OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

BACKGROUND: Disease-modifying therapies have been approved for the treatment of relapsing multiple sclerosis (RMS). The present study aims to examine the safety of teriflunomide in Chinese patients with RMS. METHODS: This non-randomized, multi-center, 24-week, prospective study enrolled RMS patients with variant (c.421C>A) or wild type ABCG2 who received once-daily oral teriflunomide 14 mg. The primary endpoint was the relationship between ABCG2 polymorphisms and teriflunomide exposure over 24 weeks. Safety was assessed over the 24-week treatment with teriflunomide. RESULTS: Eighty-two patients were assigned to variant ( n  = 42) and wild type groups ( n  = 40), respectively. Geometric mean and geometric standard deviation (SD) of pre-dose concentration (variant, 54.9 [38.0] μg/mL; wild type, 49.1 [32.0] μg/mL) and area under plasma concentration-time curve over a dosing interval (AUC tau ) (variant, 1731.3 [769.0] μg∙h/mL; wild type, 1564.5 [1053.0] μg∙h/mL) values at steady state were approximately similar between the two groups. Safety profile was similar and well tolerated across variant and wild type groups in terms of rates of treatment emergent adverse events (TEAE), treatment-related TEAE, grade ≥3 TEAE, and serious adverse events (AEs). No new specific safety concerns or deaths were reported in the study. CONCLUSION: ABCG2 polymorphisms did not affect the steady-state exposure of teriflunomide, suggesting a similar efficacy and safety profile between variant and wild type RMS patients. REGISTRATION NCT04410965, https://clinicaltrials.gov .
Humans ; Crotonates/adverse effects* ; Toluidines/adverse effects* ; Nitriles ; Hydroxybutyrates ; Female ; Male ; Adult ; ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics* ; Middle Aged ; Multiple Sclerosis, Relapsing-Remitting/genetics* ; Prospective Studies ; Young Adult ; Neoplasm Proteins/genetics* ; East Asian People

OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

AIM: To evaluate the safety and efficacy of accelerated corneal cross-linking(A-CXL)with application of isotonic riboflavin under pedicled corneal epithelial flap in the treatment of thin keratoconus(corneal thickness less than 400 μm after epithelial removal).METHODS: Retrospective study. A total of 46 patients(74 eyes)with keratoconus treated in the refractive surgery department of Ineye Hospital of Chengdu University of TCM from September 2017 to December 2020 were enrolled. According to the preoperative thinnest corneal thickness(TCT), the patients were divided into two groups: 16 patients(20 eyes)with TCT ranging from 400 to <450 μm underwent accelerated corneal cross-linking with application of isotonic riboflavin under pedicled corneal epithelial flap, and the surgical method involves creating a pedicled corneal epithelial flap and lifting it, applying isotonic riboflavin eye drops, repositioning the corneal epithelial flap, performing ultraviolet irradiation and then using corettage of corneal epithlial flap after irradiation. Another 30 patients(54 eyes)with TCT ≥450 μm underwent epithelial-off A-CXL surgery. After follow-up for 12 mo, the results of best corrected visual acuity(BCVA), Sirius three-dimensional corneal topography, and corneal endothelial cell density were collected before and after surgery.RESULTS: At 12 months post-surgery, patients in the pedicled corneal epithelial flap group showed no significant difference in Kmax compared with pre-surgery(P>0.005), while patients in the epithelial-off group showed a significant reduction in Kmax compared with pre-surgery(P<0.005). For both groups, there were no significant differences in BCVA(LogMAR), corneal anterior and posterior surface curvatures(K1, K2), and corneal endothelial cell density at 12 mo post-surgery compared with pre-surgery(all P>0.005). The comparisons of the changes in various parameters between 12 mo post-surgery and pre-surgery for both groups showed no significant differences(all P>0.05).CONCLUSION: Accelerated corneal cross-linking with application of isotonic riboflavin under pedicled corneal epithelial flap surgery is safe and feasible for patients with thin keratoconus, achieving similar control of keratoconus progression as epithelial-off A-CXL.

Objective To explore the effect of virtual reality treadmill training on balance and gait in stroke patients. Methods From March,2022,to March,2023,40 stroke patients in Beijing Tiantan Hospital,Capital Medical University were randomly divided into control group(n = 20)and experimental group(n = 20).Both groups received rou-tine rehabilitation training.The control group received ordinary treadmill walking training,and the experimental group received treadmill walking training with virtual reality,for two weeks.They were assessed with Berg Bal-ance Scale(BBS)and Timed Up and Go Test(TUGT),and the envelope ellipse area,center of pressure(COP)av-erage speed of movement,step length,stride length and stride width were compared between two groups before and after treatment. Results After treatment,the scores of BBS and time of TUGT improved in both groups(|t|>3.508,P<0.01),and they were better in the experimental group than in the control group(|t|>3.019,P<0.01);there was no significant dif-ference in the envelope ellipse area,COP average speed,and stride width between two groups(P>0.05);howev-er,the step length and stride width improved in the experimental group(|t|>4.008,P<0.01). Conclusion Treadmill training with virtual reality can improve the balance and walking ability of stroke patients.

