OBJECTIVE:To systematically evaluate the clinical effectiveness of rehabilitation robots in treating lower limb motor dysfunction in patients with cerebral palsy,and to compare the differences in therapeutic effects among different robots.METHODS:PubMed,Web of Science,Embase,Cochrane Library,CNKI,CBM,VIP and WanFang databases were searched to collect randomized controlled trials on rehabilitation robotics for the treatment of motor dysfunction in patients with cerebral palsy,published from database inception to April 10,2024.The main outcome indicators included muscle strength,muscle tension,balance function,step speed,step frequency,step length,walking endurance,lower limb motor function,and activities of daily living.The above indicators were coded according to the International Classification of Functioning,Disability and Health.Meta-analysis was performed to evaluate the clinical efficacy and compare the therapeutic efficacy of different rehabilitation robots.Literature search and screening were performed by two researchers,and the quality of the included literature was evaluated using the Cochrane 5.1.0 risk of bias assessment tool.Meta-analysis was performed using RevMan 5.4 software and Stata 16.0 software.RESULTS:(1)Fifteen articles were finally included,involving 512 patients with 260 in the experimental group and 252 in the control group.(2)The Meta-analysis results showed that rehabilitation robots could improve body structure and function[standardized mean difference=0.41,95%confidence interval(CI):0.24-0.58,P<0.05],activities(standardized mean difference=0.53,95%CI:0.41-0.65,P<0.05)and participation ability(mean difference=7.86,95%CI:1.54-14.18,P<0.05).In particular,the rehabilitation robot improved lower limb muscle strength,balance function,step speed,walking endurance,lower limb gross motor function,and activities of daily living in patients with cerebral palsy,but showed insignificant effects on step frequency,step length,and muscle tension.(3)The network Meta-analysis results showed that:step speed:Innowalkpro>Gait Trainer>Lokomat>3DCalt;6-minute walk test score:Gait Trainer>Lokomat>Lokohelp>Innowalkro;Gross Motor Function Measure-88D score:Lokohelp>Lokomat>KidGo>Innowalkpro>3DCalt;Gross Motor Function Measure-88E score:Lokomat>Lokohelp>KidGo>3DCalt>Innowalkpro.CONCLUSION:Based on the International Classification of Functioning,Disability,and Health,rehabilitation robot training can improve the lower limb motor function and activities of daily living in patients with cerebral palsy.The Innowalkpro robot was more effective in improving step speed;the Gait trainer robot was more effective in improving 6-minute walk test scores;the Lokohelp robot was more effective in improving Gross Motor Function Measure-88D zone scores;and the Lokomat robot was more effective in improving Gross Motor Function Measure-88E zone scores.

OBJECTIVE:To systematically evaluate the clinical effectiveness of rehabilitation robots in treating lower limb motor dysfunction in patients with cerebral palsy,and to compare the differences in therapeutic effects among different robots.METHODS:PubMed,Web of Science,Embase,Cochrane Library,CNKI,CBM,VIP and WanFang databases were searched to collect randomized controlled trials on rehabilitation robotics for the treatment of motor dysfunction in patients with cerebral palsy,published from database inception to April 10,2024.The main outcome indicators included muscle strength,muscle tension,balance function,step speed,step frequency,step length,walking endurance,lower limb motor function,and activities of daily living.The above indicators were coded according to the International Classification of Functioning,Disability and Health.Meta-analysis was performed to evaluate the clinical efficacy and compare the therapeutic efficacy of different rehabilitation robots.Literature search and screening were performed by two researchers,and the quality of the included literature was evaluated using the Cochrane 5.1.0 risk of bias assessment tool.Meta-analysis was performed using RevMan 5.4 software and Stata 16.0 software.RESULTS:(1)Fifteen articles were finally included,involving 512 patients with 260 in the experimental group and 252 in the control group.(2)The Meta-analysis results showed that rehabilitation robots could improve body structure and function[standardized mean difference=0.41,95%confidence interval(CI):0.24-0.58,P<0.05],activities(standardized mean difference=0.53,95%CI:0.41-0.65,P<0.05)and participation ability(mean difference=7.86,95%CI:1.54-14.18,P<0.05).In particular,the rehabilitation robot improved lower limb muscle strength,balance function,step speed,walking endurance,lower limb gross motor function,and activities of daily living in patients with cerebral palsy,but showed insignificant effects on step frequency,step length,and muscle tension.(3)The network Meta-analysis results showed that:step speed:Innowalkpro>Gait Trainer>Lokomat>3DCalt;6-minute walk test score:Gait Trainer>Lokomat>Lokohelp>Innowalkro;Gross Motor Function Measure-88D score:Lokohelp>Lokomat>KidGo>Innowalkpro>3DCalt;Gross Motor Function Measure-88E score:Lokomat>Lokohelp>KidGo>3DCalt>Innowalkpro.CONCLUSION:Based on the International Classification of Functioning,Disability,and Health,rehabilitation robot training can improve the lower limb motor function and activities of daily living in patients with cerebral palsy.The Innowalkpro robot was more effective in improving step speed;the Gait trainer robot was more effective in improving 6-minute walk test scores;the Lokohelp robot was more effective in improving Gross Motor Function Measure-88D zone scores;and the Lokomat robot was more effective in improving Gross Motor Function Measure-88E zone scores.

