Objective: To reveal the molecular biological mechanism of a rare Dc-variant individual using PacBio third-generation sequencing technology. Methods: ABO and Rh blood type identification, DAT, unexpected antibody screening and D antigen enhancement test were conducted by serological testing. The absorption-elution test was used to detect the e antigen. RHCE gene typing was performed by PCR-SSP, and the 1-10 exons of RHCE were sequenced by Sanger sequencing. The full-length sequences of RHCE, RHD and RHAG were detected by PacBio third-generation sequencing technology. Results: Serological findings: Blood type O, Dc-phenotype, DAT negative, unexpected antibody screening negative; enhanced D antigen expression; no detection of e antigen in the absorption-elution test. PCR-SSP genotyping indicated the presence of only the RHCE c allele. Sanger sequencing results: Exons 5-9 of RHCE were deleted, exon 1 had a heterozygous mutation at c. 48G/C, and exon 2 had five heterozygous mutations at c. 150C/T, c. 178C/A, c. 201A/G, c. 203A/G and c. 307C/T. Third-generation sequencing results: RHCE genotype was RHCE 02N. 08/RHCE-D(5-9)-CE; RHD genotype was RHD 01/RHD 01; RHAG genotype was RHAG 01/RHAG 01 (c. 808G>A and c. 861G>A). Conclusion: This Dc-individual carries the allele RHCE 02N. 08 and the novel allele RHCE-D(5-9)-CE. The findings of this study provide data support and a theoretical basis for elucidating the molecular mechanisms underlying RhCE deficiency phenotypes.

Aim To investigate the effect of total glu-cosides of paeony on inflammatory injury and TLR4/NF-κB/NLRP3 pathway in autoimmune thyroiditis(AIT)rats.Methods The experiment was divided into control group,model group,total glucosides of pae-ony(TGP),TLR4 inhibitor group and TGP+TLR4 ag-onist group,with 10 animals in each group.Except for the control group,the rats in other groups were subcu-taneously injected with thyroglobulin and Freund's ad-juvant to induce the AIT rat model.After six weeks of administration,thyroid histopathological changes were observed using hematoxylin-eosin(HE)staining;ser-um levels of TPOAb,TgAb,TSH,T3,T4,TNF-α,INF-γ,IL-1 β and IL-1 β were detected by enzyme-linked immunosorbent assay(ELISA);TLR4/NF-κB/NLRP3 pathway mRNAs and proteins expression in thyroid tis-sues were detected by RT-qPCR and Western blot.Re-sults Compared with the control group,the thyroid follicular epithelium of rats was significantly damaged,and the serum levels of TPOAb,TgAb,TSH,T3,T4,TNF-α,INF-γ,IL-1 β and IL-1 β increased(P＜0.01).The expression of TLR4/NF-κB/NLRP3 path-way mRNAs and proteins increased in the model group(P＜0.01).Compared with the model group,the damage of thyroid follicular epithelium was alleviated,and the serum levels of TPOAb,TgAb,TSH,T3,T4,TNF-α,INF-γ,IL-1 β and IL-1 β were reduced(P＜0.01),the expression of TLR4/NF-κB/NLRP3 path-way mRNAs and proteins were down-regulated in the TGP group and TLR4 inhibitor group(P＜0.01).Compared with TGP group,the damage of thyroid follic-ular epithelium was aggravated,and the levels of serum TPOAb,TgAb,TSH,T3,T4,TNF-α,INF-γ,IL-1 β and IL-1 β were elevated(P＜0.05 or P＜0.01),the pro-tein expressions of TLR4/NF-κB/NLRP3 pathway mR-NAs and proteins were up-regulated in TGP+TLR4 ag-onist group(P＜0.05 or P＜0.01).Conclusions TGP may play a protective role in thyroid by inhibiting the TLR4/NF-κB/NLRP3 pathway and improving the inflammatory injury of thyroid tissues.

