Increasing evidence has revealed an unexpected localization of mature microRNA(miRNA)in the nucleus,exerting additional and broader functions.These findings differ from the traditional view that miRNAs in the cyto-plasm inhibit gene expression through their canonical regulation via post-transcriptional mechanisms.Metabolic diseases involve multiple organs throughout the body and rely highly on miRNA regulation.Notably,recent studies have demon-strated the newly discovered nuclear function of miRNA in regulating metabolic dysfunction-associated pathological pro-cesses.In this review,according to the classification of metabolic diseases,we summarize the current knowledge on nuclear miRNA non-canonical regulation including their working modes and potential targets,such as directly binding on gene pro-moters,noncoding RNA,or proteins,etc.,to suggest a viable strategy for the treatment of metabolic diseases.

Objective To identify genetic regulators of ionizing radiation(IR)sensitivity through clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)genome-wide screening.Methods A specialized single guide RNA(sgRNA)library was developed targeting 987 stress-response regulatory genes identified through Kyoto Encyclopedia of Genes and Genomes(KEGG),Reactome and gene ontology(GO)databases,followed by construction of sgRNA plasmids and packaging into a lentiviral library.Using colon adenocarcinoma Caco-2 cells as a model system,the Cas9-stable transgenic line was established via lentiviral transduction and antibiotic selection.Library-transduced cells underwent puromycin selection,followed by γ-irradiation(dose optimized via preliminary experiments).Post-irradiation phenotypic selection was conducted after 14 days,with subsequent next-generation sequencing of surviving cell populations to identify enriched/depleted sgRNAs.Candidate genes were functionally validated through orthogonal assays:cell counting kit-8(CCK-8)proliferation analysis,clonogenic survival assays and flow cytometric quantification of reactive oxygen species(ROS)and apoptotic markers.Results The optimized screening platform identified 281 radiation response genes(165 radiosensitive and 116 radioprotective candidates).Functional validation revealed Bcl2-associated athanogene 3(BAG3)knockdown significantly enhanced radioresistance.Proliferation was increased 1.2-1.5 fold,clonogenic survival improved 1.5-2.0 fold,and ROS was reduced by 13％-25％coupled with 32％-73％apoptosis attenuation compared to control groups.Conclusion The integrated CRISPR/Cas9 screening platform effectively identifies novel radiation response regulators,as evidenced by the discovery of BAG3 as a critical radiosensitivity determinant.This high-throughput functional genomics approach provides a robust methodology for systematically mapping molecular determinants of cellular radiation response,with potential applications in both mechanistic studies and therapeutic target discovery.

Objective To explore the role of ocular vestibular evoked myogenic potential (oVEMP) and unilateral centrifugation subjective visual vertical (UC-SVV) tests in evaluating the utricular function of patients with Meniere’s disease (MD) at different clinical stages. Methods A total of 97 unilateral MD patients at Shandong Provincial ENT Hospital from July 2019 to September 2021 were selected. All patients underwent oVEMP, UC-SVV, and pure tone audiometry tests. MD patients were classified into clinical stages 1, 2, 3, and 4, with stages 1 and 2 defined as early stage and stages 3 and 4 as late stage. The results of utricular function tests (abnormal rates of oVEMP, UC-SVV, and oVEMP＋UC-SVV) were compared among patients at different stages. Spearman correlation analysis was used to evaluate the correlation between utricular function and clinical staging. Results Among the 97 MD patients, the abnormal rate of oVEMP was 66.0% (64/97), and the abnormal rate of UC-SVV was 55.7% (54/97). The abnormal rates of oVEMP and oVEMP＋UC-SVV in early-stage patients were significantly lower than those in late-stage patients (P＜0.05), while the difference in UC-SVV abnormal rates between the two groups was not statistically significant. Intra-group comparisons showed that the abnormal rate of oVEMP＋UC-SVV in stage 1 patients was significantly lower than that in stage 2 patients (P＜0.05), without significant difference in the other indices. There were no significant differences among the three indices in stages 3 and 4 patients. Spearman correlation test results indicated that the abnormal rate of oVEMP (r＝0.336, P＝0.001) and the abnormal rate of oVEMP＋UC-SVV (r＝0.301, P＝0.003) were weakly positively correlated with clinical staging, while there was no correlation between the abnormal rate of UC-SVV and clinical staging (r＝0.022, P＝0.832). Conclusions Both oVEMP and UC-SVV tests can assess utricular function in MD patients at different clinical stages. Their combination is helpful of early-stage (stages 1 and 2) MD diagnosis.

