Objective:To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods:The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δ wza were detected by measuring the optical density OD 600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δ wza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δ wza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δ wza mutant strain. Results:The PCR results revealed that the Δ wza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δ wza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δ wza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δ wza mutant strain (45.963±2.795) μg/ml compared with wild-type strain W14 (138.800±5.201) μg/ml. There was a statistical difference between the two groups ( t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion:Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

Objective:To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hv Kp) serotype K1 and validate its function. Methods:Phage that infected serotype K1-type hv Kp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results:A lytic phage (phiA2) that infected serotype K1-type hv Kp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates. Conclusion:Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hv Kp, which has potential application value in treating bacterial infection.

Objective:To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity. Methods:The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination. The growth curves of W14 and Δ wza were detected by measuring the optical density OD 600. In order to analyze the effect of gene wza on bacterial capsule formation, wild-type strain W14 and Δ wza mutant strain were detected by transmission electron microscope, and their capsule contents were measured by quantifying the uronic acid contents. The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 and Δ wza mutant strain. The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 and Δ wza mutant strain. Results:The PCR results revealed that the Δ wza mutant strain was successfully constructed. Compared with wild-type strain W14, the growth curves of Δ wza on the solid plates demonstrated a slightly slower growth. However, no difference in growth was observed among wild-type strain W14 and Δ wza mutant strains in LB broth. The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria. The uronic acid content assay suggested that the capsule content was significantly decreased in Δ wza mutant strain (45.963±2.795) μg/ml compared with wild-type strain W14 (138.800±5.201) μg/ml. There was a statistical difference between the two groups ( t=27.233, P<0.001). The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted. Conclusion:Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

Objective:To express and purify the phage depolymerase from hypervirulent Klebsiella pneumoniae (hv Kp) serotype K1 and validate its function. Methods:Phage that infected serotype K1-type hv Kp was isolated from hospital sewage. The biology and morphology of the phage were determined by plaque assay and transmission electron microscopy. The whole genome of the phage was sequenced by the Illumina HiSeq 2500 platform. The presence of depolymerase was determined by observing the plaque halo. Bioinformatic analysis and prokaryotic protein expression system were further used to predict and identify phage depolymerase. The depolymerase gene fragment was obtained by PCR and cloned into the pET28a expression vector, and the expression and purification of the depolymerase were completed in strain BL21. The depolymerase activities on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates were detected by plaque assay and low-speed centrifugation assay. Results:A lytic phage (phiA2) that infected serotype K1-type hv Kp clinical isolate was isolated from hospital sewage. It was typical of the Caudovirales order and Autographiviridae family, and its whole genome was 43 526 bp in length and contained 51 coding domain sequences. The phage phiA2-derived depolymerase phiA2-dep was predicted, expressed and purified. The plaque assay and low-speed centrifugation assay indicated that the depolymerase phiA2-dep had good lytic activity on the capsular polysaccharide of serotype K1-type hv Kp clinical isolates. Conclusion:Depolymerase phiA2-dep can specifically degrade the capsular polysaccharide of serotype K1-type hv Kp, which has potential application value in treating bacterial infection.

Objective To analyze the situation and changing trend of free medication service in Beijing from 2013 to 2020, and to propose policies and service improvement measures. Methods A retrospective survey was conducted in Beijing to collect and analyze the service subjects, service process, drug catalog, fiscal investment and patient participation of free medication in each district in recent years. Results Among the 16 districts of Beijing, 81.25% of the districts included the patients listed in Item (2) of Paragraph 2 of Article 30 of the Mental Health Law into the scope of free medication, and 75% of the districts broke the restriction that the service targets were registered residents in Beijing. 56.25% of the districts provided a variety of free medication modes, and 81.25% of the districts provided a mode of nearby community service. The catalog of free medications in various areas was still based on the Beijing catalog, and only 18.75% of the districts supplemented the list. In 2020, the total investment of free medicines and subsidies was 88.3768 million RMB, and the per capita investment was 1 616.02 RMB. The total cost showed an increasing trend year by year. From 2013 to 2020, the difference of per capita annual financial investment in drugs and subsidies was statistically significant (F=9.248, P<0.001). The per capita cost basically showed an increasing trend year by year except for individual years. In 2020, 54,688 patients participated in the free medication service in Beijing, and the participation rate was 67.23%. From 2013 to 2020, the difference of annual participation rate of patients was statistically significant (χ²=37098.759, P<0.001), and the number and proportion of patients increased year by year. Conclusion The practice of the district level has proved feasible to expand the scope of free medication service and to optimize the service process, which should be paid more attention in the future. With the continuous advancement of policies and services, the level of patient participation in various areas will tend to be relatively balanced after rising year by year.

