Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

BACKGROUND:Patients with chronic ankle instability tend to overpronate when walking,which increases the risk of ankle sprain during the stance phase of the walking cycle.Clinicians often use Kinesio taping or athletic taping for ankle taping.Since Kinesio taping is elastic and athletic taping cannot be stretched,there are differences in technical applications and physiological mechanisms between them,which may result in different rehabilitation effects.OBJECTIVE:To compare the effects of Kinesio taping and athletic taping on the frontal plane of the foot motion and the horizontal plane of the tibia motion in patients with chronic ankle instability during the stance phase of walking.METHODS:Forty patients with chronic ankle instability were randomly divided into Kinesio taping group and athletic taping group.In the Kinesio taping group,two patches were pasted from the inside to the outside of the hind foot to generate pulling tension that facilitates foot eversion;in the athletic taping group,ankle locking basket taping was used.Walking tests were conducted on an electric treadmill before and after taping.A three-dimensional motion analysis system was used to obtain the kinematic parameters of the subjects'foot and tibia.RESULTS AND CONCLUSION:After taping,in the Kinesio taping group,the foot valgus angle increased in the early stance phase(P<0.05),but there was no effect on foot position in the late stance phase(P>0.05).After taping,in the athletic taping group,internal rotation of the tibia increased in the late stance phase(P<0.05);however,there was no significant change in tibial position in the early stance phase(P>0.05).To conclude,compared with athletic taping,Kinesio taping can provide a flexible pulling force that is beneficial for foot eversion in the early stance phase of the gait cycle,while not restricting normal foot inversion in the late stance phase.Therefore,Kinesio taping may be a practical rehabilitation therapy for patients with chronic ankle instability,correcting abnormal motion of the ankle joint without restricting its natural motion.

Objective:To analyze the clinical features and prognosis of acute B lymphoblastic leukemia (B-ALL) children carrying a TCF3: : PBX1 fusion gene and to evaluate the prognostic value of this gene.Methods:Retrospective cohort study.A total of 2 164 B-ALL children aged 0-18 years diagnosed and treated at 19 pediatric centers from October 2016 to June 2022 were enrolled.They were divided into the positive group and the negative group according to whether they carried a TCF3: : PBX1 fusion gene.The clinical characteristics, treatment response, adverse reactions, and prognosis of the 2 groups of patients were analyzed.The rank sum and Kruskal-Wallis tests were used to compare two and more than two groups of numerical variables, respectively.Fisher′s exact test was used to compare categorical variables.Results:Among the 2 164 patients, 116 (5.4%) were TCF3: : PBX1 positive, of which 70 patients were female, accounting for 60.3%.There were 840 female patients in the TCF3: : PBX1-negative group, accounting for 41.0%.There was a significant difference in the ratio of females between the TCF3: : PBX1-positive and TCF3: : PBX1-negative groups ( P<0.001).No significant difference was observed in age of onset between the two groups( P>0.05).The proportion of bone marrow naive cells [54.00 (14.00, 76.50)% vs.29.00 (3.00, 68.00)%], white blood cell counts [25.30 (10.46, 60.94)×10 9/L vs.9.03 (4.38, 30.73)×10 9/L] and hemoglobin counts [82.00(63.00, 101.00) g/L vs.74.00(60.00, 90.00) g/L] in the TCF3: : PBX1-positive group were significantly higher than those in the negative group at the onset (all P<0.05).In terms of treatment response, the proportion of peripheral blood naive cells on Day 8 in the TCF3: : PBX1-positive group was significantly higher than that in the negative group [2.00 (0, 9.00)% vs.0 (0, 2.00)%, P<0.001].The proportion of minimal residual disease <0.1% on Day 15 in the TCF3: : PBX1-positive group was significantly higher than that in the negative group ( P=0.038).There were no significant differences in cumulative recurrence rate, treatment-related mortality (TRM), and overall survival (OS) between the TCF3: : PBX1-positive group and TCF3: : PBX1-negative group (all P>0.05).The cumulative recurrence risk of TCF3: : PBX1-positive patients was 9.646 times higher than that of ETV6: : RUNX1-positive patients with better prognosis( HR=9.646, 95% CI: 1.026-90.700, P=0.047).There were no significant differences in TRM and OS between TCF3: : PBX1-positive and ETV6: : RUNX1-positive patients (all P>0.05).A significant enrichment of PAX5 mutations was detected in TCF3: : PBX1-positive patients.Among the 7 high-risk TCF3: : PBX1-positive patients in a single center, 4 patients had PAX5 mutations, and this proportion was significantly higher than that in other patients ( P<0.001). Conclusions:B-ALL children carrying a TCF3: : PBX1 fusion gene have a high remission rate and good long-term prognosis after intensive chemotherapy.It is suggesting that TCF3: : PBX1-positive B-ALL patients should be rated at intermediate risk to receive intensive chemotherapy.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

