Cell viability of Pichia pastoris was detected by flow cytometry (FCM) with two reagents fluorescein diacetate (FDA) and propidium iodide (PI). Compared with FDA/PI double-stained dot plots and PI single-stained dot plots,the latter could divide dead and living cells into two separate zones,and get the correct proportion. Then PI single-stained method was used to detect the change of cell viability in Pichia patoris fermentation. At glycerol batch and fed-batch phase,little dead cells were detected. At methanol fed-batch phase,cell viability decreased when cell weight increased,and was only 73.8% at 88 h.

BACKGROUND:A new open“C”-shaped molar band with nanocoating (patent number:ZL201110057699.1) has been invented to overcome some weakness of traditional bands and buccal tubes, which is used for molar fixation. OBJECTIVE:Using three-dimension finite element analysis software to theoretical y prove that the new band comes up to clinic requirement. METHODS:We established a finite element model of alveolar bone-tooth-band, and node domains tensile and shear loading were given. Stress distribution of the new bands through numerical simulation was predicted and calculated. Simulation of 20 N orthodontic force was applied in medial direction, distal direction and vertical direction in the three-dimensional model of a new band buccal tube and traditional band, to analyze the stress state in different directions under the force of the binder. RESULTS AND CONCLUSION:By numerical simulation of three-dimensional finite element, the stress of the new band was higher than that of the traditional band, but it could not reach the clinical cracking limit of resin reinforced glass ionomer cement. So the new band could theoretical y satisfy the clinical requirements.

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Proteolytic degradation has been a severe problem when Pichia pastoris is employed to express recombinant proteins.One alternative method to circumvent this problem is to construct protease gene disruptant.However,the main study of gene disruption is focused on nonrecombinant Pichia pastoris rather than recombinant strain.In our study,we established two different methods to directly disrupt PRC1 and KEX1 gene in recombinant Pichia pastoris.On the basis of this,we further discussed and compared the application and advantages of both methods.

The kinase domain of receptor tyrosine kinase(RTK) ErbB2 was expressed fused with GFP in Pichia pastoris. Recombinant expression vector pPIC3.5K was constructed in Escherichia coli TOP10. The right P. pastoris transformants were screened on his-deficient plates and YPD-G418 plates by turns after electroporation of recombinant vector, and then induced by methanol in baffled shake bottles. The strain with highest protein yield was scaled up in a 5 L fermentor. Recombinant protein was analyzed with tyrosine kinase assay after Ni2+ affinity chromatograph. Results showed that the 100 kD recombinant protein with tyrosine kinase activity was successfully expressed in P. pastoris.

The bioreactor production of recombinant Lateolabrax japonicus growth hormone (rljGH) expressed intracellularly by Pichia pastoris was investigated. A strategy of feeding methanol at the exponential rate was established and the effect of specific growth rate on the rljGH production was examined. The results indicated that the average specific production rate increased and the rljGH production duration decreased as the specific growth rate increased. The maximum specific rljGH production (0.58 mg/g WCW) was achieved at a specific growth rate of 0.029/h. The effect of supplementing ammonium sulfate, peptone and yeast ex- tract on the rljGH production was further investigated. The results indicated that the effects of ammonium sulfate and peptone were not significant. Supplementing yeast extract of 2.5 g/L was advantageous for the rljGH production. The duration of the rljGH production was increased to 23 h from 17 h and the fermenta- tion stability of run-to-run could be improved.

A fusion expression vector pPIC3.5K-PDGFR? was constructed to express recombinant receptor tyrosine kinase PDGFR? and the right Pichia pastoris transformants were screened on his-deficient plates and YPD-G418 plates by turns after electroporation of strain GS115, a high yield strain named M3 was screened. The strain M3 was cultured in a 5 L fermentor and His-GFP-PDGFR? fusion protein was purified by Ni2+ chelating affinity chromatography. One distinct peak was obtained after elution with 250 mmol/L imidazole. Fusion protein was proved to be 90.08 kD by western blotting, and have tyrosine kinase activity by ELISA. Results showed that the receptor tyrosine kinase PDGFR? was successfully expressed in P. pastoris and could be used as a target for small molecule selective inhibitors screening.

