OBJECTIVE To analyze the etiological and clinical characteristics of the neonates with early-onset and late-onset purulent meningitis.METHODS A total of 140 neonates who were diagnosed with purulent meningitis and were hospitalized in neonatal intensive care unit(NICU)of Xuzhou Children's Hospital Affiliated to Xuzhou Medical University from Jan.2018 to Dec.2022 were recruited as the research subjects and were divided into the early-onset group with 32 cases and the late-onset group with 108 cases according to the onset time.The clinical data were collected from the enrolled neonates and were retrospectively analyzed.The etiological and clinical char-acteristics of the neonates with purulent meningitis were observed.RESULTS Totally 140 neonates with purulent meningitis were included in the study,the total morbidity rate was 1.03％(140/13644),and it was more common among the male neonates and full-term neonates,dominated by late-onset cases.The incidence rates of perinatal infection,premature rupture of fetal membranes,lethargy/irritability,cyanosis,abnormal muscle tone and septic shock were higher in the early-onset group than in the late-onset group(P＜0.05).There were significant differ-ences in serum PCT,cerebrospinal fluid cell counts,percentage of neutrophils and proteins between the early-on-set group and the late-onset group(P＜0.05).The positive rate of etiological culture of the early-onset group was 43.75％(14/32),higher than 20.37％(22/108)of the late-onset group(P＜0.05);Escherichia coli was the pre-dominant species of pathogen isolated from the two groups.The total proportion of the neonates with abnormal imaging findings was 22.85％among the two groups of neonates;the intracranial hemorrhage,subdural effusion and ventricular enlargement/hydrocephalus were the major abnormal types;the proportion of the neonates with abnormal imaging findings of the early-onset group was 43.75％,higher than 16.67％of the late-onset group(P＜0.05).CONCLUSIONS The neonates with late-onset purulent meningitis are dominant among the neonates with purulent meningitis enrolled in the study.The neonates with early-onset purulent meningitis present with more se-vere clinical manifestations and higher proportion of abnormal imaging findings;there is significant difference in the clinical characteristics between the early-onset neonates and the late-onset neonates.The result of etiological culture shows that E.coli is the predominant species of pathogen among both the early-onset neonates and the late-onset children.

The structure and potential antigenic epitopes of FliCEcN were analysed using bioinformat-ics technology.With the help of ClonExpress? homologous recombination technology,primers were designed to deletion of different structural domains in the highly variable region of FliCEcN and cloned into pET-28a(+)expression vector for expression.The expressed flagellin variants were identified by SDS-PAGE and Western blot.Endotoxin residues in the flagellin variants were detected by horseshoe crab reagent chromatography,and Caco-2 cells were stimulated with differ-ent concentrations of flagellin variants.The biological activity of each flagellin variant was assessed by detecting the secretion level of IL-8.Bioinformatic analysis showed that most of the structural domains in the highly variable region of FliCEcN were predicted to contain potential antigenic epitopes.PC R results showed that fliC△820-1 518,fliC△736-963,fliC△985-1 200,fliC △748-828,fliC△1 114-1 191,andfliC△1 225-1311 were approximately 1 095,1 566,1 578,1 713,1 716 and 1 707 bp,respectively.SDS-PAGE results showed that the sizes of the flagellin variants treated with nickel column purifi-cation and dialysis replication were about 41.36,57.06,57.50,61.97,61.95 and 61.56 kDa,respec-tively.Western blot results showed that all six flagellin variants reacted specifically with anti-His monoclonal antibody and E.coli H7 antigen diagnostic serum.The results of TLR5 activity assay showed that the flagellin variants missing different structural domains retained their TLR5 agonist function.In this study,six flagellin variants with different structural domains of FliCEcN deletion hypervariable region were successfully constructed,and all of them retained their TLR5 agonist function and showed good biological properties.This provides a reference for further research on the adjuvant effect of flagellin after deletion of different structural domains and the effect of flagel-lin antibody titer on its adjuvant effect.