BACKGROUND:Patients with chronic ankle instability tend to overpronate when walking,which increases the risk of ankle sprain during the stance phase of the walking cycle.Clinicians often use Kinesio taping or athletic taping for ankle taping.Since Kinesio taping is elastic and athletic taping cannot be stretched,there are differences in technical applications and physiological mechanisms between them,which may result in different rehabilitation effects.OBJECTIVE:To compare the effects of Kinesio taping and athletic taping on the frontal plane of the foot motion and the horizontal plane of the tibia motion in patients with chronic ankle instability during the stance phase of walking.METHODS:Forty patients with chronic ankle instability were randomly divided into Kinesio taping group and athletic taping group.In the Kinesio taping group,two patches were pasted from the inside to the outside of the hind foot to generate pulling tension that facilitates foot eversion;in the athletic taping group,ankle locking basket taping was used.Walking tests were conducted on an electric treadmill before and after taping.A three-dimensional motion analysis system was used to obtain the kinematic parameters of the subjects'foot and tibia.RESULTS AND CONCLUSION:After taping,in the Kinesio taping group,the foot valgus angle increased in the early stance phase(P<0.05),but there was no effect on foot position in the late stance phase(P>0.05).After taping,in the athletic taping group,internal rotation of the tibia increased in the late stance phase(P<0.05);however,there was no significant change in tibial position in the early stance phase(P>0.05).To conclude,compared with athletic taping,Kinesio taping can provide a flexible pulling force that is beneficial for foot eversion in the early stance phase of the gait cycle,while not restricting normal foot inversion in the late stance phase.Therefore,Kinesio taping may be a practical rehabilitation therapy for patients with chronic ankle instability,correcting abnormal motion of the ankle joint without restricting its natural motion.

