Objective:To analyze the methylation characteristics of the lymphocyte-specific protein-tyrosine kinase (LCK) promoter region in the peripheral blood circulation of rheumatoid arthritis (RA) patients and its correlation with clinical indicators.Methods:Targeted methylation sequencing was used to compare the methylation levels of 7 CpG sites in the LCK promoter region in the peripheral blood of RA patients with healthy controls (HC) and osteoarthritis (OA) patients. Correlation analysis and ROC curve construction were performed with clinical information.Results:Non-parametric tests revealed that compared with HC [0.53(0.50, 0.57)] and OA patients [0.59(0.54, 0.62), H=47.17, P<0.001], RA patients [0.63(0.59, 0.68)] exhibited an overall increase in methylation levels. Simultaneously, when compared with the HC group [0.38(0.35, 0.41), 0.59(0.55, 0.63), 0.60(0.55, 0.64), 0.59(0.55, 0.63), 0.58(0.53, 0.62), 0.45(0.43, 0.49), 0.57(0.54, 0.61)], the RA group [0.46(0.42, 0.49), 0.70(0.65, 0.75), 0.70(0.66, 0.76), 0.70(0.65, 0.75), 0.69(0.64, 0.74), 0.55(0.51, 0.59), 0.68(0.63, 0.73)] showed a significant elevation in methylation levels at CpG sites cg05350315_60, cg05350315_80, cg05350315_95, cg05350315_101, cg05350315_104, cg05350315_128, and cg05350315_142, with statistically significant differences ( Z=-5.63, -5.89, -5.91, -5.89, -5.98, -5.95, -5.95, all P<0.001). Compared with the OA group [0.65(0.59, 0.69), 0.65(0.60, 0.69), 0.64(0.58, 0.68), 0.50(0.45, 0.54), 0.63(0.58, 0.67)], the RA group [0.70(0.66, 0.76), 0.70(0.65, 0.75), 0.69(0.64, 0.74), 0.55(0.51, 0.59), 0.68(0.63, 0.73)] exhibited a significant increase in methylation levels at CpG sites cg05350315_95, cg05350315_101, cg05350315_104, cg05350315_128, and cg05350315_142, with statistically significant differences ( Z=-3.56, -3.52, -3.60, -3.67, -3.62; P=0.036, 0.042, 0.031, 0.030, 0.030). Furthermore, Pearson correlation coefficient analysis revealed a positive correlation between the overall methylation level in this region and C-reactive protein (CRP) ( r=0.19, P=0.004) and erythrocyte sedimentation rate ( r=0.14, P=0.035). The overall methylation level of the LCK promoter region in the CRP (low) group [0.63 (0.58, 0.68)] was higher than that in the CRP (high) group [0.65(0.61, 0.70)], with statistically significant differences ( Z=2.60, P=0.009). Finally, by constru-cting a ROC curve, the discriminatory efficacy of peripheral blood LCK promoter region methylation levels for identifying RA patients, especially seronegative RA patients, from HC and OA groups was validated, with an AUC value of 0.78 (95% CI: 0.63, 0.93). Conclusion:This study provides insights into the methylation status and methylation haplotype patterns of the LCK promoter region in the peripheral blood of RA patients. The overall methylation level in this region is positively correlated with the level of inflammation and can be used to differentiate seronegative RA patients from the HC and OA patients.

Objective: To construct an evaluation index system of oral health literacy for children's parents, so as to provide an index system for the evaluation of oral health literacy for children's parents in China. Methods: The evaluation index system of oral health literacy for children's parents was designed based on literature review and semi-structured interview. Experts from oral prevention and pediatric oral medicine were invited to participate in two-round Delphi consultations. The indicators were scored and screened according to the importance, and the weight determined using analytic hierarchy process. The effectiveness of the consultation was evaluated by positive coefficient, authority coefficient and coordination coefficient. Results: Twenty-four experts participated in the consultation, including 6 males and 18 females. There were 21 experts with a master degree, 3 experts with a doctor degree, and 20 experts with vice senior professional titles and above. The recovery rates of the two rounds of consultations were 96.00% and 100.00%, the authority coefficients were 0.866 and 0.917, the Kendall's coefficient of concordance were 0.120 and 0.156 (both P<0.05), and the coefficient of variation was 0.15-0.38 and 0.03-0.17, respectively. The final evaluation index system included 3 primary indicators, 11 secondary indicators and 40 tertiary indicators. The primary indicators were basic knowledge and concepts related to oral health, promoting lifestyle and behaviors related to oral health, and maintaining basic skills related to oral health. Conclusion The evaluation index system of oral health literacy for children's parents has been established in this study and needs to be further applied and evaluated.

