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Objective:To compare the detection rate of genital Chlamydia trachomatis (CT) DNA between urine and urethral/cervical swab samples. Methods:From December 2018 to December 2019, a total of 1 475 outpatients were collected from sexually transmitted disease clinics in 7 medical institutions, such as Department of Venereology, Guangzhou Institute of Dermatology, including 1 118 males and 357 females. One urethral/cervical swab sample and one urine sample were collected successively from each patient. Real-time fluorescence-based PCR was performed to detect CT DNA in urine and urethral/cervical swab samples, and paired chi-square test was used to compare the positive rate of CT DNA between the 2 kinds of samples. Random- or fixed-effect meta-analysis was conducted for the test of heterogeneity and merging of positive rates of CT DNA in the urine and urethral/cervical swabs among 7 medical institutions.Results:The positive rate of CT DNA in the urine samples was significantly higher than that in the swab samples from 4 medical institutions (all P < 0.05) , while there was no significant difference in the positive rate of CT DNA between the 2 kinds of samples from 3 medical institutions (all P > 0.05) . The heterogeneity ( I2) estimates of the CT-DNA positive rate in urine and swab samples among different medical institutions were 78.6% (95% CI: 55.9% - 89.6%) and 73.7% (95% CI: 43.7% - 87.7%) , respectively; meta-analysis showed that the total merged positive rate of CT DNA in the urine samples was 10.8% (95% CI: 7.2% - 15.9%) , which was significantly higher than that in the swab samples (7.8%, 95% CI: 4.9% - 12.1%; χ2 = 39.2, P < 0.05) . Compared with the swab sample-based CT-DNA detection method, the sensitivity, specificity, positive predictive value, negative predictive value and consistency rate of the urine sample-based CT-DNA detection method were 97.0% (128/132) , 96.3% (1 293/1 343) , 71.9% (128/178) , 99.7% (1 293/1 297) , and 96.3% (1 421/1 475) , respectively. The positive rate of CT DNA in the urine samples from 1 118 male patients was 11.0% (95% CI: 7.2% - 16.5%) , which was significantly higher than that in the swab samples (7.6%, 95% CI: 4.9% - 11.8%; χ2 = 34.3, P < 0.05) . There was no significant difference in the positive rate of CT DNA between the urine (11.9%, 95% CI: 7.7% - 17.9%) and cervical swab samples from 357 female patients (10.4%, 95% CI: 7.6% - 14.0%; χ2 = 3.2, P > 0.05) . Conclusions:The positive rate of CT DNA in urine samples is higher than or similar to that in urethral/cervical swab samples. The urine sample-based CT-DNA detection method has characteristics of convenience, non-invasiveness, painlessness and low cost, and is worthy of clinical promotion.
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Objective To investigate the regulatory role of cathepsin D (CatD) in the degradation of intracellular advanced glycation end products (AGEs) endocytosed by human dermal fibroblasts (HDFs).Methods Cultured HDFs were treated with 1 μnol/L CA074Me (an inhibitor of CatB and CatL),75 μmol/L pepstatin A (an inhibitor of CatD) and 1 μmol/L MG-132 (an inhibitor of20S proteasome) separately for 4 hours,and then cell counting kit 8 (CCKS) assay and fluorometric assay were performed to determine the cellular viability and protease activity,respectively.The cells in the CA074Me group,pepstatin A group and MG-132 group were additionally treated with AGE-bovine serum albumin (BSA) for 8 hours,and the cells in the blank control group were treated with phosphate-buffered saline (PBS) alone.After 8-hour cultivation,the cells in the above groups were subsequently reincubated with fresh culture medium containing the corresponding inhibitors for 24 hours.Then,flow cytometry was performed to assess the mean fluorescence intensity of intracellular AGE-BSA at different time points.Some other HDFs were treated with 37.5,75 and 150 μmol/L pepstatin A and PBS separately for 4 hours,and then the cells in the 4 groups were treated with 200 mg/L AGE-BSA for 8 hours,followed by the removal of AGE-BSA from the medium and the treatment with 37.5,75 and 150 μmol/L pepstatin A and PBS respectively.Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the mean concentration of intracellular AGE-BSA at different time points,and the degradation rate of AGE was calculated.Some HDFs were divided into 3 groups:blank control group receiving no treatment,NC group transfected with an empty vector,and CatD group transfected with a CatD-overexpressing lentiviral vector.Fluorescence microscopy was conducted to estimate the transfection efficiency.Reverse transcription-PCR,Western blot analysis and fluorometric assay were performed to determine the mRNA and protein expression,and activity of CatD respectively.Then,the cells in the above 3 groups were incubated with AGE-BSA for 8 hours,followed by the removal of AGE-BSA from the medium and the treatment with fresh culture medium.The detection methods were same as the above experiment,and the degradation rate was calculated.Results The cellular proliferative activity in the 1-μmol/L CA074Me group,75-μmnol/L pepstatin A group and 1-μ mol/L MG-132 group was more than 90％,and there was no significant difference between the 3 groups and the control group (100％,F =1.525,P ＞ 0.05).Twenty-four hours after the removal of AGE-BSA from the medium,the fluorescence intensities of intracellular AGE-BSA in the CA074Me + AGE-BSA group (275.00 ± 10.15) and MG-132 + AGE-BSA group (259.00 ± 11.14) significantly decreased compared with those at the 8-hour time point (295.00 ± 6.56 and 285.67±8.74 respectively;paired t test,t =4.778,6.154 respectively,both P ＜ 0.05),while no significant difference was observed in the fluorescence intensities of intracellular AGE-BSA in the pepstatin A + AGE-BSA group between the 8-hour time point and 32-hour time point (P ＞ 0.05).The degradation rates of intracellular AGE-BSA within 24 hours in the 37.5,75 and 150 μmol/L pepstatin A groups were 9.64％ ± 1.27％,5.62％ ± 0.47％ and 3.21％ ± 0.73％ respectively;there were significant differences among the 3 groups (F =45.876,P ＜ 0.05),and the degradation rate significantly decreased along with the increase of pepstatin A concentration (P ＜ 0.05).Fluorescence microscopy showed no fluorescent cells in the blank control group,while the NC group and CatD group both showed a high proportion (＞ 80％) of fluorescent cells.The mRNA and protein expression as well as the activity of CatD were significantly higher in the CatD group than in the blank control group and NC group (all P ＜ 0.05).The CatD + AGE-BSA group showed a significantly higher degradation rate of intracellular AGE-BSA within 24 hours compared with the AGE-BSA group and NC + AGE-BSA group (both P ＜ 0.05).Conclusion CatD can promote the degradation of intracellular AGE-BSA endocytosed by HDFs.
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Objective To evaluate the effect of photoaging on the degradation of advanced glycation end products (AGEs) by human dermal fibroblasts.Methods Some cultured human dermal fibroblasts were subjected to repetitive ultraviolet A (UVA) radiation (UVA radiation group) to establish a photoaging cell model,which was then evaluated by cell counting kit 8 (CCK-8) assay,senescenceassociated β-galactosidase staining and detection of apoptosis rate.Moreover,fibroblasts receiving no treatment served as control group.Some other primary fibroblasts were divided into 4 groups:photoaged group receiving UVA radiation,non-photoaged group receiving no treatment,AGE-treated photoaged group treated with UVA radiation followed by the treatment with 200 mg/L AGE-bovine serum albumin (BSA),and AGE-treated non-photoaged group treated with 200 mg/L AGE-BSA alone.After the treatment with AGE-BSA for 4-72 hours,flow cytometry was performed to determine the fluorescence intensity of AGE-BSA in fibroblasts of the above groups.After 8-hour treatment with AGE-BSA,confocal laser scanning microscopy was performed to localize and semiquantitatively detect AGE-BSA in fibroblasts,and enzymelinked immunosorbent assay (ELISA) was conducted to detect AGE-BSA levels in fibroblasts,as well as changes in the intracellular AGE-BSA level within 24 hours after the removal of AGE-BSA.Results Compared with the control group,the UVA radiation group showed significantly decreased cellular proliferative activity (t =7.559,P ＜ 0.05),but significantly increased apoptosis rate and percentage of β-galactosidase-positive fibroblasts (t =14.075,43.524 respectively,both P ＜ 0.05).Flow cytometry revealed that the average fluorescence intensities of AGE-BSA after 4-,8-,16-,24-,48-and 72-hour treatment with AGE-BSA were significantly higher in the AGE-treated photoaged group (293.00 ± 8.19,359.67 ± 11.59,347.00 ± 12.29,338.00 ± 12.77,334.67 ± 14.22 and 336.30 ± 10.21,respectively) than in the photoaged group (all P ＜ 0.05),as well as in the AGE-treated non-photoaged group (222.33 ± 8.74,276.33 ± 6.11,256.33 ± 5.51,243.00 ± 10.15,236.33 ± 1.53 and 240.33 ± 1.52,respectively) than in the non-photoaged group (all P ＜ 0.05).Moreover,the average fluorescence intensities of AGE-BSA at different time points were all significantly higher in the AGE-treated photoaged group than in the AGE-treated non-photoaged group (all P ＜ 0.05).Confocal laser scanning microscopy showed that AGE-BSA was mainly localized in lysosomes after endocytic uptake into the fibroblasts,and the AGE-treated photoaged group showed significantly increased fluorescence intensity of AGE-BSA compared with the AGE-treated non-photoaged group (P ＜ 0.05).ELISA revealed that the intracellular AGE level in the AGE-treated non-photoaged group at 24 hours after the removal of AGE-BSA was decreased by (14.6 ± 1.2)％ compared with that before the removal,and the degradation rate of AGE-BSA was significantly higher in the AGE-treated non-photoaged group than in the AGE-treated photoaged group (7.6％ ± 1.4％,t =6.604,P ＜ 0.05).Conclusion The internalized AGE-degradating ability decreases in photoaged fibroblasts,which may induce the accumulation of AGEs in photoaged skin.
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Objective To evaluate the effect of miRNA-29 (miR-29) family on the synthesis of collagen Ⅰ and Ⅲ in chronically photodamaged (photoaged) skin.Methods Some cultured human dermal fibroblasts (HDFs) were divided into 2 groups:non-irradiated group receiving no treatment,and chronic photodamage group treated with repetitive ultraviolet A (UVA) radiation,which served as a chronically photodamaged cell model and was verified by flow cytometry and β-galactosidase staining.Western blot analysis was performed to determine the protein expression of collagen Ⅰ and Ⅲ,and real-time fluorescence-based quantitative PCR (qRT-PCR) to measure expression of 3 members of the miR-29 family (miR-29a-3p,miR-29b-3p and miR-29c-3p) in the above 2 groups.The differentially expressed miR-29c-3p between the above 2 groups was chosen for further functional tests.Some HDFs were divided into 4 groups to be transfected with fluorescein-labelled miR-29c-3p mimics (overexpression group),inhibitors (inhibition group),and their control RNA oligonucleotides (negative control group and inhibitor control group) respectively.The transfection efficiency was evaluated by the proportion of fluorescent cells,and the relative expression of miR-29c-3p in the above 4 groups was measured by qRT-PCR for evaluating the RNA interference efficiency,qRT-PCR was conducted to determine the mRNA expression of collagen type Ⅰ α1 (COL1A1) and collagen type Ⅲ α1 (COL3A1) genes,and Western blot analysis to measure the protein expression of collagen Ⅰ and Ⅲ.Results Compared with the non-irradiated group,the chronic photodamage group showed significantly increased proportion of senescent cells (36.47％ ± 3.20％ vs.12.56％ ± 1.46％,P ＜ 0.01) and G1-phase cells (71.70％ ± 2.43％ vs.41.89％ ± 1.86％,P ＜ 0.01),but significantly decreased proportion of S-phase cells (10.63％ ± 0.36％ vs.36.48％ ± 1.31％,P ＜ 0.01),which indicated that the chronically photodamaged cell model was established successfully.The protein expression of collagen Ⅰ and Ⅲ was significantly lower in the chronic photodamage group (0.40 ± 0.19 and 0.52 ± 0.10) than in the non-irradiated group (1.00 ± 0.12 and 1.00 ± 0.10,respectively,both P ＜ 0.01).The expression of miR-29c-3p was significantly higher in the chronic photodamage group than in the non-irradiated group (4.42 ± 2.05 vs.0.89± 0.10,P ＜ 0.05),while there were no significant differences in the expression of miR-29a-3p or miR-29b-3p between the 2 groups (both P ＞ 0.05).Twenty-four hours after transfection,the overexpression group and inhibition group both showed more than 90％ transfection efficiency which met the interference requirements.The expression of miR-29c-3p was significantly higher in the overexpression group than in the negative control group (224.17 ± 2.00 vs.2.45 ± 0.34,P ＜ 0.01),but significantly lower in the inhibition group than in the inhibitor control group (0.20 ± 0.08 vs.2.24± 0.14,P ＜ 0.01),suggesting that a RNA interference model was successfully established.The mRNA expression of COL1A1 and COL3A1 and the protein expression of collagen Ⅰ and Ⅲ were significantly lower in the overexpression group than in the negative control group and inhibition group (all P ＜ 0.05),and significantly higher in the inhibition group than in the inhibitor control group (all P ＜ 0.01).Conclusion The expression of miR-29c-3p is up-regulated in chronically photodamaged HDFs,likely by regulating the mRNA expression of COL1A1 and COL3A1 and the protein expression of collagen Ⅰ and Ⅲ.
