Objective To explore the role of neogambogic acid in the characteristics of colorectal cancer stem cells (CRC-CSCs) through the Wnt/β-catenin signaling pathway. Methods The colorectal cells SW480 and HCT166 were divided into control group and neogambogic acid groups (1.5, 3, 6, and 12 μmol/L). The viability of CRC-CSCs was determined by MTT method, and spheroid and clone formation assays were used to assess the capacity of spheroid formation and self-renewal ability of the cells. The effects of neogambogic acid on the apoptosis and cell cycle of CRC-CSCs were evaluated by flow cytometry assays. Real-time quantitative PCR was used to detect the mRNA expression levels of relative markers (CD133, CD44, ALDH1, Oct4, and Nanog) of CRC-CSCs, and the protein expression levels of the self-renewal marker (PCNA), apoptosis markers (cleaved caspase-3 and cleaved caspase-9), and Wnt/β-catenin signaling pathway markers (p-GSK3β, GSK3β, β-catenin, and Wnt) were analyzed using Western blot. Results Compared with the control group, after neogambogic acid treatment, the viability of SW480 and HCT116 cells decreased (P<0.05), the spheroid forming ability and the clone numbers of CRC-CSCs decreased (P<0.001, P<0.01) but the cell apoptosis rate increased (P<0.01), and cell cycle was arrested in G₀/G₁ phase. Moreover, neogambogic acid downregulated the mRNA and protein expression of relative markers of CRC-CSCs (CD133, CD44, ALDH1, Oct4, and Nanog), PCNA, p-GSK3β, β-catenin, and Wnt (P<0.05) and upregulated the expression of cleaved caspase-3, cleaved caspase-9, and GSK3β (P<0.01). Conclusion Neogambogic can inhibit the stem cell properties of colorectal cells via inhibition of Wnt/β-catenin signaling pathway. As a result, neogambogic acid may be an attractive agent against colorectal cancer.

Objective To analyze the effect of baseline cytokines in aqueous humor in predicting the improvement of the central macular thickness(CMT)and best corrected visual acuity(BCVA)of patients with macular edema secondary to retinal vein obstruction(RVO-ME)after anti-vascular endothelial growth factor(VEGF)treatment for 3 months.Meth-ods Totally 30 eyes of 30 patients initially diagnosed with RVO-ME in the Affiliated Eye Hospital of Nanjing Medical Uni-versity from October 2021 to January 2023 were enrolled.Aqueous humor was collected by anterior chamber puncture be-fore the anti-VEGF drug injection.The levels of 11 cytokines in aqueous humor,including VEGF,intercellular adhesion molecule(ICAM)-1,interleukins(ILs)and interferon-γ(IFN-γ),were measured by Luminex xMAP technology.The cor-relation between baseline aqueous humor cytokine levels and CMT,BCVA(logMAR)as well as their changes(difference between baseline and anti-VEGF treatment for 3 months)in RVO-ME patients was analyzed;the correlation between CMT and BCVA(logMAR)was analyzed at baseline and 3 months after anti-VEGF treatment.CMT and BCVA responders were defined as patients with 50％or more reduction in CMT and BCVA(logMAR)from baseline to the third month.A Logistic regression model was used to analyze the relationship between baseline aqueous humor cytokines and RVO-ME patients be-coming CMT or BCVA responders after 3 months of anti-VEGF treatment.Results Compared with baseline,the CMT sig-nificantly decreased and BCVA significantly improved in RVO-ME patients after anti-VEGF treatment for 3 months(both P＜0.001).The CMT was positively correlated with BCVA(logMAR)of RVO-ME patients at baseline and after anti-VEGF treatment for 3 months(P=0.026,0.002).At baseline,the levels of VEGF and IL-8 of RVO-ME patients were positively correlated with CMT(P=0.032,0.035);the levels of IL-6 and IL-8 were positively correlated with BCVA(logMAR)(P=0.018,0.002).The baseline IL-8 level in RVO-ME patients was negatively associated with the change in CMT(P=0.024).Logistic regression analysis showed that elevated baseline ICAM-1 level could increase the risk of RVO-ME patients becom-ing BCVA non-responders after 3 months of anti-VEGF treatment(P=0.023).Conclusion Baseline IL-8 and ICAM-1 may be predictors of CMT and BCVA improvement after anti-VEGF treatment of RVO-ME.

