Objective To investigate the role and mechanism of the zinc finger protein Kruppel-like transcription factor 9 (KLF9) in the stimulation of type I interferon expression induced by herpes simplex virus type 1 (HSV-1) in macrophages. Methods Agarose Gel electrophoresis, quantitative real-time PCR (qRT-PCR) and western blot analyses were employed to detect the KLF9 relative expression in bone marrow-derived macrophages (BMDMs) from Klf9^-/- (gKO) mice and wild-type (WT) mice. RNA-seq analysis was utilized to identify the potential targeted genes upon HSV-1 stimulation in BMDMs. ELISA was used to measure the potent of IFN-β in the supernatant of BMDMs derived from gKO and WT mice after HSV-1 stimulation. qRT-PCR analysis was employed to further confirm the changes of Ifnb1 and interferon-stimulated gene (ISG) such as interferon-induced protein with tetratricopeptide repeats 1 (Ifit1), interferon-stimulated exonuclease gene 20 (Isg20), cholesterol 25-hydroxylase (Ch25h) and 2'-5' oligoadenylate synthetase-like 1 (Oasl1). Western blot was used to detect the expression of phosphorylated interferon regulatory factor-3 (p-IRF3), IRF3, phosphorylated interferon regulatory factor-7 (p-IRF7), IRF7, phosphorylated nuclear factor-kappa B p65 (p-NF-κB p65) and NF-κB p65. CUT-Tag and ChIP-qPCR assay were utilized to confirm the binding region of KLF9 in Ifnb1. Results The KLF9 expression was significantly decreased in BMDMs from gKO mice compared with that from WT mice. The RNA-seq analysis showed that Klf9 deletion in BMDMs resulted in an impaired type I interferon signaling pathway. The qRT-PCR analysis revealed that Klf9 deletion in BMDMs led to a significant decrease of Ifnb1 and ISG such as Ifit1, Ch25h and Oasl1 except Isg20. Moreover, ELISA revealed that Klf9 knockout in BMDMs resulted in a significant decrease of IFN-β secreted from BMDMs. Mechanistically, KLF9 directly binds to the promoter of Ifnb1. Conclusion KLF9 is essential for macrophages to resist HSV-1 infection.
Animals ; Kruppel-Like Transcription Factors/physiology* ; Interferon-beta/metabolism* ; Macrophages/virology* ; Mice ; Herpesvirus 1, Human/physiology* ; Mice, Knockout ; Signal Transduction ; Mice, Inbred C57BL ; Interferon Regulatory Factor-3/genetics* ; Interferon Regulatory Factor-7/genetics* ; Gene Expression Regulation

To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cm-FAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rF Ad V4-Cap/GoAstV-2.After 15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GoAstV-2 infected LMH cells as template.LMH cells were in-fected with 15th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant vi-rus rF AdV4-Cap/Go Ast V-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-in-fection of FAdV-4 and GoAstV-2 in goose.

Objective This study aimed to investigate the antimicrobial resistance profiles of bacterial strains isolated from pediatric intensive care units(PICU)in China for better antimicrobial therapy.Methods Clinical isolates were collected from 17 institutions,including tertiary care children's hospitals and pediatric department of tertiary general hospitals in China from January 1,2020 to December 31,2022.Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or automated systems.Results were interpreted according to the breakpoints released by the Clinical and Laboratory Standards Institute(CLSI)in 2020.Results A total of 10 688 isolates were collected,including gram-positive organisms(39.2％)and gram-negative organisms(60.8％).The top three organisms were S.aureus(13.6％,1 453/10 688),A.baumannii(10.0％,1 067/10 688),and coagulase-negative Staphylococcus(9.9％,1 058/10 688).Multi-drug resistant organisms(MDROs)were very common in children.The prevalence of methicillin-resistant Staphylococcus aureus(MRSA),carbapenem-resistant Enterobacterales(CRE),carbapenem-resistant E.coli,carbapenem-resistant K.pneumoniae(CRKP),carbapenem-resistant A.baumannii(CRAB),and carbapenem-resistant P.aeruginosa(CRPA)was 41.1％,19.4％,8.8％,30.9％,67.4％,and 28.8％,respectively.Overall,more than 50％of Enterobacteriales isolates were resistant to cephalosporins,while nearly 25％of Enterobacteriales isolates were resistant to carbapenems.MDROs were highly resistant to commonly used antibiotics.More than 80％of CRE and CRAB strains were resistant to all beta-lactam antibiotics.CRE and CRAB showed low resistance rates to tigecycline and polymyxin.CRPA showed lower resistance rates to piperacillin,beta-lactamase inhibitor combinations than the resistance rates to third and fourth generation cephalosporins.All of the Staphylococcus and Enterococcus isolates were susceptible to vancomycin and tigecycline.None of PRSP strains isolated from meningitis and nonmeningitis samples were resistant to rifampicin,vancomycin,or linezolid.The prevalence of β-lactamase-negative ampicillin-resistant(BLNAR)strains was 43.3％in Haemophilus influenzae.Conclusions MDROs were prevalent in PICU.It is necessary to establish an effective multidisciplinary team(MDT)to control the antimicrobial resistance.