OBJECTIVE:To systematically evaluate the efficacy of rehabilitation exoskeleton robots on the lower limb motor function of stroke patients using Meta-analysis and to compare the efficacy of different lower limb exoskeleton robots,so as to provide a theoretical basis for the scientific selection of suitable exoskeleton robots for patients with post-stroke lower limb motor dysfunction. METHODS:Computer searches of the Cochrane Library,PubMed,Web of Science,Embase,CNKI,VIP,and WanFang Data were conducted to collect randomized controlled clinical studies on exploring lower extremity rehabilitation exoskeleton robots to improve lower limb motor function in stroke patients published from database inception to November 2022.Two researchers conducted the literature search and screening.The quality of the included literature was evaluated using the Cochrane 5.1.0 risk of bias assessment tool and the Jadad scale.Meta-analysis was performed using RevMan 5.4 and Stata 17.0 software. RESULTS:(1)Finally 22 publications were included,involving 865 patients(n=436 in the test group and n=429 in the control group),and the Jadad score showed that all the included articles were of high quality.(2)Meta-analysis results showed that the exoskeleton robot significantly improved the Fugl-Meyer Assessment of Lower Extremity score(mean difference[MD]=2.63,95%confidence interval[CI]:1.87-3.38,P<0.05),Berg Balance Scale score(MD=3.62,95%CI:1.21-6.03,P<0.05),Timed Up and Go score(MD=-2.77,95%CI:-4.48 to-1.05,P<0.05)and step frequency score(MD=3.15,95%CI:1.57-4.72,P<0.05)in stroke patients compared with the control group.However,there was no significant improvement in the Functional Ambulation Category Scale score(MD=0.30,95%CI:-0.01 to 0.61,P>0.05)and 6-minute walk test score(MD=3.77,95%CI:-6.60 to 14.14,P>0.05).(3)Network Meta-analysis results showed that compared with the conventional rehabilitation therapy,both the level-walking exoskeleton(MD=10.23,95%CI:3.81-27.49,P<0.05)and the body-weight support exoskeleton(MD=33.66,95%CI:11.49-98.54,P<0.05)improved the Fugl-Meyer Assessment of Lower Extremity score.Compared with the conventional rehabilitation therapy,body-weight support exoskeleton significantly improved the Berg Balance Scale scores(MD=79.86,95%CI:2.34-2 725.99,P<0.05).In terms of Fugl-Meyer Assessment of Lower Extremity and Berg Balance Scale scores,the ranking results were body-weight support exoskeleton>level-walking exoskeleton>conventional rehabilitation therapy.Compared with the conventional rehabilitation therapy,level-walking exoskeleton significantly improved the Functional Ambulation Category Scale score(MD=1.38,95%CI:1.00-1.90,P<0.05)and body-weight support exoskeleton significantly improved the Timed Up and Go score(MD=0.07,95%CI:0.01-0.51,P<0.05).In terms of Functional Ambulation Category Scale and Timed Up and Go scores,the ranking results were level-walking exoskeleton>body-weight support exoskeleton>conventional rehabilitation therapy. CONCLUSION:Rehabilitation exoskeleton robots can improve balance,walking and activities of daily living in stroke patients,with body-weight support exoskeleton being more effective in improving lower limb motor function and balance and level walking exoskeleton being more effective in improving functional walking and transfer.

Enterotoxigenic Escherichia coli（ETEC）infection can induce watery diarrhea，leading to dehydration，electrolyte disturbance，and even death in severe cases. Recombinant B subunit/inactivated whole-cell cholera（rBS/WC）vaccine is effective in preventing ETEC infectious diarrhea. On the basis of the latest evidence on etiology and epidemiology of ETEC，as well as the effectiveness，safety，and health economics of rBS/WC vaccine，National Clinical Research Center for Child Health（The Children’s Hospital，Zhejiang University School of Medicine）and Zhejiang Provincial Center for Disease Control and Prevention invited experts to develop expert consensus on rBS/WC vaccine in prevention of ETEC infectious diarrhea. It aims to provide the clinicians and vaccination professionals with guidelines on using rBS/WC vaccine to reduce the incidence of ETEC infectious diarrhea.

