Objective:To investigate differences in bacterial and fungal microbiome between infected nails and healthy nails among patients with onychomycosis.Methods:Nail scraping samples were collected from infected nails and healthy nails of 31 patients with onychomycosis, who visited Dalian Dermatosis Hospital from August 2020 to July 2021. The total DNA of nail microbiota was extracted, and the V3-V4 regions of the bacterial 16S rDNA gene and the fungal internal transcribed spacer (ITS) region were amplified and sequenced using Illumina technology. The USEARCH and mothur softwares were used for data cluster analysis to obtain the operational taxonomic units (OTUs) , Wilcoxon rank sum test was used to analyze α diversity, analysis of similarities (ANOSIM) was performed to analyze β diversity, linear discriminant analysis of effect size (LEfSe) was performed to evaluate the species difference.Results:Among the 31 patients with onychomycosis, 16 were males and 15 were females. According to the age, they were divided into young group (18 - 35 years old, 10 cases) , middle-aged group (36 - 60 years old, 11 cases) , and elderly group (over 60 years old, 10 cases) . As the α-diversity analysis revealed, the infected nail group showed significantly decreased Shannon index ( W = 290, P = 0.007) , but significantly increased Simpson index ( W = 663, P = 0.010) compared with the healthy nail group, suggesting that the diversity and evenness of bacterial communities were lower in the infected nail group than in the healthy nail group; however, there was no significant difference in the diversity of fungal communities between the infected nail group and healthy nail group. The β-diversity analysis based on the unweighted-UniFrac distance matrix showed no significant difference in the fungal or bacterial community composition between the infected nail group and healthy nail group (bacterial communities: R = 0.0052, P = 0.331; fungal communities: R = 0.0036, P = 0.337) ; the β-diversity analysis based on the weighted-UniFrac distance matrix showed significant differences in the abundance of bacterial and fungal communities between the two groups (both P = 0.001) . In terms of the species composition, the bacterial flora with significantly decreased abundance in the infected nail group compared with the healthy nail group included Bacteroidetes, Proteobacteria, Betaproteobacteria, Burkholderiales, Ralstonia, Sphingomonas and Streptococcus, while the abundance of Bacilli, Bacillales and Staphylococcus was significantly higher in the infected nail group than in the healthy nail group. Compared with the healthy nail group, the fungal flora with significantly increased abundance in the infected nail group included Eurotiomycetes, Onygenales, Leotiomycetes-ord-incertae-sedis, Arthrodermataceae, Periconia, Erysiphe, Tilletiopsis, Trichophyton, Erysiphe cruciferarum, Trichophyton rubrum, Malassezia sympodialis, while the abundance of Saccharomycetes, Saccharomycetales, Saccharomycetaceae, Dothioraceae, Candida and Alternaria was significantly lower in the infected nail group than in the healthy nail group. Conclusion:The diversity and abundance of bacterial communities significantly differed between infected nails and healthy nails among patients with onychomycosis, while only the abundance of fungal communities differed between the two groups, and perhaps there was correlations between some bacterial and fungal communities.