Objective:To explore the clinical effect of vitamin A in the adjuvant treatment of bronchial asthma in children and its influence on serum transforming growth factor-β1 (TGF-β1), eosinophils (EOS) and interleukin-17 (IL-17) levels.Methods:A prospective study was conducted on 110 children with bronchial asthma who received treatment in Department of Pediatrics, Xi'an Central Hospital from January 2022 to December 2023. Based on the principle of balanced and comparable baseline characteristics between groups, they were randomly divided into a control group and an observation group, with 55 cases in each group, using a random number table method. The control group was treated with routine pediatric bronchial asthma therapy, while the observation group was added with vitamin A adjuvant therapy on the basis of the control group. After 15 days of continuous treatment, the scores of asthma control condition (Childhood-Asthma Control Test (C-ACT), Asthma Control Questionnaire (ACQ)) in the two groups were evaluated. The pulmonary ventilation function (forced expiratory volume in one second (FEV 1), forced expiratory volume in one second/forced vital capacity (FEV 1/FVC), peak expiratory flow (PEF)), serum inflammatory factors (TGF-β1, EOS, IL-17) and immune function indicators (T helper 17 cell (Th17), T helper 2 cell (Th2), regulatory T cell (Treg) ) were compared between groups of children before treatment and after 15 days of treatment. Measurement data with normal or approximate distribution were expressed as xˉ± s, and independent sample t test was used for comparison between groups. Enumeration data were expressed as percentage, and chi-square test was adopted for between-group comparison. Results:After 15 days of treatment, the C-ACT score with (16.20±3.14) points in observation group was higher than (14.80±2.62) points in control group while the ACQ score with (30.30±4.14) points was lower than (34.60±6.23) points in control group, with statistical differences between groups (t values were 2.54 and 4.26; P values were 0.012 and <0.001). The pulmonary ventilation function indicators in observation group and control group after 15 days of treatment (FEV 1: (1.76±0.34) L与(1.54±0.32) L, FEV 1/FVC:(76.89±5.76)%与(70.25±6.42)%, PEF(2.89±0.35) L/s与(2.68±0.39) L/s) were higher than those before treatment (FEV 1:(1.12±0.31) L与(1.20±0.33) L, FEV 1/FVC:(56.96±4.35)%与(58.12±3.48)%, PEF(2.15±0.66) L/s与(2.34±0.56) L/s), and the differences were statistically significant ( t values were 10.32, 5.49, 20.48, 10.43, 7.35, 3.70, respectively; all P<0.001), and the indicators in observation group were higher compared to control group, the differences were statistically significant ( t values were 3.49, 5.71, and 2.97; P values were 0.001, <0.001, and 0.004). After 15 days of treatment, the levels of serum inflammatory factors (TGF-β1:(6.32±1.36) ng/L与(8.75±1.81) ng/L, EOS:(3.56±0.65)%与(4.28±0.82)%, IL-17:(5.53±1.22) ng/L与(6.42±1.51) ng/L) and CD4 + T lymphocyte immune function indicators (Th17:(0.97±0.26) ng/L与(1.23±0.35) ng/L, Th2:(2.32±0.64) ng/L与(3.15±0.52) ng/L, Treg:(5.41±0.76) ng/L与(5.86±0.23) ng/L ) were lower in observation group and control group than those before treatment (TGF-β1: (14.35±2.23)与(15.26±3.05) ng/L, EOS: (6.32±1.33)%与(6.41±1.27)%, IL-17:(8.86±1.68)与(9.03±1.89) ng/L, Th17:(1.82±0.75)与(1.67±0.68) ng/L, Th2:(4.15±1.49)与(3.98±1.28) ng/L, Treg: (7.26±1.35)与(6.92±1.72) ng/L), and the differences were statistically significant ( t values were 22.80, 13.61, 13.83, 10.45, 11.90, 8.08, 7.94, 4.27, 8.37, 4.46, 8.86, 4.58, respectively; all P<0.001). However, the above indicators in the observation group were lower than those in the control group ( t values were 7.96, 5.10, 3.40, 4.42, 7.47, 4.20, and P-values were <0.001, <0.001, 0.001, <0.001, <0.001, and <0.001 respectively). Conclusion:Vitamin A adjuvant therapy is helpful to the control of bronchial asthma, and it can effectively improve the pulmonary function, reduce the inflammatory reaction and enhance the body's immunity.

