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Objective:To investigate changes in skin microecological structures and functions between acute and remission phases in adult patients with severe atopic dermatitis (AD) .Methods:From October 2019 to November 2020, skin scale specimens were collected from 5 body sites (cheeks, cubital fossa, back of the hand, abdomen, lower limbs) of 4 adult patients with severe AD in the acute and remission phases, who visited the outpatient clinic of Guangzhou Institute of Dermatology. The next-generation high-throughput sequencing was performed for metagenomic sequencing to construct the microbial gene catalogue of these specimens, followed by gene annotation and bioinformatics analysis for each sample.Results:A total of 18 phyla, 37 classes, 73 orders, 142 families, 237 genera, and 331 species were identified in the skin specimens from the 4 patients with severe AD. The patients with AD in the remission phase showed significantly increased diversity of skin microbiota and markedly different relative abundance of skin microorganisms compared with those in the acute phase (both P < 0.05). At the microbial species level, Staphylococcus aureus showed the highest impact on the acute phase of AD, while Staphylococcus epidermidis, Moraxella osloensis, Francisella sp., Staphylococcus cohnii, Staphylococcus warneri, Malassezia globosa and Malassezia restricta were enriched in the remission phase of AD with the absolute value of the common logarithm of the linear discriminant analysis score > 2 (Kruksal-Wallis test, all P < 0.05). As KEGG pathway enrichment analysis showed, the differentially abundant genes were annotated into a total of 355 functional pathways, of which 38 pathways were significantly enriched (all P < 0.05), mainly involving Staphylococcus aureus infection, tryptophan metabolism, histidine metabolism, nitrogen metabolism, metabolism of arginine and proline, biosynthesis and degradation of valine, leucine and isoleucine, fatty acid degradation, peroxisome proliferator-activated receptor signaling pathway, etc. Conclusion:The skin microecological structure significantly differed between the acute and remission phases among the patients with severe AD, which may be related to multiple functional pathways, such as Staphylococcus aureus infection, tryptophan metabolism, histidine metabolism and nitrogen metabolism.
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MicroRNAs (miRNAs) are a class of non-coding RNA molecules that regulate gene expression after transcription and participate in various pathophysiological processes in the skin. In recent years, it has been reported that changes in miRNA expression profiles are related to some inflammatory skin diseases. For example, miR-203, miR-146a and miR-21 are upregulated in psoriatic lesions, miR-155 and miR-146a are upregulated in atopic dermatitis lesions, miR-21, miR-223, miR-142-3p and miR142-5p are upregulated in allergic contact dermatitis lesions; however, miR-146a and miR-155 are downregulated in peripheral blood of patients with systemic lupus erythematosus, and miR-223 is downregulated in dermatomyositis lesions. This review summarizes relationships of miRNAs with the occurrence and development of some inflammatory skin diseases.