Objective To investigate the clinical application of chemokine receptor 4 (CXCR4)-targeted PET/CT imaging in breast cancer using 68Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-TN14003 (NOTA-NFB) and the correlation between 68Ga-NOTA-NFB uptake and pathology.Methods From June 2014 to December 2014,11 female patients (age range:38-68 years) with non-specific invasive breast cancer were recruited in this study.All patients underwent neoadjuvant chemotherapy before surgery.68GaNOTA-NFB and 18F-fluorodeoxyglucose (FDG) PET/CT imaging were performed before the chemotherapy.Three patients also underwent 68Ga-NOTA-NFB PET/CT imaging after the fourth cycle of chemotherapy.The region of interest (ROI) method was used to measure the maximum standardized value (SUVmax) and tumor/non-tumor (T/NT) ratio was calculated.Paired t test and Spearman correlation analysis were used for statistical analysis.Results The SUVmax values of primary lesions were 3.78±2.03 and 8.11±5.14 (t=-3.01,P＜0.05) respectively in 68Ga-NOTA-NFB imaging and 18F-FDG imaging.The T/NT ratios for primary lesions were not significantly different between the two imaging methods (9.36±7.81 vs 15.62±14.51;t=-1.63,P＞0.05).In the metastatic lymph nodes,SUVmax values were not significantly different between 68Ga-NOTA-NFB imaging and 18F-FDG imaging (t=-2.02,P＞0.05),but T/NT ratios were significantly different (t=-2.43,P＜0.05).After neoadjuvant chemotherapy,T/NT ratios were decreased in the 3 patients.Correlation was not found between T/NT in 68Ga-NOTA-NFB imaging and Ki-67,but the P value was close to 0.05 (rs =0.600,P=0.051).Conclusion 68Ga-NOTA-NFB PET/CT can be used as a new CXCR4-targered imaging in diagnosis of breast cancer,and it may be beneficial to evaluate the effect of neoadjuvant chemotherapy.

Objective To analyze kinematic characteristics of children with spastic cerebral palsy during walking based on the method of gait analysis. Methods The gait of 14 children with spastic cerebral palsy and 16 healthy children, who were required to walk back and forth on level ground at normal speed, was tested using portable gait analyzer. The gait differences between diseased side and healthy side of lower limbs for children with spastic cerebral palsy, as well as the gait differences between children with spastic cerebral palsy children and healthy children were compared. Results For children with spastic cerebral palsy, single step time, swing time and toe-off time of diseased side were significantly longer than those of healthy side (P＜0.05), while step frequency, velocity and terminal stance were significantly shorter than those of healthy side (P＜0.05). Compared with healthy children, gait cycle time, single step time, stance time, swing time, percentage of stance phase, mid stance phase, pre-swing stage and toe-off time for diseased side of children with spastic cerebral palsy were significantly longer (P＜0.05). Stride, velocity, step frequency and terminal stance of the children with spastic cerebral palsy were significantly lower than those of healthy children (P＜0.05). Pulling acceleration for children with spastic cerebral palsy also decreased compared with healthy children (P=0.05). Conclusions The stability of children with spastic cerebral palsy decreased during walking, and their single step time, swing time, toe-off time and pulling acceleration might be considered as the sensitive indicators.

Objective To evaluate the cause and the treatment of the vagus nerve reflex in patients with hemoptysis during bron-chial artery embolization (BAE).Methods 1 12 patients with much hemoptysis were enrolled,9 of whom represented vagus nerve reflex in the process of interventional embolization.Results In 9 patients with mixed vagal reflex,5 occurred in the process of bron-chial artery embolization,1 in removing of sheath,1 in hemostasis by compression and 2 in returning to the ward.The intraoperative vagus reflex during BAE was related to over tension and unnormolized operation,and it improved by block of vagus nerve,raising blood pressure and fluid expansion without serious complications.Conclusion Vagus nerve reflex during BAE should be noticed, and early detection and timely intervention may improve its prognosis.

Objective To discuss the value of combining superselective mesenteric angiography (SSMA)with methylene blue in-jection for localization of the Obscure Gastrointestinal Bleeding (OGIB).Methods 12 patients presenting with OGIB had preopera-tive localization of the bleeding site by SSMA.The microcatheter was remained inside the bleeding artery branch after angiography. Then the methylene blue was injected via the microcatheter for localization during exploratory laparotomy and the dyed intestine was then removed.Results The detected rate of bleeding sites by using preoperative SSMA was 100%.The lesions in total 12 patients were found quickly during the exploratory laparotomy.The length of the removed intestine was 9 -12 cm (median,10 cm).After followed up for 10-24 months (median,18 months),all patients with OGIB had no recurrence.Conclusion The method of SSMA combined with the use of methylene blue injection is a safe and effective procedure for the localization of OGIB and could be beneficial for increasing the surgical success rate of hemostasis.

Objective To evaluate the efficacy of parecoxib sodium for preemptive analgesia.Methods PubMed,EMBASE,Cochrane Library,and CNKI database were searched for randomized placebo-controlled trials involving the efficacy of parecoxib sodium for preemptive analgesia.The modified Jadad scale was used for quality assessment.Evaluation indexes included VAS scores at 1,6,12 and 24 h after operation,consumption of morphine within 24 h after operation,and incidences of nausea and vomiting after operation.Meta-analysis was conducted using the Cochrane Collaboration's RevMan 5.0.2 software.Results Fourteen randomized placebo-controlled trials involving 1086 patients were included in our meta-analysis.The modified Jadad scale scores for the 14 studies were ≥ 4.The patients were divided into 2 groups:placebo group and parecoxib sodium 40 mg injected before operation group.The results of meta-analysis showed that VAS scores at 1,6,12 and 24 h after operation were significantly decreased,the consumption of morphine within 24 h after operation was reduced,and the incidences of nausea and vomiting after operation were decreased in parecoxib sodium 40 mg injected before operation group as compared with placebo group (P ＜ 0.05).Conclusion Intravenous injection of parecoxib sodium 40 mg before operation can produce significant preemptive analgesic efficacy and is helpful in decreasing the adverse effect of postoperative analgesia.