Objective:To investigate clinical characteristics of bullous pemphigoid (BP) developing after the treatment with dipeptidyl peptidase-Ⅳ inhibitors (DPP4i) in patients with diabetes mellitus.Methods:A total of 116 inpatients with BP complicated by diabetes mellitus were collected from the Seventh People′s Hospital of Shenyang between January 2014 and December 2020, and divided into 2 groups: DPP4i-BP group treated with DPP4i before the onset of BP, and general BP group receiving no treatment with DPP4i. General clinical data, skin lesion area, laboratory indicators, treatment regimens, and prognosis were analyzed and compared between the above 2 groups, the time interval from the administration of DPP4i to the diagnosis of BP was recorded in the DPP4i-BP group. One-way analysis of variance was used to compare measurement data among multiple groups, two-independent-sample t test was used for comparisons between two groups, and paired t-test for intra-group comparisons before and after treatment; chi-square test was used to compare enumeration data between groups. Results:There were 32 patients aged 77.17 ± 15.32 years in the DPP4i-BP group, with a male-to-female ratio being 15∶17; there were 84 patients aged 76.65 ± 19.32 years in the general BP group, with a male-to-female ratio being 43∶41. The time interval from the administration of DPP4i to the diagnosis of BP was 14.61 ± 3.93 months in the DPP4i-BP group. The time interval for vildagliptin was the shortest (5.42 ± 2.84 months) , and there was a significant difference in the time interval among vildagliptin, sitagliptin, linagliptin and saxagliptin ( F= 8.93, P < 0.001) . The proportion of patients with severe BP was significantly higher in the DPP4i-BP group (16 cases, 50%) than in the general BP group (25 cases, 29.76%; Z= 2.63, P= 0.008) . There was no significant difference in the positivity rate of anti-BP180 antibody between the two groups ( χ2= 0.03, P= 0.870) . However, the level of anti-BP180 antibody was significantly higher in the DPP4i-BP group than in the general BP group before and after treatment ( P= 0.015, < 0.001, respectively) , and the decrease in the level of anti-BP180 antibody was significantly less in the DPP4i-BP group than in the general BP group after treatment ( t= 5.11, P < 0.001) . There was no significant difference in the average effective dose of glucocorticoids required to control the disease between the two groups ( t= 1.00, P= 0.322) . However, the DPP4i-BP group showed a significant increase in the average time required to control the disease and in the proportion of patients requiring combined treatment with immunosuppressants or other drugs compared with the general BP group ( t= 6.72, 10.05, P < 0.001,= 0.002, respectively) . Within 6 months after the start of systemic treatment, the recurrence rate was significantly higher in the general BP group (17 cases, 27.86%) than in the DPP4i-BP group (2 cases, 7.69%; χ2= 4.35, P= 0.037) ; at 6 months, the average dose of glucocorticoids was also significantly higher in the general BP group than in the DPP4i-BP group ( t= 7.04, P < 0.001) . Conclusions:Among the DPP4i hypoglycemic drugs, vildagliptin was the most common drug administrated by patients before the onset of BP, with the shortest interval from the administration to the onset of BP. DPP4i-BP may be difficult to control at the early stage, but the prognosis is good.

Objective:To analyze the subcellular localization of family of serine hydrolases 1 (FSH1) protein in Microsporum canis. Methods:The FSH1 and enhanced green fluorescent protein (EGFP) genes were amplified by PCR using the previously constructed plasmid containing the FSH1 gene and the recombinant plasmid pCAMBIA-LRP-EGFP as the template; the vector DNA was obtained by double-enzyme digestion of the recombinant plasmid pCAMBIA-LRP-EGFP with SnaBI/KpnI. Then, the EGFP expression plasmid and Ptrcp-FSH1-EGHP-Ttrcp fusion plasmid were constructed by inserting the amplified EGFP gene and EGFP-FSH1 gene into the vector DNA respectively, and identified by PCR and sequencing. The two recombinant plasmids were transformed into Microsporum canis by an Agrobacterium tumefaciens-mediated method, and the gene EGFP and fusion gene FSH1-EGFP were expressed integratedly in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc. The cellular localization of the fusion protein was observed by laser scanning confocal microscopy. Results:The Agrobacterium tumefaciens-mediated transformation system and EGFP expression vector in Microsporum canis were successfully constructed; the fusion gene FSH1-EGFP was expressed integratedly in Microsporum canis. Laser confocal microscopy showed that fluorescence signals of the FSH1-EGFP fusion protein were concentrated in the cytoplasm and nuclei of Microsporum canis, with a granular or cluster-like appearance. Conclusion:The FSH1-EGFP fusion protein was successfully localized in the cytoplasm and nuclei of Microsporum canis, providing a basis for further clarifying the function and pathogenic mechanisms of the FSH1 gene in Microsporum canis.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.
CRISPR-Cas Systems ; DNA Ligase ATP ; genetics ; Gene Expression Regulation ; genetics ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Ku Autoantigen ; genetics