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Objective@#To identify differentially expressed genes in the transcriptome of the lesional versus nonlesional skin tissues of patients with moderate and severe atopic dermatitis (AD) , and to elucidate their roles in the pathogenesis of AD.@*Methods@#From July to October in 2016, lesional and nonlesional skin tissues were obtained from 5 outpatients of Han nationality with AD in Guangzhou Institute of Dermatology, Institute of Dermatology, Guangzhou Medical University. The next-generation high-throughput transcriptome-wide RNA sequencing (RNA-seq) was performed to identify differentially expressed genes, which were subjected to GO function annotation and KEGG pathway analysis. Real-time fluorescence-based quantitative PCR (qRT-PCR) was conducted to verify differences in candidate gene expression between lesional and nonlesional skin tissues.@*Results@#An average of 10.96 GBs sequence reads were acquired among 10 samples. A total of 21 729 genes were detected, including 19 268 known genes and 2 545 predicted novel genes. A total of 23 153 new transcripts were detected, of which 18 889 were new alternative splicing subtypes of known protein-coding genes, 2 545 were transcripts belonging to new protein-coding genes, and the remaining 1 719 belonged to long-stranded non-coding RNA. Totally, 78 differentially expressed genes were identified between the lesional and nonlesional skin tissues, including 69 upregulated and 11 downregulated genes in the lesional skin tissues. Among them, there were several genes known to be associated with AD inflammation (CXCL1/2/8, IL6/IL1β, MMP1, SERPINB4, S100A2, GZMB, OASL, OSM) and barrier (KRT16, FABP5, CYP1A1) and keratinocyte differentiation (IL-20) . GO analysis revealed that functions of 72 differentially expressed genes could be annotated. KEGG pathway analysis showed that the differentially expressed genes were grouped into 132 signaling pathways, of which 13 were significantly enriched, including the interleukin (IL) -17 pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, etc. qRT-PCR showed that the mRNA expression levels of candidate genes CXCL1, KRT6A, IL36A, SERPINB4 and PSAPL1 was consistent with the transcriptome sequencing results.@*Conclusions@#Differentially expressed genes and related important regulatory signaling pathways were identified between the lesional and nonlesional skin tissues of patients with AD at the transcriptional level, and the IL-17 pathway was found to be mostly enriched in AD lesions in patients of Han nationality. These findings provide an important basis for further study on the pathogenesis of AD..
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Objective To identify differentially expressed genes in the transcriptome of the lesional versus nonlesional skin tissues of patients with moderate and severe atopic dermatitis(AD), and to elucidate their roles in the pathogenesis of AD. Methods From July to October in 2016, lesional and nonlesional skin tissues were obtained from 5 outpatients of Han nationality with AD in Guangzhou Institute of Dermatology, Institute of Dermatology, Guangzhou Medical University. The next-generation high-throughput transcriptome-wide RNA sequencing (RNA-seq) was performed to identify differentially expressed genes, which were subjected to GO function annotation and KEGG pathway analysis. Real-time fluorescence-based quantitative PCR(qRT-PCR)was conducted to verify differences in candidate gene expression between lesional and nonlesional skin tissues. Results An average of 10.96 GBs sequence reads were acquired among 10 samples. A total of 21729 genes were detected, including 19268 known genes and 2545 predicted novel genes. A total of 23153 new transcripts were detected, of which 18889 were new alternative splicing subtypes of known protein-coding genes, 2545 were transcripts belonging to new protein-coding genes, and the remaining 1719 belonged to long-stranded non-coding RNA. Totally, 78 differentially expressed genes were identified between the lesional and nonlesional skin tissues, including 69 upregulated and 11 downregulated genes in the lesional skin tissues. Among them, there were several genes known to be associated with AD inflammation (CXCL1/2/8, IL6/IL1β, MMP1, SERPINB4, S100A2, GZMB, OASL, OSM) and barrier (KRT16, FABP5, CYP1A1) and keratinocyte differentiation (IL-20). GO analysis revealed that functions of 72 differentially expressed genes could be annotated. KEGG pathway analysis showed that the differentially expressed genes were grouped into 132 signaling pathways, of which 13 were significantly enriched, including the interleukin(IL)-17 pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, etc. qRT-PCR showed that the mRNA expression levels of candidate genes CXCL1, KRT6A, IL36A, SERPINB4 and PSAPL1 was consistent with the transcriptome sequencing results. Conclusions Differentially expressed genes and related important regulatory signaling pathways were identified between the lesional and nonlesional skin tissues of patients with AD at the transcriptional level, and the IL-17 pathway was found to be mostly enriched in AD lesions in patients of Han nationality. These findings provide an important basis for further study on the pathogenesis of AD. .