ObjectiveTo explore the clinical effectiveness and safety of stick-point sinew-soothing and bone-setting manipulation for scapulohumeral periarthritis. MethodsUsing prospective randomised controlled trial method， 60 cases of patients with scapulohumeral periarthritis were collected and randomly divided into 30 cases each in control group and trial group. Both groups of patients were orally treated with celecoxib， on the basis of which the control group was treated with traditional bonesetting manipulation once every other day for 14 days， while the trial group was treated with stick-point sinew-soothing and bone-setting manipulation once every 3~5 days for 14 days. Both groups were treated for 2 courses. The main observation indexes were pain visual analogue scale （VAS） score and shoulder pain and dysfunction index （SPADI）， which were evaluated once before treatment and after 2 and 4 weeks of treatment. The secondary effectiveness indicators included the university of California at Los Angeles shoulder rating scale （UCLA）， traditional Chinese medicine syndrome score （including symptom scores as joint pain， pain in a fixed place， activity limitation， local stiffness）， and serum interleukin （IL-6） and tumour necrosis factor α （TNF-α） levels before and after the treatment， in order to evaluate the clinical effectiveness， and to record the adverse reactions that occurred in the process of diagnosis and treatment. ResultsCompared with the groups before treatment， the pain VAS score， SPADI and scores of joint pain， pain with a fixed place， activity limitation and local stiffness were lower， UCLA score was higher， and serum IL-6 and TNF-α levels were lower at 4 weeks of treatment in the two groups （P＜0.05）. When comparing the two groups between the groups at 2 and 4 weeks of treatment， the pain VAS score， SPADI and TCM scores of each symptom in the study group were lower than those in the control group， the UCLA score was higher than those in the control group （P＜0.01）， and the serum IL-6 and TNF-α levels were lower than those in the control group at 4 weeks of treatment （P＜0.01）. The clinical effectiveness rate of the study group was 66.67%， which was significantly higher than that of the control group， which was 40.00% （P = 0.038）. No adverse reactions were seen in both groups during the study. ConclusionCompared with the traditional massage manipulation， the treatment of scapulohumeral periarthritis with stick-point sinew-soothing and bone-setting manipulation has more advantages in relieving pain symptoms， reducing inflammatory reaction， and promoting the recovery of shoulder joint function.