Objective:To explore the correlation between the body mass index (BMI) trajectories and new-onset non-alcoholic fatty liver disease (NAFLD) so as to provide a scientific basis for the prevention and treatment of NAFLD.Methods:A total of 16388 observation subjects that met the inclusion criteria in the Kailuan study were used to form a cohort study. According to the BMI values of the observed subjects during annual physical examinations from 2006 to 2007, 2008 to 2009 and 2010 to 2011, SAS Proc Traj was used to determine four different BMI trajectories groups, namely, the low-stable medium-stable, medium-high and high-stable group. NAFLD incidence in each group was followed up during annual physical examinations from 2012 to 2013, 2014-2015 and 2016-2017. A total of 14998 observation subjects were finally included in the statistical analysis. The cumulative incidences of NAFLD differences in the four groups were compared. The Cox’s proportional hazards regression model was used to analyze the correlation between different BMI trajectories and new-onset NAFLD. One-way analysis of variance was used to compare the intergroup difference of measurement data, and pairwise comparisons were conducted. LSD test was used for the homogeneity of variance. Dunnett's T3 test was used for heterogeneity of variances. χ2 test was used to compare the count data, and the difference of NAFLD cumulative incidence rate between the different BMI trajectories groups was compared by log-rank test. Results:(1) the cumulative incidence of NAFLD was increased with the increase of BMI trajectories, which were 31%, 47%, 63%, 77%, respectively, and the difference was statistically significant ( P < 0.01). (2) after adjusting for multiple confounding factors such as age and gender with the Cox’s proportional hazards regression model, the risk of NAFLD in the BMI medium stable, medium-high, and high stable group was still 1.757 times [95% confidence interval ( CI): 1.589 ~ 1.942], 2.612 (95% CI: 2.353 ~ 2.900), 3.566 (95% CI: 3.129 ~ 4.064) of the low-stable group ( P < 0.01). Conclusion:The risk of NAFLD increases with increase of BMI trajectories, and long-term high levels of BMI are independent risk factors for the onset of NAFLD.

Objective To determine the location of PQ-LRP protein in Microsporum canis using the enhanced green fluorescent protein(EGFP)as a marker.Methods The total RNA was extracted from Microsporum canis,and reversely transcribed into cDNA.The PQ-LRP gene was amplified by PCR using the above cDNA as the template.The fusion gene of PQ-LRP gene and EGFP gene was linked to the plasmid pCAMBIA 1300.Microsporum canis was subjected to Agrobacterium tumefaciens-mediated transformation,in order to achieve the integrated expression of the fusion gene LRP-EGFP in Microsporum canis under the regulation by the fungal universal promoter Ptrpc and terminator Ttrpc.Laser-scanning confocal microscopy was conducted to determine the cellular localization of the fusion protein.Results The expression vector pCAMBIA-LRP-EGFP was successfully constructed,and the fusion gene LRP-EGFP was expressed integratedly in Microsporum canis.Laser-scanning confocal microscopy showed that fluorescence signals of LRP-EGFP were concentrated on the cell membrane of Microsporum canis,giving a granular or cluster-like appearance.Conclusion The infusion gene LRP-EGFP can be successfully expressed in Microsporum canis,and PQ-LRP protein is located on the cell membrane of Microsporum canis.

Morphea complicated by Hashimoto's thyroiditis is reported in two sisters.Case 1:a 64-year-old female presented with skin rashes on the anterior neck,trunk and bilateral anterior shins for 5 years,itching skin rashes on the perineum for 4 years,and Hashimoto's thyroiditis for 9 years.Physical examination revealed grade 1 enlargement of firm thyroid gland without exophthalmos or pretibial myxedema.Dermatological examination showed pink patches on the neck and breast,sclerosis and atrophy of skin over the back,porcelain-white patches on the perineum.Histopathological findings suggested the diagnosis of morphea on the breast and lichen sclerosus et atrophicus on the perineum.Case 2:a 55-year-old female,who was the younger sister of case 1,suffered from gradual sclerosis and atrophy of skin in the left inframammary region and abdominal region for 4 years,as well as Hashimoto's thyroiditis for 3 years.Similarly,physical examination revealed grade 1 enlargement of firm thyroid gland without exophthalmos or pretibial myxedema.Hypopigmentation,sclerosis and atrophy of skin were observed in the left inframammary region,abdominal region and central back region.Histopathological examination suggested a diagnosis of morphea.According to the clinical and histopathological manifestations,periodic acid-Schiff staining and thyroid gland function test results,the 2 cases were both diagnosed as morphea complicated by Hashimoto's thyroiditis.