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Objective To evaluate the effect of curcumin on phagocytic function of macrophages after the stimulation with exosomes from patients with systemic lupus erythematosus (SLE).Methods From February 2016 to November 2017,18 patients with active SLE and 18 healthy controls were enrolled from Guangzhou Institute of Dermatology and Sun Yat-sen Memorial Hospital,Sun Yat-sen University,and serum exosomes were extracted from these subjects.The exosomes from the healthy controls (CON-exo) and SLE patients (SLE-exo) as well as different concentrations (1,5,20 μmol/L) of curcumin were used alone or in combination to stimulate the acute monocytic leukemia cell line THP-1-derived macrophages.After coincubation of the stimulated macrophages with pHrodo TM Red indicator,flow cytometry was performed to determine the average fluorescence intensity,immunofluorescence staining to calculate the proportion of pHrodo TM Red-positive macrophages,and then the phagocytic activity of macrophages was assessed.Western blot analysis was conducted to determine the protein expression of CD 14 in the stimulated macrophages.Statistical analysis was carried out by Student-t test for comparison between two groups,oneway analysis of variance for comparison among several groups,and least significant difference (LSD)-t test for multiple comparisons.Results Flow cytometry showed that there were significant differences in the relative fluorescence intensity of macrophages among the CON-exo group,CON-exo + 20 μmol/L curcumin group,SLE-exo group and SLE-exo + 20 μmol/L curcumin group (101.3％ ± 14.05％,94.27％ ± 14.13％,41.02％ ± 9.54％ and 87.33％ ± 15.01％,respectively;F =10.81,P ＜ 0.01),and immunofluorescence staining revealed that the proportion of pHrodo Red-positive macrophages significantly differed among the above 4 groups (82.16％ ± 5.20％,81.33％ ± 4.51％,54.20％ ± 9.31％ and 71.23％ ± 5.43％ respectively;F =12.42,P ＜ 0.01).The fluorescence intensity of macrophages and proportion of pHrodo-positive macrophages were both significantly lower in the SLE-exo group than in the CON-exo group (t =5.26,5.35 respectively,both P ＜ 0.01) and SLE-exo + 20 μmol/L curcumin group (t =3.97,3.26 respectively,both P ＜ 0.05).Western blot analysis showed that there were significant differences in the protein expression ofCD14 among the CON-exo group,SLE-exo group,SLE-exo + 1 μmol/L curcumin group,SLE-exo + 5 μmol/L curcumin group and SLE-exo + 20 μmol/L curcumin group (96.33％ ± 13.65％,30.67％ ± 5.86％,45.24％ ± 8.89％,72.81％ ± 6.62％ and 90.67％ ± 12.66％ respectively;F =24.57,P ＜ 0.01).The protein expression of CD14 was significantly lower in the SLE-exo group than in the CON-exo group(t =8.06,P ＜ 0.001),SLEexo + 5 μmol/L curcumin group and SLE-exo + 20 μmol/L curcumin group(t =5.08,7.38,both P ＜ 0.001),and the CD14 level showed an increasing trend along with the increase in the concentration of curcumin.Conclusion Exosomes from SLE patients markedly inhibit the phagocytosis of macrophages,while curcumin can reverse this inhibitory effect.
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Objective To analyze the characteristics of genotyping and gene polymorphism of Neisseria gonorrhoeae(N.go) with azithromycin(AZM)-resistance(AZM-R) and decreased susceptibility to ceftriaxone(CROD).Methods The minimum inhibitory concentration(MIC) of AZM and CRO were determined.AZM-R isolates were detected for mutations in 23S rRNA,mtrR and penA genes.Genotypes were analyzed by using N.go multi-antigen sequence typing(NG-MAST).Results All total of 485 isolates of N.go were detected.77(15.9%) strains were AZM-R(MIC≥1 mg/L),including 33(6.8%) isolates of AZM low-level resistant(AZM-LLR,MIC=1 mg/L) strains and 44(9.1%) isolates of AZM middle-level resistant(AZM-MLR,MIC≥2 mg/L) strains.There were more CROD(MIC≥0.125 mg/L) strains in AZM-MLR isolates(43.2%),compared with those in AZM-LLR isolates(18.2%,P0.05).Similar results were found between combined AZM-LLR/CROD isolates and combined AZM-MLR/CROD isolates(P>0.05).No mutation of A2059G and AZM high-level resistant(AZM-HLR,MIC≥256 mg/L) isolate were found.Among 77 AZM-R isolates,67 sequence types(ST) were identified by NG-MAST,of which 30 types were novel.Most ST were represented by a single isolate.Conclusion AZM-R and CROD isolates,presented in this area,might be deserved continuous surveillance to identify the mechanism of concurrent resistance.