Aim To investigate the impact of Cinobu-facini on the proliferation,invasion,and apoptosis of HepG2 cells and the underlying mechanism.Methods The proliferation of HepG2 cells was assessed using the CCK-8 method following treatment with Cinobufaci-ni.The invasion capability of HepG2 cells was evalua-ted through Transwell assay after exposure to Cinobufa-cini.The apoptosis rates of HepG2 cells post Cinobufa-cini intervention were measured using flow cytometry,and the expression levels of VEGF in the culture medi-um of HepG2 cells were determined using enzyme-linked immunoassay.Furthermore,qRT-PCR and Western blot analyses were conducted to assess the im-pact of Cinobufacini on mRNA and protein expression levels related to the CXCL5/FOXD1/VEGF pathway.The interaction between CXCL5 and FOXD1 was inves-tigated via co-immunoprecipitation.Results Cinobufa-cini treatment led to a gradual decrease in HepG2 cell viability in a dose-dependent manner compared to the control group(P＜0.05).Moreover,Cinobufacini sig-nificantly suppressed HepG2 cell invasion(P＜0.05)while enhancing cell apoptosis(P＜0.05).Notably,Cinobufacini exhibited inhibitory effects on the CX-CL5/FOXD1/VEGF pathway,as evidenced by re-duced expression of related mRNA and proteins(P＜0.05).FOXD1 was identified as the binding site of CXCL5.Overexpression of CXCL5 resulted in in-creased proliferation and VEGF secretion by HepG2 cells(P＜0.05),and increased expression of FOXD1 and VEGF(P＜0.05).However,Cinobufacini inter-vention effectively inhibited liver cancer cell prolifera-tion and invasion(P＜0.05),promoted apoptosis(P＜0.05),reduced VEGF secretion by HepG2 cells(P＜0.05),and downregulated the expression of CXCL5 and FOXD1 in HepG2 cells(P＜0.05);but com-pared with the unexpressed group of Cinobufacini,its ability to inhibit cell activity was weakened(P＜0.05),and its ability to inhibit the expression of CX-CL5,FOXD1,and VEGF was weakened(P＜0.05).Conclusion Cinobufacini may inhibit HepG2 cell pro-liferation and invasion and promote HepG2 cell apopto-sis by regulating the CXCL5/FOXD1/VEGF pathway.

Objective:To improve the method for the determination of related substances in vidarabine monophos-phate.Methods:The analysis was performed on an ChromCore AQC18 column(4.6 mm × 150 mm,5 μm)with mobile phase A of an aqueous solution containing 0.01 mol·L-1 tetrabutylammonium hydroxide and 0.1 mol·L-1 potassium dihydrogen phosphate and mobile phase B of methanol by gradient elution at the flow rate of 1.0 mL·min-1.The column temperature was maintained at 30 ℃ and the UV detection wavelength was set at 258 nm.Results:Related substances were effectively separated from the principal component.Vidarabine mono-phosphate and its four impurities showed a good linear relationship in the self-concentration ranges(r＞0.999 9).The average recoveries and were 95.0％-99.2％with RSDs(n=9)of 1.0％-4.4％related substances in vidar-abine monophosphate and 91.8％-102.1％with RSDs of 0.5％-4.8％(n=9)for vidarabine monophosphate for injection,respectively.Conclusion:The improved method is simple,rapid and specific,and can be used for the determination of related substances in vidarabine monophosphate.