Increasing evidence has revealed an unexpected localization of mature microRNA(miRNA)in the nucleus,exerting additional and broader functions.These findings differ from the traditional view that miRNAs in the cyto-plasm inhibit gene expression through their canonical regulation via post-transcriptional mechanisms.Metabolic diseases involve multiple organs throughout the body and rely highly on miRNA regulation.Notably,recent studies have demon-strated the newly discovered nuclear function of miRNA in regulating metabolic dysfunction-associated pathological pro-cesses.In this review,according to the classification of metabolic diseases,we summarize the current knowledge on nuclear miRNA non-canonical regulation including their working modes and potential targets,such as directly binding on gene pro-moters,noncoding RNA,or proteins,etc.,to suggest a viable strategy for the treatment of metabolic diseases.

Objective To monitor the susceptibility of clinical isolates to antimicrobial agents in tertiary hospitals in major regions of China in 2022.Methods Clinical isolates from 58 hospitals in China were tested for antimicrobial susceptibility using a unified protocol based on disc diffusion method or automated testing systems.Results were interpreted using the 2022 Clinical &Laboratory Standards Institute(CLSI)breakpoints.Results A total of 318 013 clinical isolates were collected from January 1,2022 to December 31,2022,of which 29.5％were gram-positive and 70.5％were gram-negative.The prevalence of methicillin-resistant strains in Staphylococcus aureus,Staphylococcus epidermidis and other coagulase-negative Staphylococcus species(excluding Staphylococcus pseudintermedius and Staphylococcus schleiferi)was 28.3％,76.7％and 77.9％,respectively.Overall,94.0％of MRSA strains were susceptible to trimethoprim-sulfamethoxazole and 90.8％of MRSE strains were susceptible to rifampicin.No vancomycin-resistant strains were found.Enterococcus faecalis showed significantly lower resistance rates to most antimicrobial agents tested than Enterococcus faecium.A few vancomycin-resistant strains were identified in both E.faecalis and E.faecium.The prevalence of penicillin-susceptible Streptococcus pneumoniae was 94.2％in the isolates from children and 95.7％in the isolates from adults.The resistance rate to carbapenems was lower than 13.1％in most Enterobacterales species except for Klebsiella,21.7％-23.1％of which were resistant to carbapenems.Most Enterobacterales isolates were highly susceptible to tigecycline,colistin and polymyxin B,with resistance rates ranging from 0.1％to 13.3％.The prevalence of meropenem-resistant strains decreased from 23.5％in 2019 to 18.0％in 2022 in Pseudomonas aeruginosa,and decreased from 79.0％in 2019 to 72.5％in 2022 in Acinetobacter baumannii.Conclusions The resistance of clinical isolates to the commonly used antimicrobial agents is still increasing in tertiary hospitals.However,the prevalence of important carbapenem-resistant organisms such as carbapenem-resistant K.pneumoniae,P.aeruginosa,and A.baumannii showed a downward trend in recent years.This finding suggests that the strategy of combining antimicrobial resistance surveillance with multidisciplinary concerted action works well in curbing the spread of resistant bacteria.

Objective To monitor the susceptibility of clinical isolates to antimicrobial agents in healthcare facilities in major regions of China in 2023.Methods Clinical isolates collected from 73 hospitals across China were tested for antimicrobial susceptibility using a unified protocol based on disc diffusion method or automated testing systems.Results were interpreted using the 2023 Clinical & Laboratory Standards Institute (CLSI) breakpoints.Results A total of 445199 clinical isolates were collected in 2023,of which 29.0％ were gram-positive and 71.0％ were gram-negative.The prevalence of methicillin-resistant strains in Staphylococcus aureus,Staphylococcus epidermidis and other coagulase-negative Staphylococcus species (excluding Staphylococcus pseudintermedius and Staphylococcus schleiferi) (MRSA,MRSE and MRCNS) was 29.6％,81.9％ and 78.5％,respectively.Methicillin-resistant strains showed significantly higher resistance rates to most antimicrobial agents than methicillin-susceptible strains (MSSA,MSSE and MSCNS).Overall,92.9％ of MRSA strains were susceptible to trimethoprim-sulfamethoxazole and 91.4％ of MRSE strains were susceptible to rifampicin.No vancomycin-resistant strains were found.Enterococcus faecalis had significantly lower resistance rates to most antimicrobial agents tested than Enterococcus faecium.A few vancomycin-resistant strains were identified in both E.faecalis and E.faecium.The prevalence of penicillin-susceptible Streptococcus pneumoniae was 93.1％ in the isolates from children and and 95.9％ in the isolates from adults.The resistance rate to carbapenems was lower than 15.0％ for most Enterobacterales species except for Klebsiella,22.5％ and 23.6％ of which were resistant to imipenem and meropenem,respectively .Most Enterobacterales isolates were highly susceptible to tigecycline,colistin and polymyxin B,with resistance rates ranging from 0.6％ to 10.0％.The resistance rate to imipenem and meropenem was 21.9％ and 17.4％ for Pseudomonas aeruginosa,respectively,and 67.5％ and 68.1％ for Acinetobacter baumannii,respectively.Conclusions Increasing resistance to the commonly used antimicrobial agents is still observed in clinical bacterial isolates.However,the prevalence of important crabapenem-resistant organisms such as crabapenem-resistant K.pneumoniae,P.aeruginosa,and A.baumannii showed a slightly decreasing trend.This finding suggests that strengthening bacterial resistance surveillance and multidisciplinary linkage are important for preventing the occurrence and development of bacterial resistance.