Objective:To investigate the role and mechanism of PM(2.5)exposure on airway inflammation in juvenile rats based on macrophage recruitment induced by JNK/CCl2 signaling pathway.Methods:A total of 50 juvenile SD rats were randomly divided into 5 groups(n=10).The control group received no treatment,the PM(2.5)group received PM(2.5)particulate matter expo-sure,and the PM(2.5)+Anisomycin group received PM(2.5)exposure and Anisomycin,an activator of JNK,intravenously.Rats in the PM(2.5)+SP600125 group received PM(2.5)exposure and intravenous administration of the JNK inhibitor SP600125,and rats in the PM(2.5)+Pirfenidone group received PM(2.5)exposure and intravenous administration of Pirfenidone,a CCl2 inhibitor.The rats were euthanized and lung tissue was harvested.JNK,phosphorylated JNK(p-JNK)and CCl2 protein expressions were detected by Western blot.Hematoxylin-eosin(HE)staining was used to detect the pathological changes of lung airway tissue and score the pulmo-nary bronchial inflammation.The number of macrophages in alveolar lavage fluid was analyzed by flow cytometry.The levels of IL-6,IL-1β,and TNF-α in alveolar lavage fluid were determined by ELISA.Results:The expression levels of JNK,p-JNK,and CCl2 among all groups(F=205.296,950.408,260.019;all P<0.001)and macrophage content(F=48.414;P<0.001),pulmonary bronchial inflammation score(F=101.703;P<0.001)and IL-6(H=44.890;P<0.001),IL-1β(H=42.071;P<0.001),TNF-α(F=297.154;P<0.001)were statistically significant.Compared with the control group,the expressions of JNK/CCl2 pathway proteins JNK,p-JNK,and CCl2 in PM(2.5)group were significantly up-regulated(all P<0.05),while the content of macrophages was increased(P<0.05),and the pulmonary and bronchial inflammation score was significantly increased(P<0.05).The levels of IL-6,IL-1β,and TNF-α were up-regulated(all P<0.05).Compared with PM(2.5)group,the content of macrophages in PM(2.5)+Anisomycin group was sig-nificantly increased(P<0.05),the pulmonary bronchial inflammation score was significantly increased(P<0.05),and the levels of IL-6,IL-1β,and TNF-α were increased(all P<0.05).The expression levels of JNK,p-JNK,and CCl2 were increased(all P<0.05).Compared with PM(2.5)group,the content of macrophages in PM(2.5)+SP600125 group and PM(2.5)+Pirfenidone group were signifi-cantly decreased(P<0.05),and the pulmonary bronchial inflammation score was significantly decreased(P<0.05).In addition,the levels of IL-6,IL-1β,and TNF-α were significantly decreased(all P<0.05).Compared with PM(2.5)group,the expression levels of JNK,p-JNK,and CCl2 in PM(2.5)+SP600125 group were down-regulated(all P<0.05),and the expression level of CCl2 in PM(2.5)+Pirfenidone group was down-regulated(all P<0.05).Conclusion:JNK/CCl2 pathway induces macrophage recruitment and pro-motes allergic airway inflammation induced by PM(2.5)particulate matter exposure in juvenile rats.

Objective:To investigate the effects of mild SARS-CoV-2 infection on hematological parameters of adult blood donors and the suitability of apheresis platelet donation,the changes of the hematological parameters in blood donors with mild infection of the SARS-CoV-2 Omicron variant strain were evaluated.Methods:Seventy-two blood donors with mild COVID-19 symptoms who donated consecutive apheresis platelets for 3 times from December 2022 to January 2023,42 cases among which were included in the infection-positive group,and 30 cases in the suspected infection group.Forty-two donors un-vaccinated against SARS-CoV-2,un-infected,and donated three consecutive apheresis platelets from October to November 2022 were included in the control group.The changes of blood routine testing in the positive group and the suspected infection group were retrospectively compared before(Time1)and after(Time2 and Time3)the onset of symptoms,three consecutive times(Time1,Time2,Time3)in the control group by repeated measures analysis of variance.The Bayesian discriminant method was used to establish a discriminant equation to determine whether the recent infection of SARS-CoV-2 occurred or not.Results:Simple effect of the number times of tests in the positive and suspected infection groups was significant(Finfection-positive group=6.98,P＜0.001,partial η2=0.79,Fsuspected infection group=4.31,P＜0.001,partial η2=0.70).The positive group and the suspected infection group had lower RBC,HCT,and HGB,and higher PLT and PCT at Time2 compared to Time1 and Time3(P＜0.05).The positive group and the suspected infection group showes RDW-CV and RDW-SD at Time3 higher than Time1 and Time2(P＜0.001).The simple effect of the number times of tests in the control group was not significant(F=0.96,P=0.55,partial η2=0.34).The difference of the whole blood count parameters in the control group for three times was not statistically significant(P＞0.05).We established a discriminant equation to determine whether the recent infection of SARS-CoV-2 occurred or not.The equation had an eigenvalue of 0.22,a canonical correlation of 0.43(x2=27.81,P＜0.001),and an analysis accuracy of 72.9％.Conclusion:The hematological indicators of RBC,HCT,HGB,PLT,PCT,RDW-CV and RDW-SD in blood donors who had infected with mild COVID-19 showed dynamic changes.The discriminant equation for whether they are infected recently with COVID-19 has a high accuracy rate.

