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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.
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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.
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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.
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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.
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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.
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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.
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Objective To screen differentially expressed proteins between granuloma- and tinea corporis-derived Trichophyton mentagrophytes strains,and to explore the pathogenesis of T.mentagrophytes in deep and superficial infection.MethodsFour T.mentagrophytes isolates from granuloma and 4 isolates from tinea corporis were cultured in agar plates and small steel rings at 27 ℃ and 37 ℃ respectively, followed by morphologic observation.Eight guinea pigs were immunocompromised by glucocorticoids,and superficially and subcutaneously inoculated with the same amount of fungal suspension to develop an animal model of tinea corporis and granuloma,respectively.Two weeks later,the infection of guinea pigs was confirmed by microscopy,fungal culture and histopathology.Proteins were extracted from a highly toxic granuloma-derived T.mentagrophytes strain and a lowly toxic tinea corporis-derived T.mentagrophytes strain,and subjected to twodimensional electrophoresis,mass spectrometry and identification by using the National Center for Biotechnology Information (NCBI) database.ResultsThe granuloma-derived T.mentagrophytes isolates grew better at 37 ℃ than tinea corporis-derived T.mentagrophytes isolates did,while no significant difference was observed in the morphology of colonies between the two kinds of T.mentagrophytes isolates at 27 ℃.Tinea corporis models were successfully established in guinea pigs with the 8 T.mentagrophytes strains,and the granuloma-derived isolates induced a more intense inflammation than tinea corporis-derived isolates.Granuloma model was constructed only with 3 granuloma-derived strains,which was proved by microscopy,fungal culture and histopathology.A total of 463 ± 20 and 398 ± 17 protein spots were detected,with 62 and 21 upregulated proteins,from granuloma-derived and tinea corporis-derived T.mentagrophytes strains,respectively.Bioinformatics analysis revealed the following meaningful proteins from differentially expressed proteins in granuloma-derived T.mentagrophytesstrains, includingenolase, heatshockprotein, serine/threonineprotease, cellularsignal transduction proteins,energy metabolism-related proteins,cytoskeleton proteins and some hypothetical proteins.ConclusionsThe granuloma-derived T.mentagrophytes strains are more heat-resistant with a higher potency to cause infection compared with tinea corporis-derived T.mentagrophytes strains.Differences exist in the expression of proteins between granuloma- and tinea corporis-derived T.mentagrophytes strains.
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Objective To renovate angiography in identifying portal vein anatomy during transjugular intrahepatic portosystemic shunt (TIPS) procedures,saving the time of TIPS procedures,decreasing the risk of the complications of the post-procedure.MethodsThe difference between the Wedge hepatic venography with Carbon Dioxide in 6 cases and Inferior Mesenteric artery angiography in 7 cases during TIPS procedures were compared in the identification of portal vein anatomy.The quality of images,their effects on the procedures,the complications and the recovery post-procedure were evaluated.Results Using CO2,the portal veins were opacified in all 6 cases.TIPS procedures succeeded in all cases except 1 case because of poor coagulation function.Using Inferior Mesenteric artery angiography,the portal veins were opacified in al1 7 cases.TIPS procedure succeeded in all cases except 1 case because of chronic portal occlusion.Puncture-site hematoma occurred in 1 case after TIPS procedure.ConclusionWedge hepatic venography with Carbon Dioxide is superior,safer and more convenient than Inferior Mesenteric Artery angiography in identifying portal vein anatomy during TIPS.
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ObjectiveTo clone the full-length cDNA of Microsporum canis membrane protein PQ-loop repeat protein(PQ-LRP) gene,so as to investigate the roles of PQ-LRP in the pathogenesis of tinea capitis.MethodsA Microsporum canis strain(A518) from a patient with tinea capitis served as the experimental strain.Rapid cDNA end amplification(RACE) was performed to clone the full length cDNA sequence of PQLRP gene.Bioinformatics methods were used to make a preliminary functional analysis of the gene.Results The cDNA of PQ-LRP gene was obtained with a full length of 1522 bp,including the 5' untranslated region (49 bp),coding region(1080 bp) and 3' untranslated region(393 bp).The coding region encoded a protein precursor including 359 amino acid residues.The cloned cDNA of PQ-LRP gene shared an 81％ nucleotide identity with that of Trichophyton tonsurans and a 79％ nucleotide identity with that of Trichophyton rubrum.Conclusions The full-lengthcDNA of Microsporumcanis membraneproteinPQ-LRP gene hasbeen successfully cloned,which will provide an important basis for further researches into the roles of PQ-LRP in Microsporum canis-associated diseases.