Objective To study mine and identify the key genes on the biosynthetic pathway of indigo and indirubin in Baphicacanthus cusia(Nees)Bremek.(B.cusia).Methods A weighted co-expression network analysis of transcriptome and metabolome data was conducted to screen out candidate genes.Selected genes were further cloned by homologous recombination in Escherichia coli and Saccharomyces cerevisiae to confirm their function.The LC-MS analysis was used to test the metabolic products.Results The enzyme encoded by gene FMO-EVM0009245 recombined in Escherichia coli could oxidize indole to indigo and indirubin,While the enzyme encoded by gene CYP-EVM0022856 and CYP-EVM0028891 recombined in Saccharomyces cerevisiae could oxidize indole to indigo.Conclusion This article lays a foundation for further elucidating the molecular mechanism of indigo and indirubin biosynthesis and provided research basis for increasing the content of medicinal components of B.cusia.

Liver diseases cannot be easily detected in the early stage， and although invasive diagnostic methods， such as liver biopsy， are relatively accurate， they tend to have a low degree of acceptance， which greatly limits the improvement in diagnosis and treatment techniques for liver diseases. Therefore， it is of great importance to search for new biomarkers and therapeutic targets. As an emerging biomarker for liquid biopsy， tRNA-derived small RNA （tsRNA） is abnormally expressed in various liver diseases including viral hepatitis， fatty liver disease， liver injury， and liver cancer， and it can affect the development and progression of liver diseases by regulating the biological functions such as gene expression， epigenetic regulation， and protein translation. This article reviews the origin， classification， and biological function of tsRNA， as well as the research advances in tsRNA as biomarkers and potential therapeutic targets for liver diseases， so as to provide ideas for the early diagnosis and treatment of liver diseases.

Objective To investigate the effect of Modified Renshen Wumei Decoction on the TLR4/MyD88/pNF-κBp65 signaling pathway and elucidate the potential mechanism by which this formula repairs the intestinal mucosal barrier in diarrheal rats.Methods Twelve rats were randomly selected from a total of 48 rats to serve as the blank control group(CK),while the remaining 36 rats were used to establish a disease model via a compound method.After 14 days of model preparation,the rats were randomly divided into three groups:the model group(MC),the western medicine group(MV),and the traditional Chinese medicine group(MRWD).Each of the four groups(including CK)received corresponding interventions for 7 days.The concentrations of serum diamine oxidase(DAO),D-lactic acid(D-Lac),interleukin-1β(IL-1β),IL-6,IL-10,tumor necrosis factor-α(TNF-α),mucin 2(MUC2),MUC4,MUC6,and colonic homogenate secretory immunoglobulin A(SIgA)were measured using ELISA.Additionally,the protein and gene expressions of colonic toll-like receptor 4(TLR4),myeloid differentiation primary response 88(MyD88),phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells p65(pNF-κBp65),occludin,claudin-1,and zonula occludens-1(ZO-1)were analyzed by Western blot and RT-PCR.Results(1)Intestinal mucosal injury markers:Compared with the blank group,the serum levels of DAO and D-Lac in the model group were significantly increased(P<0.05).Compared with the model group,both the Chinese medicine group and Western medicine group significantly decreased the serum levels of DAO(P<0.001),while the traditional Chinese medicine group also significantly reduced the serum levels of D-Lac(P<0.05).There was no significant difference in the changes of DAO and D-Lac serum levels between the Chinese medicine group and Western medicine group(P>0.05).(2)Inflammatory indicators:Compared with the blank group,the model group exhibited significant upregulation of TLR4,MyD88,pNF-κ Bp65 protein and gene expression,as well as serum levels of IL-1β,IL-6,and TNF-α(P<0.05),along with a significant decrease in IL-10 serum levels(P<0.05).Compared with the model group,both the Chinese medicine group and Western medicine group significantly downregulated TLR4,MyD88,pNF-κBp65 protein and gene expression,as well as serum levels of IL-1β,IL-6,and TNF-α(P<0.05),and significantly upregulated IL-10 serum levels(P<0.05).There was no significant difference in serum levels of TLR4,MyD88,pNF-κBp65 protein,gene expression,and IL-1β,IL-6,IL-10,and TNF-α between the Chinese medicine group and Western medicine group(P>0.05).(3)Intestinal mucosal barrier factors:Compared with the blank group,the model group exhibited significant downregulation in MUC2,MUC6,SIgA content,as well as Claudin-1,ZO-1 protein and gene expression,and Occludin protein expression(P<0.05).Compared with the model group,both Chinese and Western medicine groups significantly upregulated the content of MUC2 and SIgA,as well as the protein and gene expression of Claudin-1 and ZO-1(P<0.05).The traditional Chinese medicine group also significantly increased the content of MUC6 and Occludin protein expression(P<0.05).No significant differ-ences were observed between the Chinese and Western medicine groups in terms of MUC2,MUC6,SIgA serum content,and Claudin-1 and ZO-1 gene expression(P>0.05).However,the Western medicine group showed better Claudin-1 protein expression than the Chinese medicine group(P<0.05),while the ZO-1 protein expression was higher in the traditional Chinese medicine group compared to the Western medicine group(P<0.05).Conclusion Modified Renshen Wumei Decoction exerts an intestinal mucosal barrier repair effect in diarrhea rats by modulating the TLR4/MyD88/pNF-κBp65 signaling pathway.