Objective To express and purify mammalian glycosylation modified trimeric Ebola virus trimeric glycoprotein (EBOV-GP) using novel glycoengineered Pichia pastoris.Methods The EBOV-GP and EBOV-GPΔMLDΔTM genes were cloned into the pPICZ-αA vector,electrochemically converted to glycoengineered Pichia pastoris,and compared with EBOV-GP expressed in HEK-293T cells.Glycosylation was analyzed by PNGaseF and EndoH digestion,while the target protein was purified by affinity chromatography and ion exchange chromatography.N-terminal sequencing was used to determine whether the signal peptide was correctly cleaved during protein translation and gel column analysis was used to find out whether the trimeric structure was formed.Results The results of PNGaseF showed that EBOV-GP expressed by glycoengineered Pichia pastoris and HEK-293T cells had the same relative molecular mass and N-glycosylation degree.EndoH digestion showed that the N-glycosylation modification of EBOV-GPΔMLDΔTM was in a non-high mannose form.N-terminal sequencing showed that the signal peptide of the GP protein itself was correctly excised.Gel column analysis showed that the purified protein was in a trimeric form.Conclusion An EBOV-GP is obtained with complex glycosylation modification based on Glycoengineered Pichia pastoris.

Objective To prepare recombinant human prothrombin-2 expressed in Pichia pastoris, and assay the enzymatic and clotting activities of prothrombin-2 activated by prothrombin activator ecarin.Methods Human prothrombin-2 gene and Echis carinatus ecarin gene were synthesized separately on the basis of the cDNA sequences published in GenBank.The gene of prothrombin-2 was cloned into the expression vector pPICZαA.The expression vector pPICZαA/prothrombin-2 was transformed into glycoengineered P.pastoris, and then prothrombin-2 engineered P.pastoris was screened.The expression products were induced by methanol, purified by two-step chromatography and identified by diges-tion by PNGase F and analysis of pepetide fingerprint.The ecarin gene was cloned into the expression vector pcDNA3.1. The expression vector pcDNA3.1/Ecarin was transformed into HEK 293T cells and the culture supernatant of HEK 293T/Ecarin was collected.The reaction product of HEK 293T/Ecarin cell culture supernatant and purified prothrombin-2 was analyzed by S-2238,which was the chromogenic substrate for thrombin.Fibrinogen was used to measure blood clotting time. Results The purified protein of P.pastoris expressed prothrombin-2 culture supernatant was 37 ×103 .The relative molecular mass(Mr) of the purified protein was reduced to 35 ×103, which was consistent with the theoretical Mr of prothrombin-2 molecular weight.The purified protein was proved to be prothrombin-2 by peptide fingerprint identification. The purified prothrombin-2 processed by HEK 293T/Ecarin culture supernatant could hydrolyze S-2238 to produce yellow pNA, and D405 of pNA increased with the volume of the processed prothrombin-2 that could promote the plasma coagulation.The blood clotting time was close to that of the thrombin kit.Conclusion Prothrombin-2 is prepared by P.pastoris and activated toα-thrombin by ecarin.This technique may replace the method of extraction of prothrombin from plasma and can be used for the treatment of war wounds or for future clinical research.

Objective To obtain a strain of glycoengineering yeast with higher N-glycosylation efficiency by overexpressing N-glycosyltransferase.Methods Through the selecting marker URA3 gene, a new glycoengineering yeast strain named 4-32-STT3D was constructed, which could overexpress the Leishmania major N-glycosyltransferase staurosporine and temperature sensitivity3 D subunit(STT3D) under the control of an inducible alcohol oxidase 1(AOX1) promoter.We analyzed the N-glycosylation status of anti-human epidermal growth factor receptor 2 ( HER2 ) antibody and granulocyte macrophage colony stimulating factor (GM-CSF) expressed in 4-32-STT3D using SDS-PAGE,Western blotting and peptide-N-asparigineamidase F(PNGase F).Finally the effect of STT3D on the growth rate of glycoengineering yeast was detected.Results SDS-PAGE showed that anti-HER2 antibody expressed in 4-32-HL had two components:the first one with a relative molecular mass 55 ×103 was glycosylated,while the second one with 50 ×103 was non-glycosylated,but anti-HER2 antibody expressed in 4-32-HL-STT3D had the component of 55 ×103 only without any non-glycosylated 50 ×103 .The above components became 50 ×103 with the digestion of PNGaseF.All of them proved to be antibodies by Western blotting.As a report protein,GM-CSF expressed in 4-32-GM-CSF had two components: 22 ×103 and 20 ×103, while in 4-32-GM-CSF-STT3D there was only one with 22 ×103 .All these components became 18 ×103 with the digestion of PNGase F.Statistical analysis showed that without induction,STT3D had no effect on the growth rate of glycoengineering yeast, while great effect was observed when STT3D was induced.Conclusion Glycoengineering yeast with the overexpression of N-glycosyltransferase has higher N-glycosylation efficiency.