Objective:The authors introduced the change point analysis of normal distribution based on the likelihood ratio principle, analyzed the number of outpatients in a hospital of Luohu District, Shenzhen, to provide scientific basis for rational allocation of health resources.Methods:The authors collected totally 636 number of outpatients' data from 8: 00 to 12: 00 a.m. at 48 time windows at 5 minutes intervals in a hospital of Luohu District, Shenzhen, and analyzed it with single change point analysis of simultaneous change of mean and variance, and discussed when the change point occurred.Results:The average number of outpatient was 13.250 0 for every time window, the change point occurred at 8: 50, the probability was 0.000 025, the average number of outpatient per 5 minutes from 8: 00 to 8: 50 was 7.000 0, the average number of outpatient per 5 minutes from 8: 50 to 12: 00 was 14.897 4, and the ratio of number of outpatients before and after the change point occurred was 1∶2.Conclusion:In the case of no need to determine the base period or compare the data, the change point analysis of normal distribution based on the principle of likelihood ratio provides a new way of statistical analysis and statistical monitoring for the rational allocation of health resources based on the number of patients.

Objective:To observe the corneal morphology and visual quality after transepithelial photorefractive keratectomy (Trans-PRK) with smart pulse technique (SPT) and 1 050 Hz cutting frequency in the correction of myopia and astigmatism.Methods:A self-controlled case series study was conducted.Sixty five eyes of 33 patients who underwent Trans-PRK surgery in Ineye Hospital of Chengdu University of TCM from July 2017 to June 2018 were followed up for 6 months.The uncorrected visual acuity (UCVA) converted to logarithm of the minimum angle of resolution (LogMAR) unit, best corrected visual acuity (BCVA) (LogMAR), and spherical equivalent (SE) of the subjects were recorded.The anterior corneal surface symmetry index (SI), the anterior corneal surface Q value in the range of 6, 7, 8, and 9 mm diameter, the spherical aberration, coma, trefoil and total higher-order aberration of the anterior corneal surface, the strehl ratio (SR), and the modulation transfer function (MTF) of 10, 20, 30, and 40 c/d in the horizontal and vertical meridian directions before and after surgery were measured with Sirius corneal topography analyzer.The differences of each index among different time points were compared, and the correlation between indexes was analyzed by Pearson correlation analysis.This study followed the Declaration of Helsinki.The study protocol was approved by the Medical Ethics Committee of Ineye Hospital of Chengdu University of TCM (No.2020yh-004). All patients signed the informed consent form before surgery.Results:The average preoperative BCVA and SE were -0.09±0.06 and (-4.24±1.24)D.The mean UCVA and SE at 7 days, 1, 3 and 6 months postoperatively were -0.10±0.08 and (0.03±0.63)D, -0.12±0.06 and (0.08±0.53)D, 1.16±0.06 and (0.02±0.79)D, -0.18±0.05 and (0.08±0.37)D, respectively.The SI at different time points after the surgery were significantly higher than that before operation (all at P<0.05). At 1, 3 and 6 months after surgery, the Q value of anterior corneal surface in different diameter ranges increased from negative to positive, showing statistically significant differences (all at P<0.05). At each time point after surgery, the trefoil and total higher-order aberrations of the anterior corneal surface increased to varying degrees.Coma at 7 days and 6 months after surgery were significantly higher than that before surgery, and spherical aberration at 3 and 6 months after surgery were significantly higher than that before surgery (all at P<0.05). The SR values at 3 and 6 months after operation were significantly higher than that before operation (all at P<0.05). At 6 months after operation, the MTF values at different spatial frequencies of the horizontal meridian and the MTF values at 30 and 40c/d spatial frequencies of the vertical meridian were lower than those before operation, and the differences were statistically significant (all at P<0.05). The correlation analysis showed that the Q value of different diameter ranges was positively correlated with spherical aberration ( r=0.798-0.925, P<0.05), total higher-order aberration ( r=0.596-0.630, P<0.05), SI ( r=0.235-0.303, P<0.05) and corneal ablation depth ( r=0.583-0.659, P<0.05) at 6 months after surgery.SI was positively correlated with spherical aberration ( r=0.307, P<0.05), coma ( r=0.424, P<0.05), total higher-order aberration ( r=0.300, P<0.05), corneal ablation depth ( r=0.227, P<0.05), and eccentric cutting amount ( r=0.281, P<0.05). There was no correlation between SR and aberration, corneal ablation depth, eccentric cutting amount, etc.(all at P≥0.05). Conclusions:Trans-PRK using SPT to correct myopic astigmatism can improve vision, stabilize diopter, enhance retinal imaging quality, increase the asymmetry of the anterior corneal surface, and introduce different degrees of higher-order aberrations.