Objective To establish a UV method for the determination of the functional compositions(cinnamic acid derivatives,total flavonoids)in Ligustrum robustum,to predict the contents of trans-p-hydroxycinnamic acid,trans-p-hydroxycinnamic acid esters,and rhoifolin based on UV/HPLC,and to optimize the technological conditions of ultrasonic-assistant extraction of L.robustum.Methods ①In the determination of the functional compositions in L.robustum,total cinnamic acid derivatives were determined by dual-wavelength isobestic point UV method(detection wavelength 316 nm,reference wavelength 268 nm),and total flavonoids were determined by single-wavelength UV method(detection wavelength 268 nm),while rhoifolin was determined by HPLC(C 18 column,eluting with methanol-0.1％acetic acid=40∶60,detection wavelength 310 nm).②The conversion factors were used to establish a UV/HPLC-based prediction model,and the precision of the predicted results was evaluated with other 3 test samples.③ The technological conditions of ultrasonic-assisted extraction were optimized by an orthogonal test.Results ① The linear ranges of total cinnamic acid derivatives,total flavonoids,and rhoifolin were 4.64-20.88,8.24-28.84,5.15-51.50 μg·mL-1(r≥0.999 5),respectively.RSDs of the precision,stability,and reproducibility tests were no more than 1.4％.The recoveries were 97.9％-100.5％(RSD≤ 1.2％,n=6).②The relative errors of the values predicted by UV/HPLC from the results measured by HPLC were from-5.3％to 1.7％.There was no significant difference between the UV/HPLC predicted values and the HPLC determined results(P＞0.05)for the contents of trans-p-hydroxycinnamic acid and rhoifolin.③The optimal technological conditions of ultrasonic-assistant extraction of L.robustum were as follows:ethanol concentration 80％,temperature 50 ℃,liquid-solid ratio 20 mL·g 1,extraction frequency 2 times,extraction time 20 min.The total yield of functional compositions under above conditions was 42.0％.Conclusion The above determination methods are accurate and rapid,and the above extraction technology is effective,energy-saving and rapid,which provide an experimental base for the quality control and application of L.robustum.

The 16S rRNA sequencing,whole genome sequencing and drug sensitivity tests were used to identify the isolates molecularly and to detect and analyse their virulence genes,resistance genes and drug resistance.The results showed that the isolate was highly homologous to Klebsiella pneumoniae X4 and located on the same branch by 16S rRNA sequence analysis,and it was named as KLKp10.Whole genome sequencing results showed that the KLKp10 genome was 5 342 841 bp in length,containing 5 138 genes,346 repetitive segments,6 rRNAs and 81 tRNAs,with a GC con-tent of 57.30％.MLST analysis showed that KLKp10 belongs to the ST-4263 type.The functions of 4 097 of the genes encoding proteins were classified and annotated by COG,and there were also 382 genes with unknown functions.A total of 50 functional classifications were involved in the an-notation results based on the GO database;33 kinds of signaling pathways were covered based on the signaling pathway annotations in the KEGG database.A total of 443 virulence genes were screened in the VFDB database,of which 339 belonged to the Set A database and could encode 124 virulence factors.The 101 resistance genes were predicted by comparing with the CARD database,among which there were more resistance genes against β-lactam antibiotics.The results of drug sensitivity test showed that KLKp10 was highly sensitive to ceftazidime,gentamicin,azithro-mycin,chloramphenicol,norfloxacin,ofloxacin,and enrofloxacin;moderately sensitive to ceftriax-one,neomycin,kanamycin,and streptomycin;and resistant to ciprofloxacin,tetracycline,amoxicil-lin,and penicillin.In this study,we systematically revealed the gene-wide characterization,virulence factors and drug resistance of Klebsiella pneumoniae KLKp10 of Kole pig origin,which provides important data support for the study of Klebsiella pneumoniae at the overall level of its genome.

