77) MPa (after 24 hours) but dropped markedly to (6.80+/-1.24) MPa, (3.38+/-2.32) MPa after 30 days (P0.05).Resin luting cement containing phosphate monomer (MDP) can provide zirconia ceramics a strong and long-lasting bonding. Resin-reinforced glass ionomer can get good bonding strength too, but can't last long.

To study the influence on the abutment movement with three kinds of precision attachments designed for distal-extension removable partial dentures (RPDs).Snap attachment, Ceka attachment, Mini-SG attachment were used to retain RPDs. Then food chip was masticated between the upper and lower dentitions on the attachments side. Placement of abutment was measured by LB-72 high-sensitive-laser-traneferring-detector and the data were statistically analyzed for random block analysis of variance.The displacement, inclination, and torsion of the abutment movements in the mesial-distal, bucco-lingual and occluso-gingival directions all had statistical significance (P0.05). The largest mesial-distal inclination occurred with the Mini-SG attachment. The Ceka attachment produced more abutment displacement in mesial distal and apical directions. Occlusal displacement and anti-clockwise rotation were the least with the Snap attachment.The most equitable distribution of stresses on the supporting structures was achieved by Snap attachment with its resilent assemble, Ceka attachment transfers most of the loading forces to the abutments, Mini-SG attachment can not reduce the excessive torque applied to the abutments effectively.

Objective To explore effect of srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons. Methods In this study, expression of Slit1 was observed predominantly in the glia, while expression of Robo2 and srGAP3 was detected in sensory neurons of postnatal rat cultured dorsal root ganglion (DRG). Furthermore, upregulation of srGAP3 following sciatic nerve transection was detected by immunohistochemistry and Western blotting. Results It was observed that inhibition of neurite outgrowth in cultured adult DRG neurons following treatment with anti-srGAP3 or anti-Robo2 was more effectively (1.5-fold higher) than that following treatment with an anti-BDNF positive control antibody. It demonstrated that srGAP3 interacted with Robo2 and Slit1 protein to decrease Rac1-GTP activity in cultured adult rat DRG neurons and the opposite effect on Rac1-GTP activity was detected by co-immunoprecipitation and immunoblotting analyses following treatment with anti-Robo2 or anti-srGAP3. These data demonstrated a role for srGAP3 in neurite outgrowth of DRG sensory neurons. Conclusions Our observations suggest that srGAP3 promotes neurite outgrowth and filopodial growth cones by interacting with Robo2 to inactivate Rac1 in mammalian DRG neurons.