Objective To investigate the relationship between baseline aqueous humor cytokines and optical coher-ence tomography angiography(OCTA)changes in eyes with macular edema secondary to retinal vein occlusion(RVO-ME)after anti-vascular endothelial growth factor(VEGF)treatment.Methods From October 2021 to January 2023,30 eyes of 30 patients diagnosed with RVO-ME at the Affiliated Eye Hospital of Nanjing Medical University were included in this study.All eyes were treated with the intravitreal injection of anti-VEGF drugs once a month for 3 consecutive months and then treated as needed.The aqueous humors before the first and third injections were collected.The expression levels of VEGF,interleukins(IL),γ-interferon(IFN-y),intercellular adhesion molecule-1(ICAM-1),and platelet-derived growth factor(PDGF)were detected using the Luminex platform.The central macular thickness(CMT),fovea avascular zone(FAZ)area,perimeter(PERIM),acircularity index(AI),and the vessel density in a 300 μm wide region around FAZ(FD-300)were measured using OCTA.The best corrected visual acuity(BCVA)was examined using the international standard logarithmic visual acuity chart,which was converted into the logarithm of the minimum angle of resolution(log-MAR)for statistics.Aqueous humor cytokines and OCTA parameters were compared before and after anti-VEGF treatment for RVO-ME,and the relationship between OCTA changes(the difference between before and after treatment)and baseline aqueous humor cytokines was analyzed.Results After treatment,the levels of VEGF,ICAM-1,PDGF,IL-8,and IL-10 in RVO-ME patients decreased(all P＜0.05),the BCVA improved(P＜0.001),and CMT,AI and FD-300 decreased(all P＜0.05).Correlation analysis results showed that baseline IL-13 was negatively correlated with the AI change(r=-0.388,P＜0.05),and baseline IL-8 was negatively correlated with the FD-300 change(r=-0.421,P＜0.05).Conclusion Baseline IL-13 and IL-8 may be related to the improvement of the retinal macular microvascular structure after anti-VEGF treatment for RVO-ME.For the treatment and management of RVO-ME,in addition to anti-VEGF therapy,targeted anti-in-flammatory therapy should also be considered.

Objective:To investigate the clinical and imaging features of population receiving opportunistic screening for lung cancer and in convalescent stage of COVID-19.Methods:Cross-sectional study and analysis was performed on the patients who underwent chest low-dose CT examination for cancer prevention in Cancer Hospital of Chinese Academy of Medical Sciences from December 28, 2022 to January 19, 2023. All the patients completed the COVID-19 questionnaire. A total of 334 questionnaires were sent out, all of which were recovered, and 321 valid questionnaires were collected, among them, 293 questionnaires were included in the analysis. Statistical analysis was conducted according to the questionnaire information, clinical symptoms and chest CT imaging results. The potential influencing factors of COVID-19 infection were explored, and the imaging characteristics of COVID-19 infection and early stage of lung cancer were compared.Results:A total of 293 patients underwent lung cancer screening during the convalescent stage of COVID-19 infection. A total of 231 (78.8%) cases had cough and 228 (77.8%) cases had fever. 214 (73.0%) cases still had clinical symptoms within 2 weeks after nucleic acid or antigen test showing negative, especially for cough in 171 (58.4%) cases, fatigue in 79 (25.3%) cases and sputum in 73 (24.9%) cases. There were 54 (18.4%) cases with positive chest CT changes. The main imaging findings showed that there were 46 cases with new patchy shadows, 25 cases with linear opacities, 10 cases with ground-glass opacities, 10 cases with nodular like shadows and 2 cases with consolidation, and most lesions were in the subpleural area of both lungs. Univariate analysis showed that positive CT findings were correlated with the time from positive detection of COVID-19 to screening ( P=0.026), age ( P<0.001) and underlying diseases ( P=0.006). Multivariate analysis showed that age≥65 years old ( OR=6.425, 95% CI: 2.688-15.358; P<0.001) and underlying diseases ( OR=2.292, 95% CI: 1.120-4.691; P=0.023) were risk factors for pulmonary imaging changes of COVID-19 infection. For lung cancer opportunistic screening, 36 (12.3%) cases showed ground-glass opacities in bilateral or unilateral lung lobes, among which 4 cases were suspected to be atypical adenomatous hyperplasia and 2 cases s were suspected to be early stage of lung cancer. Conclusions:Abnormal clinical symptoms and chest CT findings are still observed in the patients during the convalescent stage of COVID-19 infection. Age≥65 years, underlying diseases were the high-risk factors for the changes in chest CT imaging after COVID-19 infection. Attention should be paid to the differential diagnosis of chest CT findings between changes in the convalescent stage of COVID-19 and early stage of lung cancer.