Objective:To explore the regulation and mechanism of human placental extract(HPE)on lipopolysaccharide(LPS)-induced BV2 microglial cell inflammation.Methods:Microglia cell lines(BV2)were cultured in vitro and divided into PBS group,HPE group,LPS group and LPS+HPE group.BV2 cell viability was measured by CCK-8 analysis.A fluorescent probe targeting reactive oxygen species(ROS)was to detect the level of intracellular ROS.The mRNA levels of TNF-α,IL-1β and IL-6 were detected by RT-qPCR.The supernatants of different treatment groups were collected.The content of nitric oxide(NO)was detected by the Griess method,and the secretion levels of TNF-α,IL-1β and IL-6 were detected by the flow magnetic bead microarray(CBA)method.The indirect contact co-culture system between BV2 and mouse hippocampal neurons cell line HT22 cells was established to evaluate the neurotoxicity of HPE by the assessment of the cell viability and apoptosis of HT22 cells using CCK-8 or flow cytometry.The potential signaling molecules of NF-κB signaling pathway was detected by flow cytometry.Results:Compared with the PBS group,1.2 μg/ml LPS and 50 ng/ml HPE significantly inhibited the activity of BV2 cells.Compared with the PBS group,the mRNA levels of TNF-α,IL-1β and IL-6 in BV2 cells of the LPS group were significantly increased(P<0.05),and the secretion levels of NO,TNF-α,IL-1βand IL-6 in the supernatant were also significantly upregulated(P<0.05).The expressions of related signaling pathway molecules Toll-like receptor 4(TLR4),pIκBα and pNF-κB p65 were significantly increased(P<0.05).Compared with the LPS group,the mRNA levels of TNF-α,IL-1β and IL-6 in BV2 cells,the secretion levels of NO,TNF-α,IL-1β and IL-6,the neurotoxicity and neuronal apoptosis induced by microglial conditional medium,and the expressions of TLR4,pIκBα and pNF-κB p65 in the LPS+HPE group were significantly downregulate(P<0.05).Conclusion:HPE may alleviate the microglial inflammation,possibly through the TLR4/NF-κB signaling pathway.

The 16S rRNA sequencing,whole genome sequencing and drug sensitivity tests were used to identify the isolates molecularly and to detect and analyse their virulence genes,resistance genes and drug resistance.The results showed that the isolate was highly homologous to Klebsiella pneumoniae X4 and located on the same branch by 16S rRNA sequence analysis,and it was named as KLKp10.Whole genome sequencing results showed that the KLKp10 genome was 5 342 841 bp in length,containing 5 138 genes,346 repetitive segments,6 rRNAs and 81 tRNAs,with a GC con-tent of 57.30％.MLST analysis showed that KLKp10 belongs to the ST-4263 type.The functions of 4 097 of the genes encoding proteins were classified and annotated by COG,and there were also 382 genes with unknown functions.A total of 50 functional classifications were involved in the an-notation results based on the GO database;33 kinds of signaling pathways were covered based on the signaling pathway annotations in the KEGG database.A total of 443 virulence genes were screened in the VFDB database,of which 339 belonged to the Set A database and could encode 124 virulence factors.The 101 resistance genes were predicted by comparing with the CARD database,among which there were more resistance genes against β-lactam antibiotics.The results of drug sensitivity test showed that KLKp10 was highly sensitive to ceftazidime,gentamicin,azithro-mycin,chloramphenicol,norfloxacin,ofloxacin,and enrofloxacin;moderately sensitive to ceftriax-one,neomycin,kanamycin,and streptomycin;and resistant to ciprofloxacin,tetracycline,amoxicil-lin,and penicillin.In this study,we systematically revealed the gene-wide characterization,virulence factors and drug resistance of Klebsiella pneumoniae KLKp10 of Kole pig origin,which provides important data support for the study of Klebsiella pneumoniae at the overall level of its genome.

