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.

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%,P<0.05).The detected rates of 23S rRNA,mtrR,penA single or combined mutations were without significant differences between AZM-LLR isolates and AZM-MLR isolates(P>0.05).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.

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.

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.

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.

Objective To investigate the risk factors and prognosis of acute kidney injury (AKI) after orthotopic liver transplantation (OLT).Methods The retrospective case-control study was conducted.The clinical data of 127 patients who underwent OLT in the First Affiliated Hospital of Xi'an Jiaotong University from January 2013 to December 2015 were collected.Of 127 patients,24 were complicated with postoperative AKI,including 17 in grade 1,5 in grade 2 and 2 in grade 3,and 103 were not complicated with AKI.AKI after OLT was treated according to the diagnostic criteria of AKI from 2012 guidelines of Kidney Disease:Improving Global Outcomes (KDIGO).Observation indicators:(1) risk factors analysis affecting AKI after OLT;(2) comparison of postoperative recovery in patients with different AKI grade;(3) follow-up and survival situations.Follow-up using outpatient examination and telephone interview was performed up to July 2017.Measurement data with nornal distribution were represented as x±-s,and measurement data with skewed distribution were described as M (range).Univariate analysis was done using the t test and rank sum test.Comparisons of count data and univariate analysis were done using chi-square test or Fisher exact probability.Multivariate analysis was done using the logistic regression model.The survival rate and curve were respectively calculated and drawn by the KaplanMeier method,and Log-rank test was used for survival analysis.Results (1) Risk factors analysis affecting AKI after OLT:results of univariate analysis showed that age,combined hypertension,preoperative Child-Pugh score,preoperative model for end-stage liver disease score (MELD),preoperative hemoglobin,preoperative serum albumin,preoperative blood urea nitrogen,preoperative glomerular filtration rate,preoperative prothrombin time,operation time,inferior vena cava occlusion time,duration of anhepatic phase,volume of intraoperative blood loss,total volume of intraoperative blood transfusion,volumes of intraoperative plasma and red blood cells transfusion,duration of postoperative ICU stay,use time of postoperative vasoactive drugs,time of postoperative mechanical ventilation,cases with postoperative infection,body mass index of donor and donor liver cold-ischemia time were related factors affecting occurrence of AKI after OLT (t =4.154,x2 =8.482,t =5.129,3.694,1.294,9.223,5.418,Z=4.287,t=2.105,5.168,8.182,10.042,Z=1.074,0.664,6.274,3.712,1.289,t=1.056,x2 =10.617,t=2.447,3.371,1.476,P＜0.05).Results of multivariate analysis showed that age,preoperative MELD score,preoperative serum albumin,volume of intraoperative blood loss and donor liver cold-ischemia time were independent factors affecting occurrence of AKI after OLT [odds ratio (OR) =0.812,0.866,1.392,1.001,0.516,95％ confidence interval:0.717-0.919,0.751-0.997,1.104-1.755,1.000-1.001,0.282-0.944,P＜0.05].(2) Comparison of postoperative recovery in patients with different AKI grade:cases with complete recovery,partial recovery and chronic renal failure were respectively 14,3,0 in 17 patients with grade 1 of AKI and 3,2,0 in 5 patients with grade 2 of AKI and 0,1,1 in 2 patients with grade 3 of AKI,with a statistically significant difference (x2=14.140,P＜0.05).(3) Follow-up and survival situations:127 patients were followed up for 9-44 months,with a median of 23 months.The 1-year overall survival rate of 127 patients was 95.3％.During the follow-up,22 patients died,including 9 with multiple organ failure,8 with primary disease recurrence and 5 with respiratory complication.The median overall survival time after OLT was 36 months in 24 patients with AKI and 40 months in 103 patients without AKI,with no statistically significant difference (x2=3.033,P＞0.05).Conclusion Age,preoperative MELD score,preoperative serum albumin,volume of intraoperative blood loss and donor liver cold-ischemia time are independent factors affecting occurrence of AKI after OLT,and there is better recovery in patients with grade 1 of AKI.

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..