Objective To monitor the susceptibility of clinical isolates to antimicrobial agents in healthcare facilities in major regions of China in 2023.Methods Clinical isolates collected from 73 hospitals across China were tested for antimicrobial susceptibility using a unified protocol based on disc diffusion method or automated testing systems.Results were interpreted using the 2023 Clinical & Laboratory Standards Institute (CLSI) breakpoints.Results A total of 445199 clinical isolates were collected in 2023,of which 29.0％ were gram-positive and 71.0％ were gram-negative.The prevalence of methicillin-resistant strains in Staphylococcus aureus,Staphylococcus epidermidis and other coagulase-negative Staphylococcus species (excluding Staphylococcus pseudintermedius and Staphylococcus schleiferi) (MRSA,MRSE and MRCNS) was 29.6％,81.9％ and 78.5％,respectively.Methicillin-resistant strains showed significantly higher resistance rates to most antimicrobial agents than methicillin-susceptible strains (MSSA,MSSE and MSCNS).Overall,92.9％ of MRSA strains were susceptible to trimethoprim-sulfamethoxazole and 91.4％ of MRSE strains were susceptible to rifampicin.No vancomycin-resistant strains were found.Enterococcus faecalis had significantly lower resistance rates to most antimicrobial agents tested than Enterococcus faecium.A few vancomycin-resistant strains were identified in both E.faecalis and E.faecium.The prevalence of penicillin-susceptible Streptococcus pneumoniae was 93.1％ in the isolates from children and and 95.9％ in the isolates from adults.The resistance rate to carbapenems was lower than 15.0％ for most Enterobacterales species except for Klebsiella,22.5％ and 23.6％ of which were resistant to imipenem and meropenem,respectively .Most Enterobacterales isolates were highly susceptible to tigecycline,colistin and polymyxin B,with resistance rates ranging from 0.6％ to 10.0％.The resistance rate to imipenem and meropenem was 21.9％ and 17.4％ for Pseudomonas aeruginosa,respectively,and 67.5％ and 68.1％ for Acinetobacter baumannii,respectively.Conclusions Increasing resistance to the commonly used antimicrobial agents is still observed in clinical bacterial isolates.However,the prevalence of important crabapenem-resistant organisms such as crabapenem-resistant K.pneumoniae,P.aeruginosa,and A.baumannii showed a slightly decreasing trend.This finding suggests that strengthening bacterial resistance surveillance and multidisciplinary linkage are important for preventing the occurrence and development of bacterial resistance.