Objective:To explore the regulation and mechanism of human placental extract(HPE)on lipopolysaccharide(LPS)-induced BV2 microglial cell inflammation.Methods:Microglia cell lines(BV2)were cultured in vitro and divided into PBS group,HPE group,LPS group and LPS+HPE group.BV2 cell viability was measured by CCK-8 analysis.A fluorescent probe targeting reactive oxygen species(ROS)was to detect the level of intracellular ROS.The mRNA levels of TNF-α,IL-1β and IL-6 were detected by RT-qPCR.The supernatants of different treatment groups were collected.The content of nitric oxide(NO)was detected by the Griess method,and the secretion levels of TNF-α,IL-1β and IL-6 were detected by the flow magnetic bead microarray(CBA)method.The indirect contact co-culture system between BV2 and mouse hippocampal neurons cell line HT22 cells was established to evaluate the neurotoxicity of HPE by the assessment of the cell viability and apoptosis of HT22 cells using CCK-8 or flow cytometry.The potential signaling molecules of NF-κB signaling pathway was detected by flow cytometry.Results:Compared with the PBS group,1.2 μg/ml LPS and 50 ng/ml HPE significantly inhibited the activity of BV2 cells.Compared with the PBS group,the mRNA levels of TNF-α,IL-1β and IL-6 in BV2 cells of the LPS group were significantly increased(P<0.05),and the secretion levels of NO,TNF-α,IL-1βand IL-6 in the supernatant were also significantly upregulated(P<0.05).The expressions of related signaling pathway molecules Toll-like receptor 4(TLR4),pIκBα and pNF-κB p65 were significantly increased(P<0.05).Compared with the LPS group,the mRNA levels of TNF-α,IL-1β and IL-6 in BV2 cells,the secretion levels of NO,TNF-α,IL-1β and IL-6,the neurotoxicity and neuronal apoptosis induced by microglial conditional medium,and the expressions of TLR4,pIκBα and pNF-κB p65 in the LPS+HPE group were significantly downregulate(P<0.05).Conclusion:HPE may alleviate the microglial inflammation,possibly through the TLR4/NF-κB signaling pathway.

The structure and potential antigenic epitopes of FliCEcN were analysed using bioinformat-ics technology.With the help of ClonExpress? homologous recombination technology,primers were designed to deletion of different structural domains in the highly variable region of FliCEcN and cloned into pET-28a(+)expression vector for expression.The expressed flagellin variants were identified by SDS-PAGE and Western blot.Endotoxin residues in the flagellin variants were detected by horseshoe crab reagent chromatography,and Caco-2 cells were stimulated with differ-ent concentrations of flagellin variants.The biological activity of each flagellin variant was assessed by detecting the secretion level of IL-8.Bioinformatic analysis showed that most of the structural domains in the highly variable region of FliCEcN were predicted to contain potential antigenic epitopes.PC R results showed that fliC△820-1 518,fliC△736-963,fliC△985-1 200,fliC △748-828,fliC△1 114-1 191,andfliC△1 225-1311 were approximately 1 095,1 566,1 578,1 713,1 716 and 1 707 bp,respectively.SDS-PAGE results showed that the sizes of the flagellin variants treated with nickel column purifi-cation and dialysis replication were about 41.36,57.06,57.50,61.97,61.95 and 61.56 kDa,respec-tively.Western blot results showed that all six flagellin variants reacted specifically with anti-His monoclonal antibody and E.coli H7 antigen diagnostic serum.The results of TLR5 activity assay showed that the flagellin variants missing different structural domains retained their TLR5 agonist function.In this study,six flagellin variants with different structural domains of FliCEcN deletion hypervariable region were successfully constructed,and all of them retained their TLR5 agonist function and showed good biological properties.This provides a reference for further research on the adjuvant effect of flagellin after deletion of different structural domains and the effect of flagel-lin antibody titer on its adjuvant effect.