ObjectiveTo evaluate the current situation of human resource allocation in district and county centers for disease control and prevention （CDCs） in Kashgar ， identify existing problems and influencing factors， and to provide scientific evidence for optimizing the human resource allocation. MethodsA survey was conducted among all CDCs in Kashgar in February 2022. The questionnaire included the institutional and individual questions. ResultsThe overall staff size approved for the CDCs in Kashgar was 604， with a staffing rate of 76.17%， among which the staffing rates in 5 county CDCs were less than 60%. Currently， there were a total of 524 approved staff members in all CDCs， resulting in a vacancy rate of 13.25%. In the district CDC， 85 staff members were on duty， while the median of staff on duty was 34 in each county CDC. The staff in the district CDC was ageing， of which those aged over 45 accounted for 67.06%. The staff in the county CDCs was generally young， of which those aged less than 35 accounted for 54.22%. Moreover， the proportion of staff with bachelor’s degree or above in the district and county CDCs was 31.76% and 24.95%， respectively. The proportion of staff without professional title was 32.94% and 48.03%， respectively. In contrast， the proportion of those with middle and senior professional title was 57.89% and 22.02%， respectively. In addition， in recent 3 years， 24 staff members resigned in the CDCs， all of whom had professional titles. ConclusionHuman resources are insufficient in CDCs in Kashgar. Furthermore， staff structure is unreasonable， with a serious loss of human resources. In particular， the district CDC needs to optimize the allocation of human resources.

Objective:To analyze the external root resorption of incisors caused by maxillary canine impaction, and to explore the changes of incisor external root resorption after orthodontic treatment.Methods:Twenty-four patients with impacted maxillary canines leading to external root resorption of incisors treated in Department of Orthodontics, Affiliated Hospital of Stomatology, Nanjing Medical University from July 2011 to January 2019 were selected. The age ranged from 10 to 14 years [(12.3±1.6) years], including 11 males and 14 females. The maxillary incisors with external root resorption before treatment were included in the resorption group (21 central incisors and 22 lateral incisors), and the maxillary incisors without external root resorption were used as the control group (26 central incisors and 21 lateral incisors). Cone-beam CT was taken before treatment, immediately after treatment and one year after treatment. At the one year follow-up 15 patients were collected (10 central incisors and 12 lateral incisors in the resorption group, while 16 central incisors and 12 lateral incisors in the control group). The root length and tooth volume of maxillary incisors were measured and analyzed before canine traction, after canine traction finished and at the one year follow-up.Results:After the treatment of maxillary impacted canines, the root length and volume of the central and lateral incisors in both resorption group and control group were significantly less than that before treatment ( P<0.05). At the one year follow-up, the root length and volume of the incisors (central incisors in the resorption group and the central and lateral incisors in control group) were significantly less than those immediately after impacted canine traction ( P<0.05). During the canine traction, the difference of the volume change in lateral incisors between the resorption group [(17±6) mm 3] and the control group [(12±5) mm 3] was statistically significant ( t=2.51, P<0.05). During the one year follow-up period, the difference between the changes of the root length in maxillary central incisors in the resorption group [(0.5±0.4) mm] and in the control group [(0.2±0.1) mm] was statistically significant ( t=2.35, P<0.05). Conclusions:For maxillary incisors with external root resorption caused by impacted maxillary canines, the external root resorption could not stop immediately after the treatment of impacted canines. The root resorption in maxillary lateral incisors mainly occured during the traction process, while the root resorption in maxillary central incisors existed during the orthodontic treatment and in the follow-up period.

ObjectiveTo investigate the effects of the inoculation of potassium-solubilizing bacteria on the rhizosphere soil microenvironment of Paris polyphylla var. yunnanensis. MethodThe effects of different potassium-solubilizing bacteria on the physical and chemical properties， microbial community structure， and soil enzyme activity in the rhizosphere soil of P. polyphylla var. yunnanensis were investigated by pot planting at room temperature. The correlation of various indexes was analyzed. ResultThe inoculation with different potassium-solubilizing bacteria could significantly affect the physical and chemical properties of rhizosphere soil of P. polyphylla var. yunnanensis. The mass fractions of available nitrogen， available phosphorus， and available potassium were 24.5-90.5 mg·kg^-1， 2.53-25.9 mg·kg^-1， and 132-312 mg·kg^-1， respectively， and the soil pH was 7.08-7.75， which were in line with the optimal ranges of P. polyphylla var. yunnanensis planting. The inoculation of different potassium-solubilizing bacteria could affect the number of bacteria， actinomycetes， and fungi in rhizosphere soil to varying degrees. The transformation of soil from "fungal type" to "bacterial type" marks the improvement of soil fertility. It also affected the enzyme activity of rhizosphere soil， and the activities of neutral phosphatase， protease， and polyphenol oxidase showed an increasing trend. The correlation analysis showed that the number of bacteria was negatively correlated with the number of fungi （r=-0.856， P<0.01）， positively correlated with the number of actinomycetes， the content of available nitrogen and available potassium， and negatively correlated with soil pH. ConclusionThe inoculation of potassium-solubilizing bacteria can effectively improve the content of available potassium， available nitrogen， available phosphorus， and other nutrients in the rhizosphere soil of P. polyphylla var. yunnanensis， improve soil fertility， alleviate the continuous cropping obstacles of P. polyphylla var. yunnanensis， and lay a theoretical foundation for the green and sustainable development of P. polyphylla var. yunnanensis.