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Objective To investigate genetic characteristics of Neisseria gonorrhoeae (N.gonorrhoeae) isolates from Guangzhou city in 2014,and to analyze the relationship of N.gonorrhoeae multi-antigen sequence typing (NGMAST) sequence types (STs) with ciprofloxacin resistance.Methods An agar dilution method was used to determine the minimal inhibitory concentration (MIC) of ciprofloxacin in 97 N.gonorrhoeae isolates from Guangzhou city.PCR was performed to amplify the gyrA,parC,porB and tbpB genes from these isolates,followed by gene sequencing and determination of NG-MAST STs.Results Of the 97 N.gonorrhoeae isolates,95 (97.9％) were resistant to ciprofloxacin,including 41 high-level (MIC ≥ 16 mg/L) and 54 low-level (1 mg/L ≤ MIC ＜ 16 mg/L) resistant strains.Mutations were detected at codons 91 and 95 encoding serine in the gyrA gene of all the 95 ciprofloxacin-resistant strains,and in the parC gene of 93 resistant strains.The frequency of the mutation at codon 87 in the parC gene was 85.4％ (35/41) in high-level resistant strains,significantly higher than that in low-level resistant strains (59.3％[32/54],x2 =7.64,P ＜ 0.05).MAST STs were successfully determined for all the 97 N.gonorrhoeae isolates except 1 isolate with incorrect PCR amplicons.Of the 96 genotyped isolates,50 were assigned to 35 known STs by using the NG-MAST website (www.ngmast.net),among which,10 STs each contained 2 to 4 isolates.The most common ST was ST5309.Phylogenetic tree analysis revealed that the 96 genotyped N.gonorrhoeae isolates could be classified into 2 groups,and the proportion of isolates with MIC ≥ 16 mg/L is 46.4％ (39/84) in group 1,but only 1/12 in group 2 (x2 =6.27,P=0.012).Conclusions High-level resistance of N.gonorrhoeae to ciprofloxacin may be mainly associated with the mutation at codon 87 in the parC gene.NG-MAST STs may be related to the degree of ciprofloxacin resistance.
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AIM To study the expression, purification and activity assay of recombinant mouse protein kinase CK2? subunit from Escherichia coli. METHEDS The recombinant plasmid containing mouse protein kinase CK2? subunit cDNA constructed successfully was transformed into Escherichia coli BL21 (DE3) and specifically induced by IPTG. The recombinant mouse CK2? subunit was sequentially purified by DE 52, P11 phosphocellulose and Heparin Sepharose chromatography. The purified recombinant protein was analysed by SDS PAGE. RESULTS One protein with molecular mass of 42 ku was overexpressed by inducing ITPG. The recombinant protein was composed of approximately 30 6% of the total bacterial proteins. From 278 mg soluble proteins, the yield of the CK2? protein was 4 7 mg. SDS PAGE analysis of the purified recombinant protein showed only one band in agreement with native mouse CK2? subunit. The recombinant mouse CK2? and ? subunits were mixed at the same molar ratio. The produced CK2 holoenzyme displayed full activity. The characteristics and functions of reconstituted CK2 holoenzyme were consistent with those of the given native CK2. CONCLUSION The recombinant protein is mouse protein kinase CK2? subunit.