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Objective:To evaluate the measurement agreement of Roche 25(OH)D immunoassay(evaluation method) with LC-MS/MS (reference method).Methods:A total of 909 residual serum samples from routine health check participants were collected from May to June in 2019. 25(OH)D concentrations were measured by evaluation method and LC-MS/MS, respectively. Passing-bablok regression, intraclass correlation coefficient (ICC), Bland Altman plots and Kappa test were used to analyze the consistency and bias on the results derived from the two measurement methods.Results:The 25(OH)D concentration derived from evaluation method was significantly different from those from LC-MS/MS method ( P<0.001). Slope of regression for evaluation method and LC-MS/MS was 0.962(95% CI 0.919-1.007), while intercept was -0.185 (95% CI -1.191-0.745). The ICC was 0.765 (95% CI 0.735-0.792). Altman plot showed that the average deviation between evaluation method and LC-MS/MS was -0.902 ng/ml (0.300%). The coincidence rate of evaluation method′s judgment of vitamin D sufficiency, insufficiency and deficiency with LC-MS/MS was 83.39%, and the weighted Kappa values was 0.790. Conclusion:Roche automatic 25(OH)D immunoassay shows acceptable correlation and agreement with LC-MS/MS, however, it is to note that the deviation between immunoassay and LC-MS/MS may lead to wrong judgment of vitamin D nutritional status. It is recommended that each laboratory should establish own corresponding reference values for 25(OH)D concentrations derived from these two methods.
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Objective To evaluate the inhibitory effect of geniposide on the oxidative damage to in vitro cultured human melanocytes,and to explore the role of PI3K-Akt signaling pathway in this inhibitory effect.Methods Epidermal melanocytes were isolated from the circumcised foreskin of healthy adolescent males,and then subjected to culture.Melanocytes at passage 2-3 were divided to six groups:control group receiving no treatment,geniposide group treated with 125 μmol/L geniposide alone,LY294002 group treated with 5 μmol/L LY294002 alone,H2O2 group treated with 250 μmol/L H2O2 alone,geniposide + H2O2 group firstly treated with 125 μmol/L geniposide for 24 hours followed by 4-hour treatment with 250 μmol/L H2O2,and geniposide + LY294002 + H2O2 group treated with 125 μmol/L geniposide for 24 hours,then with 5 μmol/L LY294002 for 1 hour,followed by 4-hour treatment with 250 μmol/L H2O2.After the above treatment,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the cellular proliferative activity,Western blot analysis to determine the protein expression of Akt,phosphorylated Akt (p-Akt),heme oxygenase1 (HO-1) and glutathione peroxidase (GPx-1),and flow cytometry to detect the level of cellular reactive oxygen species (ROS),and biochemical methods were used to evaluate the activity of superoxide dismutase (SOD) and catalase (CAT).Statistical analysis was carried out by one-way analysis of variance (ANOVA) for intergroup comparison,and Student-Newman-Keuls-q (SNK-q) test for multiple com-parisons.Results Compared with the control group,the H2O2 group showed significantly decreased cellular proliferative activity (P ＜ 0.01),protein expression of p-Akt (P ＜ 0.01),HO-1 (P ＜ 0.01) and GPx-1 (P ＜ 0.05),SOD activity (P ＜ 0.01) and CAT activity (P ＜ 0.01),but significantly increased ROS level (P ＜ 0.01).Compared with the H2O2 group,the geniposide + H2O2 group showed significantly increased cellular proliferative activity (72.98％ ± 8.92％ vs.50.53％ ± 10.85％,P ＜ 0.05),up-regulated protein expression of p-Akt (P ＜ 0.05),HO-1 (P ＜ 0.01) and GPx-1 (P ＜ 0.01),increased SOD activity (6.82 ±1.03 U/mg vs.1.29 ± 0.43 U/mg,P ＜ 0.05) and CAT activity (46.08 ± 4.16 U/mg vs.18.71 ± 3.09 U/mg,P ＜0.05),but decreased ROS level (1 284.33 ± 110.64 vs.2 158.00 ± 222.75,P ＜ 0.01).The proliferative activity of melanocytes was significantly lower in the geniposide + LY294002 + H2O2 group (44.35％ ±14.85％) than in the geniposide + H2O2 group (P ＜ 0.05).In addition,the geniposide + LY294002 + H2O2 group showed significantly decreased protein expression of p-Akt (P ＜ 0.01),HO-1 (P ＜ 0.05) and GPx-1 (P ＜ 0.01),SOD (1.31 ± 0.65 U/mg,P ＜ 0.05) and CAT activity (23.25 ± 5.56 U/mg,P ＜ 0.05),but significantly increased ROS level (1 668.00 ± 62.03,P ＜ 0.05)compared with the geniposide + H2O2 group.Conclusion Through the PI3K-Akt pathway,geniposide can promote the protein expression of HO-1 and GPx-1 in melanocytes,enhance SOD and CAT activity,and antagonize the oxidative damage of melanocytes.