Developmental coordination disorder (DCD) is a childhood-onset condition that primarily affects physical co-ordination.In China, DCD is not well recognized and is under-treated.Knowledge of the evaluation and intervention of DCD among physiotherapists (PT) is limited.In 2020, the Academy of Pediatric Physical Therapy of the American Physical Therapy Association published the Physical Therapy Management of Children with Developmental Coordination Disorder: An Evidence-Based Clinical Practice Guideline.From the perspective of PT, this review aims to make a comprehensive interpretation of the recommendations in the guideline regarding the physical examination and evaluation, physiotherapy planning and implementation, and family education of children at risk or diagnosed with DCD.This article aims to make DCD get more attention from domestic PT through the interpretation of the latest guidelines, and strengthen the knowledge of physiotherapy assessment and management in children with DCD to guide the clinical practice.

Objective To investigate the role and significance of opportunistic screening in cervical cancer screening for elderly women aged≥65 years old. Methods The screening data of 1 304 elderly women (≥65 years old) who underwent opportunistic screening for cervical cancer at the Cancer Hospital, Chinese Academy of Medical Sciences, between January 1, 2010, and December 31 st, 2018, were analyzed retrospectively. Women who underwent cervical cytology tests and human papillomavirus (HPV) testing were divided into two groups according to age as following 65-69 and ≥70 years old. Women with abnormal cytology or who were hrHPV-positive were followed up. The cervical cytological abnormalities and high-risk HPV infection rates in women aged≥65 years in opportunistic screening were analyzed. Results Of all cases, 175 had abnormal cytology or were hrHPV-positive. Among the 1 304 women, 69 were TCT-positive, with a positivity rate of 5.3%, including 17 cases (24.6%) of high-degree squamous intraepithelial neoplasia and 3 cases (4.4%) of squamous cell carcinoma. The total abnormality rate of TCT in the 65-to 69-year age group (6.7%, 43 cases) was significantly higher than that in the≥70 age group (3.9%, 26 cases), and the difference was statistically significant between the two groups (P=0.024). The overall prevalence of hrHPV infection was 10.7% (139/1 304). HPV58 (31/174, 17.9%) was identified as the most common high-risk HPV type, followed by HPV16, HPV52, HPV33, and HPV31. Follow-up showed that 50.3% of the women had never been screened in the past 10 years, and no statistically significant difference in TCT abnormality and hrHPV infection positivity rate were found between those who had been screened (80 cases) and those who had not been screened at least once in 5 years (87 cases) (P>0.05). Conclusion Attention should be paid to the screening for cervical cancer in elderly women aged ≥65 years old. Opportunistic screening is a supplement to the population-based organized cervical cancer screening. The termination age of cervical cancer screening for elderly women may be appropriately extended.

OBJECTIVETo investigate the epidemiology, internal opening location, and risk factors associated with recurrence of anal fistula.

METHODSClinical data of 1783 hospitalized patients admitted for anal fistula treatment to Shanghai Shuguang Hospital from January 2013 to September 2015 were retrospectively analyzed. Fistula passing through anorectal ring or locating above was defined as high anal fistula (n=125). Internal opening location was defined as follows: posterior (5 to 7 o'clock), front(11 to 1 o'clock), left (2 to 4 o'clock) and right (8 to 10 o'clock).

RESULTSAmong 1783 cases, 1526 were male with a median age of 36 years, 257 were female with a median age of 35 years, and the ratio of male to female was 5.9 vs 1.0. In high anal fistula cases, this ratio of male to female was 7.3 vs 1.0. Posterior internal opening accounted for 51.4%(884/1720), while this percentage was 66.4%(83/125) in high anal fistula cases, which was significantly higher than 50.2%(801/1595) in low anal fistula cases(P=0.002). Postoperative recurrence rate was 2.6%(45/1720) and the rates in high anal fistula and low anal fistula were 13.6%(17/125) and 1.8%(28/1595) respectively, with significant difference(P=0.000). Multivariate logistic regression analysis showed that fistula height(OR=5.475, 95%CI:2.230 to 13.445, P=0.000), treatment history(OR=2.671, 95% CI:1.315 to 5.424, P=0.007), seton placement history (OR=4.707, 95%CI:1.675 to 13.232, P=0.003) and concomitant colitis(OR=10.300, 95%CI:1.187 to 89.412, P=0.034) were independent risk factors for anal fistula recurrence. Seton placement history was an independent risk factor for high anal fistula recurrence (OR=6.476, 95%CI:1.116 to 37.589, P=0.037).

CONCLUSIONSAnal fistula occurs in young and middle-aged male patient. Internal opening locates in posterior more commonly, especially in high anal fistula patients. Postoperative recurrence rate of high anal fistula is quite high. Patient with both high anal fistula and seton placement history has significantly high rate of postoperative recurrence.