Objective:To analyze the clinical features and prognosis of acute B lymphoblastic leukemia (B-ALL) children carrying a TCF3: : PBX1 fusion gene and to evaluate the prognostic value of this gene.Methods:Retrospective cohort study.A total of 2 164 B-ALL children aged 0-18 years diagnosed and treated at 19 pediatric centers from October 2016 to June 2022 were enrolled.They were divided into the positive group and the negative group according to whether they carried a TCF3: : PBX1 fusion gene.The clinical characteristics, treatment response, adverse reactions, and prognosis of the 2 groups of patients were analyzed.The rank sum and Kruskal-Wallis tests were used to compare two and more than two groups of numerical variables, respectively.Fisher′s exact test was used to compare categorical variables.Results:Among the 2 164 patients, 116 (5.4%) were TCF3: : PBX1 positive, of which 70 patients were female, accounting for 60.3%.There were 840 female patients in the TCF3: : PBX1-negative group, accounting for 41.0%.There was a significant difference in the ratio of females between the TCF3: : PBX1-positive and TCF3: : PBX1-negative groups ( P<0.001).No significant difference was observed in age of onset between the two groups( P>0.05).The proportion of bone marrow naive cells [54.00 (14.00, 76.50)% vs.29.00 (3.00, 68.00)%], white blood cell counts [25.30 (10.46, 60.94)×10 9/L vs.9.03 (4.38, 30.73)×10 9/L] and hemoglobin counts [82.00(63.00, 101.00) g/L vs.74.00(60.00, 90.00) g/L] in the TCF3: : PBX1-positive group were significantly higher than those in the negative group at the onset (all P<0.05).In terms of treatment response, the proportion of peripheral blood naive cells on Day 8 in the TCF3: : PBX1-positive group was significantly higher than that in the negative group [2.00 (0, 9.00)% vs.0 (0, 2.00)%, P<0.001].The proportion of minimal residual disease <0.1% on Day 15 in the TCF3: : PBX1-positive group was significantly higher than that in the negative group ( P=0.038).There were no significant differences in cumulative recurrence rate, treatment-related mortality (TRM), and overall survival (OS) between the TCF3: : PBX1-positive group and TCF3: : PBX1-negative group (all P>0.05).The cumulative recurrence risk of TCF3: : PBX1-positive patients was 9.646 times higher than that of ETV6: : RUNX1-positive patients with better prognosis( HR=9.646, 95% CI: 1.026-90.700, P=0.047).There were no significant differences in TRM and OS between TCF3: : PBX1-positive and ETV6: : RUNX1-positive patients (all P>0.05).A significant enrichment of PAX5 mutations was detected in TCF3: : PBX1-positive patients.Among the 7 high-risk TCF3: : PBX1-positive patients in a single center, 4 patients had PAX5 mutations, and this proportion was significantly higher than that in other patients ( P<0.001). Conclusions:B-ALL children carrying a TCF3: : PBX1 fusion gene have a high remission rate and good long-term prognosis after intensive chemotherapy.It is suggesting that TCF3: : PBX1-positive B-ALL patients should be rated at intermediate risk to receive intensive chemotherapy.

Objective To explore the risk factors of prognosis of patients with intracerebral hemorrhage at 6 months after operation,and to study the diagnostic efficacy of event-related potential(ERP)on outcome and prognosis.Methods Forty postoperative patients with intracerebral hemorrhage were prospectively enrolled.The age,sex,preoperative blood loss and GCS were collected.The mismatch negative wave(MMN)was measured by ERP on the 1st,3rd,7th and 14th day after operation,and the modified coma recovery scale(CRS-R)was evaluated simultaneously.The Glasgow outcome scale(GOS)of patients was assessed by telephone follow-up after 6 months GOS 1～2 and 3～5 was defined as poor prognosis and good prognosis.,respectively.Multivariate Logistic regression analysis was used to analyze the risk factors affecting the outcome and prognosis of patients,to explore the correlation between MMN and CRS-R,and ROC curve was used to evaluate the predictive value of MMN on the prognosis of patients.Results There were significant differences in the preoperative blood loss,admission GCS score and MMN amplitude between patients with good prognosis and those with poor prognosis on the 3rd,7th and 14th day after operation.Multivariate Logistic regression analysis showed that MMN amplitude on the 14th day after operation was an independent risk factor for poor prognosis(OR=0.100,95%CI:0.000～0.355,P<0.05).The amplitude of MMN was strongly correlated with CRS-R and the correlation coefficient was 0.643(P<0.01).The amplitude of MMN on the 14th day after operation had a high diagnostic efficiency for the clinical prognosis at 6 months.Conclusion The lower the amplitude of MMN on the 14th day after operation,the worse the prognosis of patients.ERP monitoring of postoperative disturbance of consciousness in patients with intracerebral hemorrhage is expected to become an effective tool to predict the prognosis of patients.