OBJECTIVE To analyze the etiological and clinical characteristics of the neonates with early-onset and late-onset purulent meningitis.METHODS A total of 140 neonates who were diagnosed with purulent meningitis and were hospitalized in neonatal intensive care unit(NICU)of Xuzhou Children's Hospital Affiliated to Xuzhou Medical University from Jan.2018 to Dec.2022 were recruited as the research subjects and were divided into the early-onset group with 32 cases and the late-onset group with 108 cases according to the onset time.The clinical data were collected from the enrolled neonates and were retrospectively analyzed.The etiological and clinical char-acteristics of the neonates with purulent meningitis were observed.RESULTS Totally 140 neonates with purulent meningitis were included in the study,the total morbidity rate was 1.03％(140/13644),and it was more common among the male neonates and full-term neonates,dominated by late-onset cases.The incidence rates of perinatal infection,premature rupture of fetal membranes,lethargy/irritability,cyanosis,abnormal muscle tone and septic shock were higher in the early-onset group than in the late-onset group(P＜0.05).There were significant differ-ences in serum PCT,cerebrospinal fluid cell counts,percentage of neutrophils and proteins between the early-on-set group and the late-onset group(P＜0.05).The positive rate of etiological culture of the early-onset group was 43.75％(14/32),higher than 20.37％(22/108)of the late-onset group(P＜0.05);Escherichia coli was the pre-dominant species of pathogen isolated from the two groups.The total proportion of the neonates with abnormal imaging findings was 22.85％among the two groups of neonates;the intracranial hemorrhage,subdural effusion and ventricular enlargement/hydrocephalus were the major abnormal types;the proportion of the neonates with abnormal imaging findings of the early-onset group was 43.75％,higher than 16.67％of the late-onset group(P＜0.05).CONCLUSIONS The neonates with late-onset purulent meningitis are dominant among the neonates with purulent meningitis enrolled in the study.The neonates with early-onset purulent meningitis present with more se-vere clinical manifestations and higher proportion of abnormal imaging findings;there is significant difference in the clinical characteristics between the early-onset neonates and the late-onset neonates.The result of etiological culture shows that E.coli is the predominant species of pathogen among both the early-onset neonates and the late-onset children.

Objective To establish a UV method for the determination of the functional compositions(cinnamic acid derivatives,total flavonoids)in Ligustrum robustum,to predict the contents of trans-p-hydroxycinnamic acid,trans-p-hydroxycinnamic acid esters,and rhoifolin based on UV/HPLC,and to optimize the technological conditions of ultrasonic-assistant extraction of L.robustum.Methods ①In the determination of the functional compositions in L.robustum,total cinnamic acid derivatives were determined by dual-wavelength isobestic point UV method(detection wavelength 316 nm,reference wavelength 268 nm),and total flavonoids were determined by single-wavelength UV method(detection wavelength 268 nm),while rhoifolin was determined by HPLC(C 18 column,eluting with methanol-0.1％acetic acid=40∶60,detection wavelength 310 nm).②The conversion factors were used to establish a UV/HPLC-based prediction model,and the precision of the predicted results was evaluated with other 3 test samples.③ The technological conditions of ultrasonic-assisted extraction were optimized by an orthogonal test.Results ① The linear ranges of total cinnamic acid derivatives,total flavonoids,and rhoifolin were 4.64-20.88,8.24-28.84,5.15-51.50 μg·mL-1(r≥0.999 5),respectively.RSDs of the precision,stability,and reproducibility tests were no more than 1.4％.The recoveries were 97.9％-100.5％(RSD≤ 1.2％,n=6).②The relative errors of the values predicted by UV/HPLC from the results measured by HPLC were from-5.3％to 1.7％.There was no significant difference between the UV/HPLC predicted values and the HPLC determined results(P＞0.05)for the contents of trans-p-hydroxycinnamic acid and rhoifolin.③The optimal technological conditions of ultrasonic-assistant extraction of L.robustum were as follows:ethanol concentration 80％,temperature 50 ℃,liquid-solid ratio 20 mL·g 1,extraction frequency 2 times,extraction time 20 min.The total yield of functional compositions under above conditions was 42.0％.Conclusion The above determination methods are accurate and rapid,and the above extraction technology is effective,energy-saving and rapid,which provide an experimental base for the quality control and application of L.robustum.