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Objective To explore the significance of FSH1 in the pathogenicity of M. canis. Methods Thirty M. cam's strains from tinea capitis lesions and 30 M. canis strains from tinea corporis lesions were cultured, passaged, and induced by medium containing skin tissue of scalp or foreskin from children. Semi-quantitative reverse transcription (RT)-PCR was carried out to detect the expression of FSH1 mRNA in the firstand fifth-generation M. canis strains, as well as M. canis strains induced by the skin tissues. Results The mRNA expression of FSH1 was higher in M. canis strains derived from tinea capitis lesions than in those from tinea corporis lesions (P 0.05). The skin tissue from scalp and foreskin induced a significant elevation in the mRNA expression of FSH1 in these M. canis strains (F = 2025.713, 1833.139, both P< 0.01), and the inductive effect of the scalp tissue was different from that of the foreskin tissue (P < 0.01). Conclusions The FSH1 mRNA expression is different in M. canis isolated from different body sites. Local skin tissue has an inductive effect on the expression of FSH1 mRNA, and the inductive effect of scalp tissue is more apparent than that of foreskin tissue.
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<p><b>OBJECTIVES</b>To investigate the DNA polymorphism of Sporothrix schenckii (S. schenckii) and to find the relationship between DNA patterns and geographic areas and clinical manifestations.</p><p><b>METHOD</b>The total DNA was extracted with hexadecyltrimethyl-ammonium bromide. Random Amplification of Polymorphic DNA (RAPD) assay was used to study DNA typing of 24 strains of S. schenckii collected from different areas and isolated from different clinical types.</p><p><b>RESULTS</b>Of seven random primers used, three primers (OPAA11, OPD18 and OPB07) gave good reactions, the sequences of which were 5'-ACCCGACCTG-3', 5'-GAGAGCCAAC-3', 5'-GGTGAC~GCAG-3' respectively. The RAPD patterns of the 24 isolates were not completely identical, showing certain degrees of hereditary variability. Different isolates showed a common conserved DNA band with the same primer. Different clinical types showed different genotypes.</p><p><b>CONCLUSION</b>RAPD analysis is useful in DNA typing of S. schenckii, the DNA band type of which is related to geographic origin and Clinical manifestation.</p>
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Humans , DNA, Fungal , Random Amplified Polymorphic DNA Technique , Sporothrix , GeneticsABSTRACT
Objectives To identify genotypes of 31 Sporothrix schenckii (S.schenckii) strains by Southern blotting and to explore the relationship between genotypes and geographic distributions and clinical manifestations.Methods Total DNA was extracted by cetyltriethyl ammonium bromide (CTAB).The polymorphisms were detected by hybridization of ApaⅠ-digested S.schenckii genomic DNA with a probe amplified from the small-subunit rDNA and adjacent internal transcribed spacer (ITS) regions.The band patterns manifested by Southern blotting were employed to investigate genotypes of 31 strains of S.schenckii collected from five different areas in China.Results Of 31 strains of S.schenckii,15 individual patterns (DNA Type A-O) were recognized.Type A to C accounted for 51.61% of all strains.Conclusion The Southern blotting provides a highly sensitive and reliable means for DNA typing of S.schenckii.It is also found that there is an obvious correlation between DNA patterns and different geographic distribution and clinical manifestations.
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Objective To study the relationship between genotypes and phenotypes,geographical distribution,and the sites of infection of Trichophyton rubrum(T.rubrum).Methods The genotypes were determined by Southern blotting with a probe amplified from the small-subunit rDNA and adjacent internal transcribed spacer(ITS)regions.The phenotypes of T.rubrum were determined by conventional method.Results Twenty genotypes(DNA type A to T)and5phenotypes(villous,furrowed,granular,powdery,and woolly)were recognized among49strains of T.rubrum.Genotype A prevailed in all phenotypes except granu-lar type.Type B represented the most common genotype among the strains of villous type and furrowed type.Type A took the first place in powdery type and woolly type.All of the type A strains were from Dalian.Seven of9type B strains were from Nanjing.Six type C strains were all from Nanjing.The majority strains of21strains isolated from tinea unguium were type C,most of the16strains isolated from tinea cruris and tinea corporis were type A,8strains from tinea pedis were type B,and4strains from tinea capitis were type C.Conclusion There are certain possible relationships between genotypes and phenotypes,geographical distri-bution and sites of infection of T.rubrum.
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Objective To study the significance of AP-PCR i n identification and subtyping of der-matophytes.Methods Using a pair of random primers,OPAA11(5' -ACCCGACCTG -3' ),and OPD18(5' -GAGAGCCAAC -3' )the DNAs of 64isolates of dermatophytes(9species of 3genera),Sporothrix schenckii and Candida albicans were amplified by AP-PCR and analyse d by electrophoresis.Results Distinct DNA band patterns were observed in diffe rent dermatophyte species.Common major DNA bands were observed in Trichophyton rubrum isolated from different areas with s train difference.Conclusion Using OPAA11and OPD18as primers,AP-PCR may be applied in the identification and subtypi ng of dermatophytes.