Objective To explore clinical effect of single small incision with honeycomb titanium plate in treating acute acromioclavicular dislocation.Methods The clinical data of 40 patients with acute acromioclavicular dislocation admitted from December 2019 to December 2021 were retrospectively analyzed and divided into two groups according to different surgical methods.Among them,20 patients were fixed with single small incision with honeycomb titanium plate(titanium plate group),including 11 males and 9 females,aged from 23 to 65 years old with an average of(47.40±12.58)years old;12 patients on the left side,8 patients on the right side;11 patients with type Ⅲ,3 patients with type Ⅳ,and 6 patients with type Ⅴ according to Rockwood classification.Twenty patients were fixed with clavicular hook plate(clavicular hook group),including 8 males and 12 females,aged from 24 to 65 years old with an average of(48.40±12.08)years old;12 patients on the left side,8 patients on the right side;10 patients with type Ⅲ,2 patients with type Ⅳ,and 8 patients with type V according to Rockwood classifica-tion.Operative time,incision length,intraoperative blood loss,hospital stay,visual analogue scale(VAS)and Constant-Murley score of shoulder joint function were compared between two groups.Anteroposterior radiographs of the affected shoulder joint were recorded before,immediately and 6 months after surgery,and the coracoclavicular distance was measured and compared.Results Both groups of patients were successfully completed operation without serious complications.All patients were fol-lowed up for 6 to 15 months with an average of(11.9±4.8)months.There were no incisional infection,internal plant fracture or failure,bone tunnel fracture and other complications occurred.The incision length of titanium plate group(35.90±3.14)mm was significantly shorter than that of clavicular hook group(49.30±3.79)mm(P＜0.05).There were no significant difference in operative time,intraoperative blood loss and hospital stay between two groups(P＞0.05).At 1 and 3 months after operation,VAS of titanium plate group was lower than that of clavicular hook group(P＜0.05).Connstant-Murley scores in titanium plate group at 1,3 and 6 months after operation were(86.80±1.36),(91.60±2.32)and(94.90±2.22),respectively;and in clavicular hook group were(78.45±5.47),(85.55±2.01)and(90.25±1.92),which were higher than that of clavicular hook group(P＜0.05).There was no significant difference in coracoclavicular distance between two groups immediately and 6 months after op-eration(P＞0.05).Conclusion For the treatment of acute acromioclavicular joint dislocation,single small incision combined with honeycomb titanium plate have advantages of shorter incision,fast recovery of shoulder joint function without the second operation,and has good satisfaction of patient.

Objective To explore the difference in the effectiveness between proximal femoral nail anti-rotation(PFNA)and proximal femoral locking compression plate(PFLCP)of intertrochanteric fracture in the elderly patients combined with knee osteoarthritis.Methods The clinical data of 65 intertrochanteric femoral fractures combined with knee osteoarthritis be-tween June 2015 and February 2021 were retrospectively analyze.They were divided into two groups according to the different surgical methods.PFNA group was composed of 36 patients,12 males and 24 females,aged from 61to 88 years old with an av-erage of(77.0±6.4)years old.There were 17 cases of left injury and 19 cases of right injury.According to modified Evans clas-sification,there were 3 cases of type Ⅱ,19 cases of type Ⅲ,10 cases of type Ⅳ,and 4 cases of type Ⅴ.PFLCP group was com-posed of 29 patients,11 males and 18 females,aged from 60 to 92 years old with an average of(78.8±6.5)years old.There were 14 cases of left injury and 15 cases of right injury.According to modified Evans classification,there were 2 cases of typeⅡ,18 cases of type Ⅲ,7 cases of type Ⅳ,and 2 cases of type Ⅴ.Comparison of operation time,intraoperation blood loss,postoperative bed time,incidence of postoperative complications,Harris score at 6 months and 1 year postoperation.Results All 65 patients were followed up ranging from 12 to 24 months with an average of(16.9±3.6)months.In the PFNA and PFLCP groups,the operation time was respectively(57.6±6.8)min and(77.4±6.5)min,the intraoperative blood loss was(128.3±50.3)ml and(156.3±23.9)ml,postoperative bed time was(4.0±2.5)days and(8.1±2.0)days,Harris score at 6 months post-operative was(45.3±8.6)points and(36.3±7.0)points.There were significant differences between two groups(P＜0.05).Inci-dence of postoperative complications was 19.4％(7/36)and 34.5％(10/29),Harris score at 1 year postoperative was(60.8±6.7)points and(59.0±8.1)points.There was no significant difference between the two groups(P＞0.05).Conclusion Compared with PFLCP,PFNA treatment of elderly patients with knee osteoarthritis between the femoral intertrochanteric fractures shorter surgical time,less intraoperative blood loss,bed rest after surgery,short-term hip function recovery better,when the affected knee joint can tolerate traction,can be used as a priority.