Objective To study the feasibility and safety of right ventricular angiography(RVG)to as-sist pacing in the left bundle branch region(LBBaP).Methods The retrospective study was adopted.A total of 67 patients receiving LBBaP in this hospital from January 2019 to June 2022 were included as the study sub-jects.The basic information of the patients was collected,including the sex,age,clinical diagnosis,EKG pa-rameters,etc.The RVG three-stage adjuvant LBBaP was adopted.The specific operation was to perform RVG under the right anterior oblique perspective of 30°.A straight line was connected between the highest point of the tricuspid valvular ring and the apex of the right ventricle,and then the straight line was divided into 3 e-qual parts.The proximal junction was the LBBaP electrode implantation area.The success of LBBaP operation was defined as the simultaneous satisfaction of right bundle branch block form of QRS wave in unipolar pa-cing,QRS wave width(QRSd)＜130 ms and peak time of left ventricular excitation＜90 ms.The operation related parameters and occurrence situation of complications were recorded.The follow up was conducted in 1,3,6,12 months.The electrode parameters,electrocardiogram and color Doppler ultrasound results were recor-ded.Results Among 67 patients,43 cases were males aged(65.0±8.0)years old;twenty-two cases(32.8％)were symptomatic sick sinus syndrome,and 45 cases(67.2％)were second-degree type Ⅱ and above atrioven-tricular block.The preoperative QRS width(QRSd)was(103.0±22.0)ms.The LBBaP operation success was in 61 cases(91.0％).The operation time was(134.6±32.3)min,and the X-ray exposure time was(43.6±12.6)min.The pace-making threshold value was(0.8±0.4)V,the R wave perception amplitude was(12.1±4.7)mV,and the impedance was(741.2±130.8)Ω.At 1 V pacing,the peak time of left ventricular activation in the lead V5 was(83.4±13.7)ms.The postoperative QRSd was(116.5±18.3)ms.During the operation,8 cases developed interventricular septal perforation and 1 case developed bundle branch injury.No other serious complications occurred.After 12 months of follow-up,all patients had stable electrode parame-ters.Conclusion RVG three-stage adjuvant LBBaP is a simple,feasible and safe physiological pacing method.

Neutrophils as the first immune responders to infection can quickly identify and eliminate pathogens. The mainly rely on glycolysis to exert their killing functions. Although researches on the metabolic shifs that affect neutrophil functions began early, little is known about how neutrophils undergo metabolic transformation during the anti-infection process. It has been proven that glycogen metabolism plays an important role in regulating the functions of neutrophils. Other metabolic pathways besides glycolysis, such as mitochondrial metabolism and fatty acid oxidation during neutrophil differentiation, have potential contributions to the regulation of neutrophils′ functions. This review summaries current studies about metabolic regulatory effects of neutrophils on anti-infection responses, intending to provide reference for further study on the metabolism of neutrophils.

【Objective】 To analyze the hepatitis B virus (HBV) infection data of blood donors from 18 domestic blood stations, so as to investigate the HBV infection situation of blood donors. 【Methods】 The positive rate of HBV and its distribution characteristics of regions, the percentage of HBsAg+ ELISA in first-time vs repeated blood donors, and the percentage of HBsAg-/HBV DNA+ blood donors of 18 domestic blood stations during 2017 to 2020 were collected from the Working Platform for Practice Comparison of Blood Centers, and the HBV infection among blood donors were statistically analyzed. 【Results】 From 2017 to 2020, the positive rate of HBV in blood donors among 18 domestic blood stations was 13.48/10 000-144.02/10 000, with the average HBV positive rate in eastern, central and western region at 26.14/10 000, 51.98/10 000 and 41.00/10 000, respectively. The HBsAg+ rate by ELISA among first-time and repeated blood donors was 14.55/10 000-305.39/10 000 vs 1.04/10 000-87.43/10 000 The HBsAg-/HBV DNA+ yield was 1.80/10 000-35.31/10 000. 【Conclusion】 The distribution of HBV infection in blood donors has regional characteristics, and HBV prevalence was low in repeated blood donors. HBsAg ELISA combined with HBV DNA detection can better ensure blood safety.