BACKGROUNDIn March 2013, human cases of infection with a novel A (H7N9) influenza virus emerged in China. The epidemic spread quickly and as of 6 May 2013, there were 129 confirmed cases. The purpose of this study was to analyze the epidemiology of the confirmed cases, determine the impacts of bird migration and temperature changes on the H7N9 epidemic, predict the future trends of the epidemic, explore the response patterns of the government and propose preventive suggestions.

METHODSThe geographic, temporal and population distribution of all cases reported up to 6 May 2013 were described from available records. Risk assessment standard was established by analysing the temperature and relative humidity records during the period of extensive outbreak in three epidemic regions in eastern China, including Shanghai, Zhejiang and Jiangsu provinces. Risk assessment maps were created by combining the bird migration routes in eastern China with the monthly average temperatures from May 1993 to December 2012 nationwide.

RESULTSAmong the confirmed cases, there were more men than women, and 50.4% were elderly adults (age >61 years). The major demographic groups were retirees and farmers. The temperature on the days of disease onset was concentrated in the range of 9°C-19°C; we defined 9°C-19°C as the high-risk temperature range, 0°C-9°C or 19°C-25°C as medium risk and <0°C or >25°C as low risk. The relative humidity on the days of disease onset ranged widely from 25% to 99%, but did not correlate with the incidence of infection. Based on the temperature analysis and the eastern bird migration routes, we predicted that after May, the high-risk region would move to the northeast and inland, while after September, it would move back to north China.

CONCLUSIONSTemperature and bird migration strongly influence the spread of the H7N9 virus. In order to control the H7N9 epidemic effectively, Chinese authorities should strengthen the surveillance of migrating birds, increase poultry and environmental sampling, improve live poultry selling and husbandry patterns and move from a "passive response pattern" to an "active response pattern" in focused preventive measures.

Objective To build a RNA interference vector for PQ-loop repeat protein (LRP) gene,and to evaluate the effect of the vector on the expression of PQ-LRP gene in Microsporum canis.Methods The PUC-PLULT and PCB309-PLULT vectors were constructed sequentially by using Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis,adding multiple cloning sites,and introducing the hygromycin-resistance gene.Microsporum canis was transformed with the PCB309-PLULT vector followed by a series of passages and hygromycin selection.Real-time quantitative PCR was performed to measure the expression of PQ-LRP gene in Microsporum canis before and after transformation.Results The intermediate vectors PUC-PLUT and PUC-PLULT were constructed and identified by PCR and gene sequencing.The 8 825-bp interference vector PCB309-PLULT was successfully built and confirmed by enzyme digestion.The optimum concentration of hygromycin for screening for Microsporum canis transformants was determined as 300 mg/L.The mRNA expression level of PQ-LRP was decreased by 61％ in the transformants as compared with untransformed Microsporum canis (0.39 vs.1.00).Conclusion The constructed PCB309-PLULT interference vector can effectively inhibit the expression of PQ-LRP gene in Microsporum canis.

Objective To explore the effect of intraoperative tension adjustment and postoperative scar formation in the treatment of circular skin defects using modified purse-string suture.Methods Twenty-eight cases of circular lesions in the face region were selected and resected along the edge.We first used purse-string suture technique to reduce the circular defect area,and then closed the wound with linear suture in random direction.After 7 days,the stitches were removed and longterm follow-up effects were observed.Results All patients had no facial organ deformation and displacement,the length of the linear scar lesions was shorter than the preoperative length.In 28 cases,one patient's surgical wound was dehisced after removal of stitches in 24 h,and the rest of the skin defects healed after operation in stage Ⅰ.All cases were followed up for 3 months to 1 year,and had a satisfactory therapeutic effect.Conclusions Modified purse-string suture can linearly close the dicision in stage Ⅰ,reduce the scar area and maintain the normal morphology and relative position of the nearby organs.