Objective To express the hemagglutinin of H7N9 in Pichia pastoris and analyze its immunogenicity. Methods The HA [1 -525 amino acids(aa)] of H7N9 [A/Hangzhou/1/2013(H7N9)] lacking the C-terminal transmembrane anchor-coding sequence was amplified by PCR and cloned into the expression vector pPICZαA.The plasmid pPICZ-HA7-S-was transformed into P.pastoris and the HA1-525 was detected by ELISA.Then the HA1-525 was precipitated by PEG20000.After immunization with the HA1-525 , the anti-HA7 antibody in mouse serum was detected by ELISA and hemagglutinin inhibition ( HI) test.Results The HA1-525 was expressed in P.pastoris after being induced with methanol. Western blotting confirmed that there were specific dispersion bands and the molecular weight of HA1-525 decreased to 58 × 103 after being digested by endo H.Anti-HA7 antibody was found in serum of HA1-525 immunized mice and the hemaggluti-nation-inhibition titers reached 1∶700 after the third doses.Conclusion This study shows the HA1-525 expressed in P.pastoris can induce the neutralizing antibody in mice.

Objective To develop an interleukin-4(IL-4) antagonist named M5-IgG1Fc protein constructed by genetic engineering of antibody Fc fragment-cytokine mutein fusion protein which has a long half-life time in plasma.M5-IgG1 Fc protein binds to IL-4 receptor but cannot activate downstream signalling pathway , which provides a basis for drug develop-ment for allergic diseases .Methods The synthesized interleukin-4 mutant gene ( named M5 ) was cloned into the expres-sion vector pBV220 and transformed into E.coli DH5α.Chimeric gene M5-IgG1Fc obtained by overlap extension (SOE) method was transformed into glycoengineered Pichia pastoris GJK01 through expression vector pPICZαA .Then M5-IgGFc fusion protein was obtained by protein purification after being induced by methanol in 72 hours.The anti-IL-4 biologicial ac-tivity assay of M5 and M5-IgG1 Fc was performed with CTLL-2/IL-4R cells and detected with MTT colormetry .Finally,the half-life time of M5 and M5-IgG1 Fc protein in mice was compared by detecting the remaining amount in plasma with ELISA kit.Results The M5 protein expressed in E.coli and M5-IgG1 Fc fusion protein expressed in P.pastoris GJK01 both had IL-4 antagonistic bioactivity .The EC50 of both, which inhibited 5.6 ×10 -2 nmol/ml of IL-4, were 0.31 ±0.05 and 0.77 ± 0.03 nmol/ml,respectively.The maximum of M5 in plasma at 0.5 h was 5.8 ×10 -2 nmol/ml but the remaining amount was 2.8%of the maximum at 2 h.M5 protein could not be detected after administration at 8 h because of the detection line . The maximum of M5-IgG1 Fc fusion protein was 4.7 ×10 -2 nmol/ml,while fusion protein M5-IgG1 Fc decreased to 4.3%of its maximum at 120 h and could not be detected at 168 h.Conclusion M5 protein has IL-4 antagonistic bioactivity .M5-IgG1 Fc fusion protein expressed in glycoengineered P.pastoris GJK01 has IL-4 antagonistic bioactivity and long retention time in mice,which can be potentially used for treatment of allergic diseases .

Objective To prepare endo-beta-N-acetylglucosaminidase H (Endo-H) expressed in Pichia pastoris, and apply it to N-glycosylation analysis .Methods One complete gene was synthesized on the basis of the cDNA sequence encoding Streptomyces plicatus reported in GenBank .The gene was cloned into the expression vector pPIC 9.The expression vector pPIC9-Endo-H was transformed into P.pastoris(JC308).The expression products were induced by methanol , puri-fied by two-step chromatography , used to analyze the glycan structures of RNaseB by the DNA sequencer assisted fluoro-phore-assisted carbohydrate electrophoresis (DSA-FACE)methods, and finally compared with peptide-N-asparagine amidase F(PNGase F).Results This enzyme expressed in P.pastoris(JC308) had the ability to hydrolyze natural or denatured high-mannose type of oligosaccharide linked by β-1,4-glycosidic bonds , but not complex-type oligosaccharide .The result of DSA-FACE showed that carbohydrate chains of Man 5 GlcNAc-Man9 GlcNAc could be obtained when RNaseB was hydrolyzed by Endo-H, and that Man5 GlcNAc2-Man9 GlcNAc2 chains became available when RNaseB was hydrolyzed by PNGase F . Conclusion Endo-H expressed in P.pastoris has bioactivity which can be used to analyze N-glycosylation with the method of DSA-FACE.