Objective To investigate the distribution and antimicrobial resistance profiles of the common pathogens isolated from urine from 2015 to 2021 in the CHINET Antimicrobial Resistance Surveillance Program.Methods The bacterial strains were isolated from urine and identified routinely in 51 hospitals across China in the CHINET Antimicrobial Resistance Surveillance Program from 2015 to 2021.Antimicrobial susceptibility was determined by Kirby-Bauer method,automatic microbiological analysis system and E-test according to the unified protocol.Results A total of 261 893 nonduplicate strains were isolated from urine specimen from 2015 to 2021,of which gram-positive bacteria accounted for 23.8％(62 219/261 893),and gram-negative bacteria 76.2％(199 674/261 893).The most common species were E.coli(46.7％),E.faecium(10.4％),K.pneumoniae(9.8％),E.faecalis(8.7％),P.mirabilis(3.5％),P.aeruginosa(3.4％),SS.agalactiae(2.6％),and E.cloacae(2.1％).The strains were more frequently isolated from inpatients versus outpatients and emergency patients,from females versus males,and from adults versus children.The prevalence of ESBLs-producing strains in E.coli,K.pneumoniae and P.mirabilis was 53.2％,52.8％and 37.0％,respectively.The prevalence of carbapenem-resistant strains in E.coli,K.pneumoniae,P.aeruginosa and A.baumannii was 1.7％,18.5％,16.4％,and 40.3％,respectively.Lower than 10％of the E.faecalis isolates were resistant to ampicillin,nitrofurantoin,linezolid,vancomycin,teicoplanin and fosfomycin.More than 90％of the E.faecium isolates were ressitant to ampicillin,levofloxacin and erythromycin.The percentage of strains resistant to vancomycin,linezolid or teicoplanin was＜2％.The E.coli,K.pneumoniae,P.aeruginosa and A.baumannii strains isolated from ICU inpatients showed significantly higher resistance rates than the corresponding strains isolated from outpatients and non-ICU inpatients.Conclusions E.coli,Enterococcus and K.pneumoniae are the most common pathogens in urinary tract infection.The bacterial species and antimicrobial resistance of urinary isolates vary with different populations.More attention should be paid to antimicrobial resistance surveillance and reduce the irrational use of antimicrobial agents.

Objective To understand the distribution and changing resistance profiles of clinical isolates of Enterococcus in hospitals across China from 2015 to 2021.Methods Antimicrobial susceptibility testing was conducted for the clinical isolates of Enterococcus according to the unified protocol of CHINET program by automated systems,Kirby-Bauer method,or E-test strip.The results were interpreted according to the Clinical & Laboratory Standards Institute(CLSI)breakpoints in 2021.WHONET 5.6 software was used for statistical analysis.Results A total of 124 565 strains of Enterococcus were isolated during the 7-year period,mainly including Enterococcus faecalis(50.7％)and Enterococcus faecalis(41.5％).The strains were mainly isolated from urinary tract specimens(46.9％±2.6％),and primarily from the patients in the department of internal medicine,surgery and ICU.E.faecium and E.faecalis strains showed low level resistance rate to vancomycin,teicoplanin and linezolid(≤3.6％).The prevalence of vancomycin-resistant E.faecalis and E.faecium was 0.1％and 1.3％,respectively.The prevalence of linezolid-resistant E.faecalis increased from 0.7％in 2015 to 3.4％in 2021,while the prevalence of linezolid-resistant E.faecium was 0.3％.Conclusions The clinical isolates of Enterococcus were still highly susceptible to vancomycin,teicoplanin,and linezolid,evidenced by a low resistance rate.However,the prevalence of linezolid-resistant E.faecalis was increasing during the 7-year period.It is necessary to strengthen antimicrobial resistance surveillance to effectively identify the emergence of antibiotic-resistant bacteria and curb the spread of resistant pathogens.