Objective:To diagnose and explore the genetic etiology of a neonate with Hereditary epidermolysis bullosa.Methods:A neonate who was admitted to Suqian Hospital Affiliated to Xuzhou Medical University on July 10, 2021 was selected as the study subject. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA. And target gene capture and next-generation sequencing were carried out. Candidate variants were verified by Sanger sequencing and pathogenicity analysis.Results:The child was found to harbor compound heterozygous variants of the COL17A1 gene, namely c. 997C>T (p.Q333X) and c. 3481dupT (p.Y1161fs*2), which were respectively inherited from his father and mother. Both variants were predicted to be pathogenic. Conclusion:The child was diagnosed with Generalized atrophic benign epidermolysis bullosa due to the compound heterozygous variants of the COL17A1 gene.

177Lu-prostate specific membrane antigen(PSMA)radio-ligand therapy has been approved abroad for advanced prostate cancer and has been in several clinical trials in China.Based on domestic clinical practice and experimental data and referred to international experience and viewpoints,the expert group forms a consensus on the clinical application of 177Lu-PSMA radio-ligand therapy in prostate cancer to guide clinical practice.

Objective:To explore the clinical application value of rapid next-generation sequencing (NGS) strategy based on targeted amplicon sequencing in the diagnosis of myeloid neoplasms.Methods:In this observational study, both rapid NGS and conventional NGS on the bone marrow or peripheral blood samples of 682 patients were prospectively performed from February 2021 to August 2022 in First Affiliated Hospital of Soochow University. The sequencing results were analyzed using the local Ion Reporter software and our lab′s self-built bioinformatics platform, respectively. The timeliness of the two sequencing platforms was compared, and the Kappa consistency test was used to evaluate the consistency between the two sequencing platforms. Patients aged between 18 and 59 years with newly diagnosed acute myeloid leukemia (AML) underwent screening by rapid NGS combining multiplex RT-PCR and in situ fluorescence hybridization technique within 72 hours, from whom high-risk patients according to European LeukemiaNet (ELN) 2017 were screened for individualized induction therapy.Results:In terms of timeliness, the median time from sample receipt to report issuance were 3 (2, 4) days and 13 (11, 15) days under rapid NGS and conventional NGS testing, respectively, with a statistically significant difference ( Z=?22.636, P<0.001). Among 682 specimens with a total of 1 507 variants, rapid NGS detected a total of 1 499 variants, with a detection rate of 99.5% and 674 cases were accurate, with an accuracy rate of 98.8%; the conventional NGS detected 1 506 variants, with a detection rate of 99.9% and 681 cases were accurate, with an accuracy rate of 99.9%. In 682 specimens, there were 181 negative and 501 positive, in which 8 cases were missed under rapid NGS, and 1 case was missed under conventional NGS. The kappa value was 0.967 by Kappa consistency test, and P<0.001, suggesting good consistency and consistency between the two NGS platforms. From February 2021 to July 2022, 286 patients who were rapidly diagnosed of AML contained 78 patients screened as the ELN 2017 adverse-risk category, including 42 patients enrolled, with age 39 (33, 52) years old. After one cycle of venetoclax combined with decitabine induction therapy, 78.6%(38/42) of the patients achieved composite complete remission. Among the rest 104 additional myeloid neoplasms, rapid NGS detected mutations in 80 patients, with a detection rate of 76.9%, among which 89.0%(215/242) of the variants could serve as the basis for the diagnostic classification, prognostic evaluation, and target therapy of myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Conclusion:The rapid NGS based on targeted amplicon sequencing is in good consistency with conventional NGS, and shorters the diagnostic time, whose sensitivity and detection range meets the need for diagnostic classification, prognostic stratification, and target therapy of myeloid neoplasms.