ObjectiveTo prepare antiserum specific to aminoacy1-3″-adenylyltransferase [ AAD (3″) ],and to explore the application value of the prepared antiserum in detecting the expression levels of aadA2 gene that downstream of 8 different promoters (PcS,PcH2,PcH1,PcW,PcS-P2,PcH2-P2,PcH1P2 and PcW-P2 ) of variable regions in class 1 integron.MethodsaadA2 gene was amplified by polymerase chain reaction(PCR) and cloned into the expression plasmid pET19b.After inducing,the recombined aminoacy1-3″-adenylyltransferase[ AAD(3″)] with His-tag was expressed,purified and immunized rabbits to get anti- AAD(3″) specific serum.The prepared antiserum was used to detect the translation levels of aadA2 gene that downstream of different promoters of variable regions in class 1 integron by Western blotting (WB).Broth microdilution method was used to detect the minimal inhibitory concentrations (MIC) to streptomycin in Escherichia coli JM109 with aadA2 gene downstream of different promoters of variable regions.ResultsRecombined AAD (3″) expression plasmid pET19b-aadA2 was constructed successfully and was verified by sequence analysis.After transformed into E.coli BL21 ( DE3 ),a resoluble recombined AAD(3″) high expression strain was obtained.After fermentation,recombined AAD(3″) was purified and immunized rabbits.The anti- AAD(3″) specific serum was obtained with titer ＞ 1∶100 000.WB was used to detect the expression levels of AAD (3″),the translation product of aadA2 gene,that downstream of 8 different promoters of variable regions.The relative expression level of AAD (3″) that downstream of PcW was assumed to be 1,then the relative expression levels of AAD(3″),which all were detected 3 times independently,that downstream of PcS,PcH2,PcH1,PcS-P2,PcH2-P2,PcH1-P2 and PcW-P2 were 12.9±2.3,9.1±1.0,2.0±0.4,16.0±1.3,14.1 ±1.3,10.5±0.7 and 8.9 ±1.7 respective.Very different expression levels of AAD (3″) that downstream of different promoters of variable regions were obtained( F =32.421,P ＜ 0.01 ).The mean values of MIC,which all were detected 3 times independently,to streptomycin in E.coli JM109 with aadA2 gene downstream of PcS,PcH2,PcH1,PcW,PcS-P2,PcH2P2,PcH1-P2 and PcW-P2 were 256,256,64,128,32,128,4 and 64 mg/L respective.These results indicated the different expression levels of aadA2 gene that downstream of different promoters of variable regions can confer their host bacteria different resistance levels to streptomycin.Conclusions Resoluble recombined AAD(3″) is purified successfully and high titer anti- AAD(3″) specific antiserum is obtained from the immunized rabbits.This laid foundation for further investigation on the correlationship between the expressions of intI1 gene and the gene cassettes within variable regions.The expression levels of antibiotic gene cassettes that downstream of different promoters of variable regions are very different,so are the very different antibiotic resistance levels of the host bacteria.Therefore more attentions should be paid to the researches on the classification of promoters of variable regions when molecular epidemiology studies on the class 1 integrons in clinical isolates were conducted.

ObjectiveTo determine whether aadA2 gene can be translated from the ATG triplet,which there was no plausible ribosome binding site preceding it,and synthetized a functional protein in class 1 integron.MethodsSite-specific mutagenesis was used to construct aadA2 gene cassette with different start codons,together with their upstreamed promoters of variable regions were cloned into plasmid pACYC184 respective.The constructed plasmids were then transfored into Escherichia coli JM109,Western blot was used to detect the translation products of aadA2 gene with different start codons.Broth microdilution method was used to detect the minimal inhibitory concentrations to streptomycin in Escherichia coli JM109 containing aadA2 gene with different start codons.ResultsaadA2 gene can initiate translation from both ATG and GTG triplets in aminoacyl -3-adenylyltransferase protein synthesis,though there was no plausible ribosome binding site preceding the ATG triplet.Besides GTG and ATG triplets,there was other start codon downstream of the GTG triplet in aadA2 gene.The translated products that initiated from the start codons that described above were all functional AAD(3) proteins that can be detected by anti- aminoacyl -3-adenylyltransferase polyclonal antisera in Western blot and conferred different resistance levels to streptomycin in E.coli.ConclusionWhen inserted as the first gene cassette in class 1 integron,aadA2 gene can initiate translation from ATG triplet and synthetized a functional protein,though there was no plausible ribosome binding site preceding it.This structural characterization of class 1 integron can initiate translation of the open reading frame harbored in gene cassette that integrated into class 1 integron,though there was no plausible RBS preceding the start codon.This make class 1 integron be more convenience to express the genes that capture from environment.

An offline two-dimensional system combining a rat cardiac muscle cell membrane chromatography time-of-flight mass spectrometry (CMC-TOF/MS) with a high performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli.On the basis of the analysis of the first dimension,retention components of the mine sample were collected into 30 fractions (one fraction per minute).Then offline analysis of the second dimension was carried out.34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine,and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method.As a result,binding affinities decreased along with the process of deacylation,debenzoylation and demethylation,which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism.Moreover,some minor components in rat urine (Songorine,14-benzoylneoline,Deoxyaconitine,etc.) exerted relatively strong affinity on cell membranes are worth exploring.The results delivered by the system suggest that the CMC can be applied to in vivo study.