Objective To study mine and identify the key genes on the biosynthetic pathway of indigo and indirubin in Baphicacanthus cusia(Nees)Bremek.(B.cusia).Methods A weighted co-expression network analysis of transcriptome and metabolome data was conducted to screen out candidate genes.Selected genes were further cloned by homologous recombination in Escherichia coli and Saccharomyces cerevisiae to confirm their function.The LC-MS analysis was used to test the metabolic products.Results The enzyme encoded by gene FMO-EVM0009245 recombined in Escherichia coli could oxidize indole to indigo and indirubin,While the enzyme encoded by gene CYP-EVM0022856 and CYP-EVM0028891 recombined in Saccharomyces cerevisiae could oxidize indole to indigo.Conclusion This article lays a foundation for further elucidating the molecular mechanism of indigo and indirubin biosynthesis and provided research basis for increasing the content of medicinal components of B.cusia.

To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cm-FAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rF Ad V4-Cap/GoAstV-2.After 15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GoAstV-2 infected LMH cells as template.LMH cells were in-fected with 15th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant vi-rus rF AdV4-Cap/Go Ast V-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-in-fection of FAdV-4 and GoAstV-2 in goose.

Objective This study aimed to investigate the antimicrobial resistance profiles of bacterial strains isolated from pediatric intensive care units(PICU)in China for better antimicrobial therapy.Methods Clinical isolates were collected from 17 institutions,including tertiary care children's hospitals and pediatric department of tertiary general hospitals in China from January 1,2020 to December 31,2022.Antimicrobial susceptibility testing was carried out according to a unified protocol using Kirby-Bauer method or automated systems.Results were interpreted according to the breakpoints released by the Clinical and Laboratory Standards Institute(CLSI)in 2020.Results A total of 10 688 isolates were collected,including gram-positive organisms(39.2％)and gram-negative organisms(60.8％).The top three organisms were S.aureus(13.6％,1 453/10 688),A.baumannii(10.0％,1 067/10 688),and coagulase-negative Staphylococcus(9.9％,1 058/10 688).Multi-drug resistant organisms(MDROs)were very common in children.The prevalence of methicillin-resistant Staphylococcus aureus(MRSA),carbapenem-resistant Enterobacterales(CRE),carbapenem-resistant E.coli,carbapenem-resistant K.pneumoniae(CRKP),carbapenem-resistant A.baumannii(CRAB),and carbapenem-resistant P.aeruginosa(CRPA)was 41.1％,19.4％,8.8％,30.9％,67.4％,and 28.8％,respectively.Overall,more than 50％of Enterobacteriales isolates were resistant to cephalosporins,while nearly 25％of Enterobacteriales isolates were resistant to carbapenems.MDROs were highly resistant to commonly used antibiotics.More than 80％of CRE and CRAB strains were resistant to all beta-lactam antibiotics.CRE and CRAB showed low resistance rates to tigecycline and polymyxin.CRPA showed lower resistance rates to piperacillin,beta-lactamase inhibitor combinations than the resistance rates to third and fourth generation cephalosporins.All of the Staphylococcus and Enterococcus isolates were susceptible to vancomycin and tigecycline.None of PRSP strains isolated from meningitis and nonmeningitis samples were resistant to rifampicin,vancomycin,or linezolid.The prevalence of β-lactamase-negative ampicillin-resistant(BLNAR)strains was 43.3％in Haemophilus influenzae.Conclusions MDROs were prevalent in PICU.It is necessary to establish an effective multidisciplinary team(MDT)to control the antimicrobial resistance.