Objective To observe the effect of scalp acupuncture and rehabilitation training on the regulation of neuro-plasticity-associated protein MAP-2 in gerbils modelling cerebral ischemia and reperfusion.Methods Cerebral ischemia and reperfusion were modelled into thirty-six male gerbils.They were then randomly divided into a model group (A),a scalp acupuncture and rehabilitation training group (B) and a rehabilitation training group (C),each of 12 cases.The scalp acupuncture and rehabilitation training were administered daily to the gerbils in group B beginning 24 h after modeling,for 14 d.Any changes in Bederson scores were observed after one,7 and 14 days.On the 14th day the expression of MAP-2 around the infarct focus was detected using western blotting in all 3 groups.Results On the 7th day the average Bederson score in group B (1.81 ± 0.52) was not significantly different from that in group A (2.13 ± 0.49) or group C (2.00 ± 0.31) (P ＞ 0.05).At the end of the treatment,however,there were significant differences between the groups,with group B (0.47 ± 0.31) scoring significantly better than groups C (1.04 ± 0.63) and A (1.46 ± 0.72) (P ＜ 0.05).Group C was also significantly better than group A (P ＜0.05).The expression of MAP-2 as measured as integrated optical intensity (IOD) in group B (0.91 ±0.18) was significantly higher than that in group A (0.43 ± 0.21) and group C (0.67 ± 0.24) (P ＜ 0.05) ; so was group C compared to group A (P ＜0.05).Conclusion The scalp acupuncture combined with rehabilitation training can improve the recovery of motor function and enhance the expression of neuro-plasticity-associated protein MAP-2 in gerbils with after cerebral ischemia and reperfusion.

Objective To observe the therapeutic effects of oral placement therapy (OPT) on managing sialorrhea after stroke.Methods A total of 37 stroke inpatients with sialorrhea were enrolled from January 2011 to September 2013 in the authors' department for the study and divided into 2 group on the basis of the time of enrollment.The control group (n =18) received 30min of routine treatment (including such neuromuscular facilitation techniques as Bobath and Rood techniques,motor relearning program,neuromuscular electrical stimulation and ice stimulation),twice daily,and the treatment group (n =19) received 15 min of routine treatment plus 15 min of OPT,twice daily.Frenchay Dysarthria Assessment was used to evaluate the sialorrhea severity and clinical efficacy before and 1,2 and 4 weeks after initiation of treatment.Results Sialorrhea symptom was significantly improved in treatment group after 1 week (P ＜ 0.05),while no significant improvement was observed in control group (P ＞ 0.05).After 2 and 4 weeks of treatments,significant improvements of sialorrhea were noted in both groups.After 1,2 and 4weeks of treatment,the total effective rate were 63.16％,94.74％ and 94.74％,respectively,in the treatment group,versus 5.88％,61.11％ and 61.11％,respectively,in the control group.The total effective rate of the treatment group were significantly higher in the above three time-points than the control group (P ＜ 0.05).Conclusions Oral placement therapy can improve sialorrhea symptom after stroke more effectively than routine treatment.

Glucose was transported by the large number of hexose transporters in yeast cells. There were 18 hexose transporter genes had been identified in Saccharomyces cerevisiae. However,as an excellent expression system,there was no information of these genes had been reported in Pichia pastoris. Based on high homologous recombination efficiency in yeast,we chose G418 resistance for screening,200 bp were cloned from the up and down sequences of HXT1 ORF respectively,then ligated to the 5′ and 3′ end of G418 resis-tance gene for recombination. After electroporation of GS115 spheroplast and screened through different G418 concentration plates,finally we obtained one HXT1 gene deletion mutant named GS115?HXT1. The growth rate and glucose consumption of this mutant were both lower than the wide type.