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Objective To evaluate effectiveness of peripheral Mohs micrographic surgery for the treatment of extramammary Paget′s disease (EMPD). Methods A total of 28 patients with EMPD were treated with peripheral Mohs micrographic surgery. The depth and extent of tumor infiltration were evaluated before the surgery. One day before the surgery, 20% aminolevulinic acid hydrochloride was topically applied to determine and label surgical margins under a Wood′s lamp. After fluorescence-based localization, peritumoral skin tissues were resected and underwent frozen-section examination according to the protocol for Mohs micrographic surgery. Meanwhile, the tumor was resected. After surgery, patients were followed up every 3 - 6 months to detect local recurrence and metastasis. Results Of the 28 patients, 25 were male and 3 were female. Six patients each underwent 3 sessions of frozen-section examination, and 12 patients each received 2 sessions, with an average of 1.86 sessions for each patient. During the follow-up for 5 - 72 months, local recurrence occurred in 3 cases, and 1 patient died of tumor metastasis and uremia after 2 years of follow-up. Conclusion Peripheral Mohs micrographic surgery is a time-saving and effective treatment for EMPD.
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Objective To investigate the effects of advanced glycation end products(AGE)on the expressions and activity of cathepsin D(CatD)in ultraviolet A(UVA)?irradiated human dermal fibroblasts. Methods Human dermal fibroblasts were isolated and harvested from the circumcised foreskin of children, and subjected to a primary culture. CCK?8 assay was performed to screen non?cytotoxic concentrations of AGE?bovine serum albumin (BSA). Some fibroblasts were incubated with 50, 100 and 300 mg/L AGE?BSA separately for 24 hours, with untreated cells as the control group. Then, reverse transcription(RT)?PCR, Western?blot analysis and a fluorimetric assay were performed to measure the mRNA and protein expressions as well as activity of CatD, respectively. Some fibroblasts were classified into six groups: control group receiving no treatment, AGE?BSA group and BSA group treated with the highest non?cytotoxic concentration of AGE?BSA and the same concentration of BSA respectively for 24 hours, UVA group irradiated by 10 J/cm2 UVA, UVA?AGE?BSA group and UVA?BSA group treated with AGE?BSA and BSA at the above non?cytotoxic concentration respectively for 24 hours both before and after UVA radiation at 10 J/cm2. After the treatments, RT?PCR, Western?blot analysis and a fluorimetric assay were conducted to detect mRNA and protein expressions and activity of CatD respectively. Results AGE?BSA of 50- 200 mg/L exhibited no obvious influence on cellular proliferation of fibroblasts. The fibroblasts incubated with AGE?BSA of 50, 100 and 200 mg/L showed a significant increase in the mRNA expression(0.267 ± 0.007, 0.348 ± 0.007, and 0.418 ± 0.006 respectively), protein expression (1.403 ± 0.181, 2.233 ± 0.090 and 2.477 ± 0.111 respectively), and activity(1.760 ± 0.080, 2.330 ± 0.060 and 2.890 ± 0.080 respectively)of CatD compared with the control group(mRNA:0.161 ± 0.006;protein:0.903 ± 0.200;activity:1.100 ± 0.090, all P < 0.05). AGE?BSA increased CatD expressions and activity in a dose?dependent manner. The mRNA and protein expressions as well as activity of CatD were significantly higher in the UVA group than in the control group (mRNA expression: 0.480 ± 0.005 vs. 0.155 ± 0.005; protein expression: 2.583 ± 0.199 vs. 0.920 ± 0.235;activity:2.970 ± 0.110 vs. 1.110 ± 0.040, all P<0.05), but significantly lower in the UVA?AGE?BSA group than in the UVA group(mRNA expression:0.394 ± 0.008 vs. 0.480 ± 0.005;protein expression:2.070 ± 0.125 vs. 2.583 ± 0.199;activity: 2.560 ± 0.060 vs. 2.970 ± 0.110, all P < 0.05). Conclusion AGEs could increase CatD expressions and activity in human dermal fibroblasts not receiving UVA irradiation, but inhibit their increase in UVA?induced human dermal fibroblasts.