Abstract：Objective To improve the replication level of varicella⁃zoster virus（VZV）in human diploid cell line MRC⁃5 and increase the yield of VZV vaccine by reducing the expression of interferon（IFN）related genes via optimizing the cell line MRC⁃5. Methods Interferon receptor 1（IFNAR1）silenced MRC⁃5 cell line（MRC⁃5IFNAR1⁃）was constructed by CRISPR／Cas9 gene editing technology，which was determined for the relative expression of IFNAR1 mRNA，and for those of mRNA of IFN related genes IFNβ and OAS1 after VZV infection by qRT⁃PCR to evaluate the effect of gene silencing. Gene mutation sequences were further identified by sequencing of the silenced sites. The replication of VZV in MRC⁃5 and MRC⁃5IFNAR1⁃ cell lines was compared 168 h after VZV infection by using qRT⁃PCR and plaque formation unit（PFU）assay， to evaluate the effect of MRC⁃5IFNAR1⁃cell line on VZV replication. Results The growth status of MRC⁃5IFNAR1⁃ cell line wasconsistent with that of MRC ⁃ 5 cells，and the relative expression of IFNAR1 mRNA decreased by 73%；The relative expressions of IFNβ and OAS1 mRNA in MRC⁃5IFNAR1⁃ cell line were 61% and 90% lower than those in MRC⁃ 5 cells respectively after VZV infection；In addition，168 h after VZV infection，the level of DNA replication and the titer of VZV increased by 5. 7 folds and 4 folds respectively. Conclusion The successful establishment of MRC⁃5IFNAR1⁃ cell line may be a potential scheme to increase the yield of vaccines based on human diploid cells，and provided a reference for expanding production of VZV vaccine.

Objective To analyze the genetic subtypes of human immunodeficiency virus (HIV) and the prevalence of pretreatment drug resistance in the newly reported HIV-infected men in Guangxi. Methods The stratified random sampling method was employed to select the newly reported HIV-infected men aged≥50 years old in 14 cities of Guangxi from January to June in 2020.The pol gene of HIV-1 was amplified by nested reverse transcription polymerase chain reaction and then sequenced.The mutation sites associated with drug resistance and the degree of drug resistance were then analyzed. Results A total of 615 HIV-infected men were included in the study.The genetic subtypes of CRF01_AE,CRF07_BC,and CRF08_BC accounted for 57.4% (353/615),17.1% (105/615),and 22.4% (138/615),respectively.The mutations associated with the resistance to nucleoside reverse transcriptase inhibitors (NRTI),non-nucleoside reverse transcriptase inhibitors (NNRTI),and protease inhibitors occurred in 8 (1.3%),18 (2.9%),and 0 patients,respectively.M184V (0.7%) and K103N (1.8%) were the mutations with the highest occurrence rates for the resistance to NRTIs and NNRTIs,respectively.Twenty-two (3.6%) patients were resistant to at least one type of inhibitors.Specifically,4 (0.7%),14 (2.3%),4 (0.7%),and 0 patients were resistant to NRTIs,NNRTIs,both NRTIs and NNRTIs,and protease inhibitors,respectively.The pretreatment resistance to NNRTIs had much higher frequency than that to NRTIs (2.9% vs.1.3%;χ²=3.929,P=0.047).The prevalence of pretreatment resistance to lamivudine,zidovudine,tenofovir,abacavir,rilpivirine,efavirenz,nevirapine,and lopinavir/ritonavir was 0.8%, 0.3%, 0.7%, 1.0%, 1.3%, 2.8%, 2.9%, and 0, respectively. Conclusions CRF01_AE,CRF07_BC,and CRF08_BC are the three major strains of HIV-infected men≥50 years old newly reported in Guangxi,2020,and the pretreatment drug resistance demonstrates low prevalence.
Male ; Humans ; Middle Aged ; Reverse Transcriptase Inhibitors/therapeutic use* ; HIV Infections/drug therapy* ; Drug Resistance, Viral/genetics* ; China/epidemiology* ; Mutation ; HIV-1/genetics* ; Protease Inhibitors/therapeutic use* ; Genotype