Objective:To explore the mechanism of Honghua Xiaoyao Tablets in the treatment of premature ovarian failure (POF) using network pharmacology and molecular docking technology; To conduct further experimental verification.Methods:The active components and related targets of Honghua Xiaoyao Tablets were obtained using TCMSP and PubChem databases, and the related targets of POF were obtained by using GeneCards database. The Venn online tool was used to screen the intersection genes, and the STRING database was used to construct the PPI network. Then, GO function enrichment analysis and KEGG pathway enrichment analysis were performed on the intersecting genes through the Metescape database, and Cytoscape 3.7.1 software was used to construct the "active component-target" network and "Chinese materia medica-active component-target-key pathway-disease" network. Finally, molecular docking verification was carried out. Mice were divided into blank group, model group, and Honghua Xiaoyao Tablets group using a random number table method, with 8 mice in each group. Except for the blank group, mice in each group were treated with zona pellucida polypeptide 3 (ZP3) and Freund's Complete Adjuvant (FCA) to establish a mouse model of immune POF. Mice in the Honghua Xiaoyao Tablets group received Honghua Xiaoyao Tablets solution 0.56 g/kg for gavage, and the blank control group and the model group received saline for gavage for consecutive 4 weeks. The histopathological changes of the mouse ovary were observed by HE staining. Immunohistochemical staining was used to observe the expression of ESR1, and Western blot was used to detect the expressions of Akt and p-Akt.Results:A total of 80 intersection targets between Honghua Xiaoyao Tablets and POF were obtained, and the PPI network contained 44 core targets. The top 5 compounds in the topological analysis were formononetin, quercetin, Betulinic acid, Hydroxysafflor Yellow A and Baicalin, and the top 5 targets were PPARG, ESR1, AR, AKT1 and IL6. The molecular function of core genes was mainly receptor ligand activity, and its biological process mainly involved the positive regulation of cell migration. The cellular components mainly included membrane rafts, which were involved in signaling pathways such as cancer signaling pathway, proteoglycans in cancer, PI3K-Akt signaling pathway, and HIF-1 signaling pathway. "Chinese materia medica-component-target-pathway-disease" network showed that 8 kinds of Chinese materia medica in the Honghua Xiaoyao Tablets had important core components, among which Glycyrrhizae Radix et Rhizoma and Carthami Flos involved the most important core components. Molecular docking results showed that the active components had a good affinity with the core target. The experimental verification confirmed that Honghua Xiaoyao Tablets promoted follicular development, increased the expression of ESR1 in ovarian tissues and up-regulated the expression level of the key factor of Akt phosphorylation in the PI3K-Akt signaling pathway.Conclusions:The various active components of Honghua Xiaoyao Tablets may act on PPARG, ESR1 and other targets through multiple signaling pathways such as PI3K-Akt and HIF-1 to treat POF. The potential active components are mainly formononetin, quercetin, etc.

Objective:To evaluate the efficacy and safety of dupilumab in the treatment of prurigo nodularis (PN) in the real world.Methods:PN patients who were subcutaneously injected with dupilumab for over 12 weeks were collected from the China Type Ⅱ Inflammatory Skin Disease Clinical Research and Standardized Diagnosis and Treatment Project Database from June 2021 to October 2022. Their clinical data were retrospectively analyzed, which included demographic data, and changes in pruritus numeric rating scale (NRS), investigator global assessment for PN-Stage (IGA PN-S), dermatology life quality index (DLQI) and hospital anxiety and depression scale (HADS) scores before and after treatment. Differences in scores before and after treatment, as well as in efficacy between patients with and without a history of atopic diseases, were analyzed using Wilcoxon signed-rank test, paired t-test or chi-square test. Results:A total of 66 PN patients were collected, including 42 males and 24 females, and they were aged 8 to 89 (44.12 ± 24.17) years. Thirty-six patients had a history of atopic diseases, and 27 had a family history of atopic diseases. After 12-week treatment with dupilumab, the pruritus NRS, IGA PN-S and DLQI scores in the 66 patients significantly decreased from the baseline scores (7.00 [5.00, 8.00], 3.00 [3.00, 4.00], 12.00 [7.75, 20.25], respectively) to 3.00 [2.00, 4.25], 2.00 [2.00, 3.00], 5.00 [1.75, 8.25], respectively (all P < 0.001). Among the 66 patients, 39 continued the regular treatment with dupilumab after 12 weeks and were followed up to 16 weeks; their pruritus NRS and IGA PN-S scores at week 16 further decreased compared with those at week 12 (both P < 0.05). There were no significant differences in the proportion of patients showing an improvement of ≥ 4 points in the NRS score or the proportion of patients achieving IGA 0/1 at week 12 between the patients with history of atopic diseases and those without (both P > 0.05). Before treatment, 32 PN patients were accompanied by mild to severe anxiety and/or depression; after 12-week treatment, the HADS-A scores in the 28 patients with anxiety (HADS-A scores > 7 points) and the HADS-D scores in the 20 patients with depression (HADS-D scores > 7 points) significantly decreased compared with their baseline scores (both P < 0.001) ; 18 (56.52%) patients achieved remission in anxiety and depression (both HADS-A and HADS-D scores < 7 points). Among the 66 PN patients, there were 13 minor patients, including 7 males and 6 females, and they were aged 8 to 17 (13.77 ± 3.09) years; after 12-week treatment, their pruritus NRS, IGA PN-S, and DLQI scores significantly decreased compared with the corresponding baseline scores (all P < 0.05) ; 8 minor patients continued dupilumab treatment for 16 weeks, with a further decrease in the IGA PN-S score compared with that at week 12 ( P < 0.05), but without significant differences between the pruritus NRS and DLQI scores at week 16 and those at week 12 (both P > 0.05). Adverse reactions were observed in 7 adult patients, including eye pruritus, local injection reactions, and systemic erythema accompanied by pruritus on the day of injection. No adverse reactions were reported in minor patients. Conclusion:In the real world, dupilumab could markedly alleviate pruritus, skin lesions, anxiety and depression symptoms in PN, improve the quality of life, and exhibited a good safety profile.