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Objective To investigate whether ultraviolet A UVA)-induced CatK expression is regulated by the mitogen-activated protein kinases (MAPK) signaling pathway in human dermal fibroblasts in vitro.Methods Human dermal fibroblasts were obtained from circumcised foreskin of children,and subjected to primary culture.After several passages of subculture,some fibroblasts were irradiated with UVA at a dose of 10 J/cm2.Western blot was performed to measure the expressions of total and phosphorylated JNK (t-and p-JNK) and P38 (t-and p-P38) at 0.75,1.5,3 and 6 hours after the irradiation.Some fibroblasts were divided into six groups:control group receiving no treatment,SP group treated with SP600125 of 800 nmol/L,SB group treated with SB203580 of 10 μmol/L,UVA group irradiated with UVA at a dose of 10 J/cm2,UVA-SP group treated with SP600125 for 1 hour before and for 1.5 or 48 hours after UVA irradiation at 10 J/cm2,UVA-SB group treated with SB203580 for 1 hour before and for 1.5 or 48 hours after UVA radiation at 10 J/cm2.Subsequently,Western blot was performed to determine the expressions of p-c-Jun and p-MAPKAPK2 in these groups at 1.5 hours after the UVA irradiation,and reverse transcription (RT)-PCR and Western blot to detect the mRNA and protein expressions of CatK at 48 hours after the UVA irradiation,respectively.Statistical analysis was carried out by t test,one way analysis of variance and least significant difference (LSD)-t test.Results The expression levels (gray values) of p-JNK and p-P38 were significantly increased at 0.75 hour (4.77 ± 0.19 and 2.44 ± 0.13 respectively,both P ＜ 0.05) and 1.5 hours (4.68 ± 0.09 and 2.30 ± 0.04 respectively,both P ＜ 0.05),but showed no significant changes at 3 hours (both P ＞ 0.05) and 6 hours (both P ＞ 0.05) after the UVA irradiation compared with those before the irradiation (3.2 ± 0.27 and 1.61 ± 0.08 respectively).A significant decrease was observed in the expression of p-c-Jun in the UVA-SP group and p-MAPKAPK2 in the UVA-SB group compared with the UVA group (p-c-Jun,2.55 ± 0.48 vs.4.85 ±0.96; p-MAPKAPK2,1.16 ± 0.12 vs.2.46 ± 0.09,both P ＜ 0.05).The CatK mRNA and protein expressions were attenuated by 61.1％ and 44.3％ respectively in the UVA-SP group (both P ＜ 0.05),and by 71.3％ and 50.4％ respectively in the UVA-SB group (both P ＜ 0.05) in comparison with the UVA group.The UVA-SP group also showed a significant reduction in CatK mRNA and protein expressions as compared with the UVA-SB group (both P ＜ 0.05).Conclusion Both JNK and P38 signaling pathways,especially the JNK pathway,may contribute to the upregulation of CatK expression in dermal fibroblasts induced by UVA irradiation.