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Objective To study the biocompatibility of carboxymethyl chitosan (CMCS) membrane with melanocytes from healthy human skin,and to investigate the feasibility to transport and carry melanocytes by using CMCS membrane.Methods CMCS membrane was prepared by a casting method combined with a glutaraldehydebased cross-linking method.Melanocytes were isolated from the foreskin of healthy men,and subjected to primary culture and subculture.The third-passage melanocytes were classified into two groups to be cultured on the CMCS membrane (test group) or traditional culture plates (control group).Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of melanocytes,and a sodium hydroxide-based lysis method to determine melanin content.HMB45 staining was conducted,and tyrosinase activity was estimated for melanocytes.Results Inverted microscopy showed that melanocytes were evenly distributed on the CMCS membrane with a normal shape.The melanocytes adherent to the CMCS membrane stained positive for anti-HMB45 monoclonal antibody.The growth curve of the melanocytes on the CMCS membrane,which was obtained from MTT assay,demonstrated that CMCS membrane could support the normal growth of melanocytes.No significant difference was observed between the test group and control group in melanin content (0.083 ± 0.015 vs.0.066 ± 0.008,t =2.38,P ＞ 0.01) or tyrosinase activity (0.234 ± 0.083 vs.0.241 ± 0.061,t =0.23,P ＞ 0.05).Conclusion CMCS membrane can maintain the normal biological activity of melanocytes and have good biocompatibility with skin melanocytes.
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A 16-year-old male presented with a 11-year history of progressively enlarging erythema and crusting on the right cheek.Physical examination revealed an irregularly shaped,sharply marginated,dark erythematous patch sized 6 cm x 10 cm and plaques with mild verrucous proliferation.There were strip-like scar at the margin of lesions and multiple ulcers measuring 0.5 to 1 cm in diameter with firm crusts.No small jellycolored nodules were observed.Direct microscopy of multiple scrapings under the crusts showed many light brown,septate,branching and irregular hyphae.Olivaceous-black woolly colonies grew at 25 C and 35 C on Sabouraud's dextrose agar and potato dextrose agar; flask-shaped conidiogenous cells with funnel-shaped collarettes and ellipsoidal conidia arranged in flower-like shape were observed microscopically.PAS staining showed numerous septate and branching hyphae,pseudohyphae and yeast-like cells.There was a 99.73％ similarity in the species-specific rDNA sequence between the isolate and phialophora verrucosa standard strain CDC-B2152.The patient was diagnosed with cutaneous phaeohyphomycosis caused by Phialophora verrucosa.The lesion subsided after treatment with amphotericin B and itraconazole,but recurred after drug withdrawal.Itraconazole and terbinafine were administered for the retreatment of this patient.
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Objective To investigate the performance of DNA microarray in identifying 6 common Candida spp. and validating ERG11 mutations resulting in fluconazolc-resistance in Candida albicans. Methods Oligonucleotide probes were designed and synthesized targeting the species-specific sequence in the internal transcribed spacer 2 (ITS2) region of rDNA of Candida albicans, Candida tropicalis, Candida glabrata, Candida dubliniensis, Candida parapsilosis and Candida krusei, as well as 6 sequences embracing the following mutations respectively in ERG11 gene leading to fluconazole-resistance, i.c., T541C, A 1090G, C1361T, G1537A, G1547A, and T1559C, then arranged onto a chip. Twelve 50-base-pair oligonucleotides were artificially synthesized based on the above specific sequences, and utilized to hybridize with the DNA microarray. Thirty-lbur Candida strains, including 29 C. albicans, 1 Candida tropicalis, 1 Candida glabrata,1 Candida dubliniensis, 1 Candida parapsilosis and 1 Candida krusei, were detected with microarray. Genomic DNA was extracted from these tested strains and underwent multiple PCR for the amplification of ITS2 region and ERGI 1 gene. Sequencing was performed to analyze the sequence of ERG11 in 29 strains of C. albicans and the results were compared with those of DNA microarray hybridization. Results Multiple PCR successfully produced ITS2 fragment of 307-415 bp from all the 34 strains, as well as ERG11 fragment of 1712 bp from 29 C. albicans strains. DNA microarray hybridization offered the same results in species identification of the 34 strains with their given information, as well as in mutation detection of the 29 strains of C. albicans with ERG11 sequencing results. Also, the 6 synthesized oligonucleotides containing the muta- tions were identified precisely as T541C, A1090G, C1361T, G1537A, G1547A, and T1559C, and the 6 species specific oligonucleotieds were identified correctly as C. albicans, C. tropicalis, C. glabrata, C. dubliniensis, C. parapsilosis and C. krusei Both the sensitivity and the specificity of the microarray were 100%. Conclu- sion DNA microarray is a quite reliable method to identify Candida spp. and fluconazole resistance-associ- ated mutations in the ERG11 gene of C. albicans.
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Objective To study the effect of H_2O_2 on the cultured melanocytes and the relationship between oxidative stress and vitiligo.Method Melanocytes were cultured with H_2O_2,the proliferation and melanogenesis of melanocyte were analysed by MTT and NaOH assay.Result H_2O_2 can inhibite the proliferation and melanogenesis of melanocytes,and the inhibition was concentration and-time-dependent.Conclusion Oxidative stress can inhibite the proliferation and melanogenesis of melanocytes,but oxidative damage can not induce vitiligo.
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Objective To study the role of psychological factors in the onset of psoriasis,to investigate the role of monoamine substances in the course of psychological factors-associated psoriasis,and to reveal the psychoneurologic pathogenesis of psoriasis.Methods The psychological factors were assessed with life events scale and questionnaire of A character.The serum levels of norepinephrine(NE),dopamine(DA)and5-hydroxyindoleacetic acid(5-HIAA)were detected with high-performance liquid chromatography(HPLC).Results It was shown that there was an overall tendency of weak A character and moderate depression and anxiety in psoriasis patients.The concentrations of NE,DA and5-HIAA were higher in psoriatic patients than those in normal controls(P