Objective: To investigate the gene frequency of HLA-B* 5801 in the population of Chinese Minnan region.Methods:In this study,we enrolled 178 patients requiring allopurinol therapy( including 40 patients with gout,89 patients with hyperuricemia and 49 patients with gouty arthritis) and 100 healthy people.We isolated genomic DNA from their blood and screened for HLA-B*5801 with both PCR and gene sequencing.Results:We found 22%patients and 16%healthy people with HLA-B*5801.The frequencies of HLA-B*5801 in patients and healthy people are 0.13 and 0.09,respectively.The results from PCR and gene sequencing were consistent.Conclusion:The frequency of HLA-B*5801 in the population of Chinese Minnan region is relatively high.Therefore,it is necessary to screen for HLA-B*5801 in allopurinol users before taking the medicine.

Objective To investigate the genotype and epidemiology of plasmid‐mediated AmpC β‐lactamases produced by the clinical strains of Escherichia coli and Klebsiella pneumoniae .Methods A total of 176 clinical nonrepetitive cefoxitin non‐sensitivity isolates of Escherichia coli and Klebsiella pneumoniae was collected from July 2011 to August 2012 .Polymerase chain reaction (PCR) for AmpC enzyme gene amplification and DNA sequencing were carried out for genotype of AmpC beta‐lactamases .Results The results of PCR showed that the positive rate of ampC of the 176 strains of Escherichia coli and Klebsiella pneumoniae AmpC was 18 .2% ,mainly DHA type ,counting for 59 .4% ,CIT counting for 37 .5% ,EBC counting for 3 .1% .The positive rate of ampC of Escherichia coli was 11 .4% ,mainly CIT type ,counting for 77 .8% ,the positive rates of DHA type and EBC type both were 11 .1% .The positive rate of ampC of Klebsiella pneumoniae were 23 .7% ,mainly DHA type ,counting for 78 .3% ,CIT type count‐ing for 21 .7% .The results of DNA sequencing showed that there were 18 strains DHA‐1 type and 1 strain ampC gene type of Morganella morganii in DHA type strains ,the concordance rate was 97 .0% ,10 CIT type strains was CMY‐2 type ,1 strain was CMY‐42 ,one strain was CMY‐4 type ,EBC type was ampC gene type of Enterobacter cloacae ,the concordance rate was 99 .0% .A total of 32 strains of gene sequencing were registered as KJ127248 - KJ127279 in GenBank .Conclusion The main genotypes of plasmid‐mediated ampC enzyme produced by Escherichia coli and Klebsiella pneumoniae were CMY‐2 and DHA‐1 respectively .

Objective To construct a mutant strain of Streptococcus mutans ( S.mutans ) with clpC-deletion and to investigate the role of clpC gene in genetic competence.Methods The fragment of clpC gene and the kanamycin resistant cassette flanked by two loxP sites were amplified by PCR.The purified fragment of clpC gene was cloned into pMD-19T simple vector to construct pCKX1.The pCKX1 vector was digested with ClaⅠ/EcoRⅠ, then blunted and introduced into lox71-KMR-lox66 to obtain pCKX2 vector via homologous recombination.The pCKX2 vector was linearized with SalⅠ and transformed into S.mutans UA159 strain.The positive strains constructed via homologous recombination were screened with kanamycin and transformed with the thermosensitive plasmid pCrePA.The KMR cassette was excised after incubating at 30℃ for 48 hours.Then the pCrePA plasmid was removed after overnight incubating at 37℃for the prepara-tion of clpC-deletion mutant.Total RNA were extracted from the S.mutans UA159 strain and the clpC-dele-tion mutant strain respectively, and then reverse transcribed into first strand cDNA.The target gene frag-ments were amplified by RT-PCR and analyzed by the agarose gel electrophoresis and sequencing.After be-ing verified by PCR and sequencing, the S.mutans UA159 strain and the clpC-deletion mutant strain were re-spectively transformed with E.coli-S.mutans shuttle vector pDL276 to observe the competence development induced by the competence-stimulating peptide (CSP).Results The PCR and sequencing results showed that the pCKX2 vector and the mutant strain with clpC-deletion were constructed successfully via homologous recombination.No clpC gene was detected in the clpC-deletion mutant as indicated by RT-PCR analysis.The formation of competent clpC-deletion mutant was delayed and the competence state was prolonged as com-pared with its parent strains.Conclusion The clpC gene negatively regulated the formation of competent S.mutans.