Objective To examine the changing antimicrobial resistance profile of Enterobacter spp.isolates in 53 hospitals across China from 2015 t0 2021.Methods The clinical isolates of Enterobacter spp.were collected from 53 hospitals across China during 2015-2021 and tested for antimicrobial susceptibility using Kirby-Bauer method or automated testing systems according to the CHINET unified protocol.The results were interpreted according to the breakpoints issued by the Clinical & Laboratory Standards Institute(CLSI)in 2021(M100 31st edition)and analyzed with WHONET 5.6 software.Results A total of 37 966 Enterobacter strains were isolated from 2015 to 2021.The proportion of Enterobacter isolates among all clinical isolates showed a fluctuating trend over the 7-year period,overall 2.5％in all clinical isolates amd 5.7％in Enterobacterale strains.The most frequently isolated Enterobacter species was Enterobacter cloacae,accounting for 93.7％(35 571/37 966).The strains were mainly isolated from respiratory specimens(44.4±4.6)％,followed by secretions/pus(16.4±2.3)％and urine(16.0±0.9)％.The strains from respiratory samples decreased slightly,while those from sterile body fluids increased over the 7-year period.The Enterobacter strains were mainly isolated from inpatients(92.9％),and only(7.1±0.8)％of the strains were isolated from outpatients and emergency patients.The patients in surgical wards contributed the highest number of isolates(24.4±2.9)％compared to the inpatients in any other departement.Overall,≤ 7.9％of the E.cloacae strains were resistant to amikacin,tigecycline,polymyxin B,imipenem or meropenem,while ≤5.6％of the Enterobacter asburiae strains were resistant to these antimicrobial agents.E.asburiae showed higher resistance rate to polymyxin B than E.cloacae(19.7％vs 3.9％).Overall,≤8.1％of the Enterobacter gergoviae strains were resistant to tigecycline,amikacin,meropenem,or imipenem,while 10.5％of these strains were resistant to polycolistin B.The overall prevalence of carbapenem-resistant Enterobacter was 10.0％over the 7-year period,but showing an upward trend.The resistance profiles of Enterobacter isolates varied with the department from which they were isolated and whether the patient is an adult or a child.The prevalence of carbapenem-resistant E.cloacae was the highest in the E.cloacae isolates from ICU patients.Conclusions The results of the CHINET Antimicrobial Resistance Surveillance Program indicate that the proportion of Enterobacter strains in all clinical isolates fluctuates slightly over the 7-year period from 2015 to 2021.The Enterobacter strains showed increasing resistance to multiple antimicrobial drugs,especially carbapenems over the 7-year period.

Objective To monitor the antifungal resistance of clinical isolates of Candida spp.in the East China region.Methods MALDI-TOF MS or molecular methods were used to re-identify the strains collected from January 2018 to December 2022.Antifungal susceptibility testing was performed using the broth microdilution method.The susceptibility test results were interpreted according to the breakpoints of 2022 Clinical and Laboratory Standards Institute(CLSI)documents M27 M44s-Ed3 and M57s-Ed4.Results A total of 3 026 strains of Candida were collected,65.33％of which were isolated from sterile body sites,mainly from blood(38.86％)and pleural effusion/ascites(10.21％).The predominant species of Candida were Candida albicans(44.51％),followed by Candida parapsilosis complex(19.46％),Candida tropicalis(13.98％),Candida glabrata(10.34％),and other Candida species(0.79％).Candida albicans showed overall high susceptibility rates to the 10 antifungal drugs tested(the lowest rate being 93.62％).Only 2.97％of the strains showed dose-dependent susceptibility(SDD)to fluconazole.Candida parapsilosis complex had a SDD rate of 2.61％and a resistance rate of 9.42％to fluconazole,and susceptibility rates above 90％to other drugs.Candida glabrata had a SDD rate of 92.01％and a resistance rate of 7.99％to fluconazole,resistance rates of 32.27％and 48.24％to posaconazole and voriconazole non-wild-type strains(NWT),respectively,and susceptibility rates above 90％to other drugs.Candida tropicalis had resistance rates of 29.55％and 26.24％to fluconazole and voriconazole,respectively,resistance rates of 76.60％and 21.99％to posaconazole and echinocandins non-wild-type strains(NWT),and a resistance rate of 2.36％to echinocandins.Conclusions The prevalence and species distribution of Candida spp.in the East China region are consistent with previous domestic and international reports.Candida glabrata exhibits certain degree of resistance to fluconazole,while Candida tropicalis demonstrates higher resistance to triazole drugs.Additionally,echinocandins resistance has emerged in Candida albicans,Candida glabrata,Candida tropicalis,and Candida parapsilosis.