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Objective To observe the expression changes of cathepsin K (CatK) in human dermal fibroblasts at different time points after different doses of ultraviolet A (UVA) irradiation.Methods Dermal fibroblasts were isolated from circumcised foreskins of children,and subjected to primary culture and subculture.Cells at third-tenth passage were used in the following experiment.Some fibroblasts were irradiated with UVA of 10 J/cm2 and collected at 24,48 and 72 hours separately after the irradiation,and some fibroblasts were irradiated with UVA of 10,20 and 30 J/cm2 separately and harvested 48 hours later.The fibroblasts receiving no irradiation served as the control group.Reverse transcription PCR and Western blot were carried out to detect the mRNA and protein expressions of CatK in fibroblasts,respectively.Results Compared with the control fibroblasts,those irradiated with UVA of 10 J/cm2 showed a significant elevation in the mRNA and protein expression levels of CatK on day 1 (0.351 ± 0.038 vs.0.177 ± 0.006,1.76 ± 0.27 vs.0.82 ± 0.45,respectively,both P＜ 0.05),day 2 (0.510 ± 0.017 vs.0.176 ± 0.002,2.97 ± 0.36 vs.1.58 ± 0.15,respectively,both P＜ 0.05) and day 3 (0.313 ± 0.012 vs.0.173 ± 0.002,2.23 ± 0.14 vs.1.29 ± 0.32,respectively,both P ＜ 0.05),with the highest expressions of CatK mRNA and protein observed on day 2.Within the range of 10-30 J/cm2,UVA enhanced the CatK mRNA and protein expression levels in a dose-dependent manner.In detail,at 48 hours after the irradiation with UVA of 10,20 and 30 J/cm2,the CatK mRNA expression level in the irradiated fibroblasts was 2.34,2.91 and 3.18 times,and the CatK protein expression level 1.77,2.82 and 3.64 times,respectively,that in the control fibroblasts (all P ＜ 0.05).Conclusion The expression of CatK is up-regulated in human dermal fibroblasts after UVA irradiation.
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Objective To elucidate the molecular basis for induced resistance of N. gonorrhoeae to ceftriaxone in vitro. Methods The reference strain ATCC49226 and clinical isolate ZSSY00205 of N. gon-orrhoeae were exposed to subinhibitory concentration of ceftriaxone for the induction of resistance. Then,suppression subtractive hybridization was performed with the pre-induction parent strains as drivers and post-induction mutant strains as testers to create a subtractive cDNA library. Following that, a total of 192 clones were randomly selected from the library, and arrayed by spotting onto nylon membranes. Finally, dif-ferentially expressed genes were screened by hybridization with labeled-RsaI restriction fragments from the sensitive and resistant N.gonorrhoeae strains respectively, and analyzed by sequencing and homology research using Blast program. Results A subtractive library for these resistant N.gonorrhoeae strains was generated by SSH technique. Microarray analysis and homology research confirmed 5 genes related to ceftriaxone resistance, i.e. mtrR, mtrC, gyrB, rpsJ and PJD1. Conclusions The induced resistance of N. gonorrhoeae to ceftriaxone may be associated with mtrR, mtrC, gyrB, rpsJ and PJD1 genes which probably mediate the resistance by enhancing the activity of efflux pump system.
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Objective To investigate the incidence, clinical patterns, and species distribution of pathogenic fungi of onychomyeosis in patients with chronic viral hepatitis (CVH), and to analyze the relationship between onychomycosis and CVH. Methods From November 2005 to October 2006, direct microscopy and fungal culture were performed on nail samples from CVH patients with clinically suspected onychomycosis in the two largest institutions for communicable disease control in Guangzhou city. The incidence, clinical patterns, and species distribution of pathogenic fungi of onychomycosis were assessed based on the findings in mycologic examinations. Results The study randomly recruited 995 patients with CVH, and onychomycosis was diagnosed in 116 patients. The incidence of onychomycosis was 11.66% in total, 6.20%, 8.59%, 14.09%, 19.67% in patients with mild, moderate, severe and extremely severe CVH respectively, 7.09%, 17.29%, 19.13% and 27.27% in patients with a clinical course of CVH of 0.5-9 years, 10-19 years, 20-29 years, ≥30 years respectively. The most common clinical pattern was distal and lateral subungual onychomycosis (DLSO, 69.83%), followed by total dystrophic onychomycosis (TDO, 14.66%). Among the pathogenic fungi, dermatophytes amounted to 71.43%, yeasts 21.43%, moulds 7.14%, and Trichophyton rubrum was the most frequently isolated fungus (42.86%). Conclusions The incidence of onychomycosis in patients with CVH is correlated with the severity and course of CVH. Among these patients, the most common clinical pattern is DLSO with the most frequent fungal species being dermatophytes and predominant fungal isolate being Trichophyton rubrum.