Objective To evaluate the clinical effect and safety of regional citrate anticoagulation (RCA) in continuous renal replacement therapy (CRRT) for acute kidney injury (AKI) after hepatectomy.Methods A retrospective analysis of the clinical data of all patients with AKI after hepatectomy for CRRT admitted to surgical intensive care unit (ICU) of the First Affiliated Hospital of Xi'an Jiaotong University from January 19th, 2013 to January 19th, 2018 was performed. According to the different anticoagulants, the patients were divided into no anticoagulant group (NA group), low molecular heparin anticoagulation (LMHA) group and RCA group. The general data of patients during the perioperative period; renal function, the internal environment, electrolyte and blood coagulation function before and after CRRT; the filter time, the number of filters and adverse events (bleeding, frequent filter blood coagulation, metabolic alkalosis, metabolic acidosis, hypocalcemia, citrate accumulation, etc.) during CRRT were collected. Kaplan-Meier survival curve was used to analyze the life span of the first filter during different anticoagulation. Results A total of 67 cases were included in this study, including 11 in the NA group, 25 in the LMHA group and 31 in the RCA group. There was no significant difference in gender, age, underlying disease, etiology (tumor), Child-Pugh stage (A or B), CT angiography (CTA), basic renal function [serum creatinine (SCr), cystatin C (Cys C)], the American Society of Anesthesiologists (ASA) stage; surgical approach; intraoperative bleeding volume, blood transfusion, blood pressure, time of duration of low blood pressure; and postoperative circulatory failure, hepatic insufficiency and sepsis among three groups. However, the length of ICU stay in RCA group was significantly less than the LMHA group and NA group (days: 8.16±2.24 vs. 10.48±5.11, 13.29±6.64, bothP< 0.05). Compared with before CRRT, the levels of SCr, Cys C and Lac were significantly decreased in RCA group and LMHA group after CRRT [SCr (μmol/L): 89.02±21.90 vs. 248.30±55.32, 105.10±49.00 vs. 270.10±156.00; Cys C (mg/L): 2.18±0.95 vs. 2.94±1.26, 2.26±0.76 vs. 3.07± 0.90; Lac (mmol/L): 2.21±1.46 vs. 3.62±1.73, 2.37±1.24 vs. 4.03±1.69, allP < 0.05]; in addition, LMHA group and NA group had significant effects on hemoglobin (Hb), platelet count (PLT) and activated partial thromboplastin time (APTT) after CRRT [Hb (g/L): 85.4±5.1 vs. 99.6±23.6, 80.0±7.6 vs. 101.4±7.8; PLT (×109/L): 27.60±8.22 vs. 62.04±16.49, 21.36±3.91 vs. 61.45±17.69; APTT (s): 63.07±10.25 vs. 41.52±3.65, 49.56±5.77 vs. 41.09± 3.45, allP < 0.05]; at the same time, Cys C level and prothrombin time (PT) in the NA group after CRRT treatment were significantly increased compared with the others [Cys C (mg/L): 3.59±0.64 vs. 2.29±0.51, PT (s): 26.41±2.43 vs. 23.64±1.92 , bothP < 0.05]. Finally, the time of filters (hours: 60.52±8.82, 31.04±7.03, 13.73±6.26,F = 183.412, P < 0.001) and the number of filter during treatment (number: 2.03±0.60, 3.12±0.73, 4.64±1.29,F = 45.933,P <0.001) in the RCA group, LMHA group and NA group had statistically significant difference. Meanwhile, the incidence of adverse events such as bleeding (0 vs. 4, 7,χ2 = 23.961,P < 0.001) and frequent filter coagulation (1 vs. 10, 11,χ2 =35.413,P < 0.001) in RCA group was significantly lower than that in LMHA group and NA group. Kaplan-Meier survival analysis showed that the life time of the first filter in RCA group was significantly longer than that in LMHA group and NA group (χ2 = 139.45,P < 0.05).Conclusion The application of RCA in patients with AKI after hepatectomy during CRRT is safe and effective, which can significantly prolong the life of the filter and reduce the risk of bleeding.