Objective:To construct a recombinant gene of single-chain fragment variable(scFv)against the SARS-CoV-2 spike protein(S protein).Methods:Using genetic engineering antibody technology,BALB/c mice were immunized with SARS-CoV-2 S protein antigen.The total RNA of the spleen was extracted to obtain the heavy chain(VH)and light chain(VL)gene sequences of the antibody variable region(V).Through multiple-primer PCR amplification and screening,the corresponding VH and VL gene sequences were obtained.After purification and recovery,the VH and VL primer genes of anti-SARS-CoV-2 S protein with restriction enzyme cutting sites and the VH and VL genes were successfully connected by the overlap extension PCR method to obtain the scFv recombinant gene.The sequencing results were compared and analyzed in the gene bank by using BLAST on the NCBI website.Results:The VH and VL genes of anti-SARS-CoV-2 S protein were successfully obtained,and scFv was obtained by connecting through the overlap extension PCR technique(SOE-PCR).The BLSAT analysis of scFv showed that its gene homology was greater than 80%.The indirect ELISA detection results showed that there was obvious binding in the prokaryotic expression supernatant.Conclusion:This study provides a technical basis for the diagnosis and treatment of SARS-CoV-2 and the research and development of new related antibodies.

Objective:To construct a recombinant gene of single-chain fragment variable(scFv)against the SARS-CoV-2 spike protein(S protein).Methods:Using genetic engineering antibody technology,BALB/c mice were immunized with SARS-CoV-2 S protein antigen.The total RNA of the spleen was extracted to obtain the heavy chain(VH)and light chain(VL)gene sequences of the antibody variable region(V).Through multiple-primer PCR amplification and screening,the corresponding VH and VL gene sequences were obtained.After purification and recovery,the VH and VL primer genes of anti-SARS-CoV-2 S protein with restriction enzyme cutting sites and the VH and VL genes were successfully connected by the overlap extension PCR method to obtain the scFv recombinant gene.The sequencing results were compared and analyzed in the gene bank by using BLAST on the NCBI website.Results:The VH and VL genes of anti-SARS-CoV-2 S protein were successfully obtained,and scFv was obtained by connecting through the overlap extension PCR technique(SOE-PCR).The BLSAT analysis of scFv showed that its gene homology was greater than 80%.The indirect ELISA detection results showed that there was obvious binding in the prokaryotic expression supernatant.Conclusion:This study provides a technical basis for the diagnosis and treatment of SARS-CoV-2 and the research and development of new related antibodies.

Objective To investigate the effects of Huanglian Jiedu Decoction Aqueous Extract(HJDAE)on the gut microbiota structure and colon metabolites of rats using 16S rRNA technology and non targeted metabolomics technology.Methods SD rats were continuously gavaged with HJDAE for 7 days and then Euthanized under anesthesia to extract the colon contents of the rats,,the contents of the rats'colon were taken,and 16S rRNA high-throughput sequencing was performed on the gut microbiota of the rats'colon segments using the Illumina Miseq platform.The differential microbiota,differential metabolites,and related pathways were analyzed in combination with the fecal non targeted metabonomics method.Results The results showed that HJDAE could affect the structure of gut microbiota in rats while maintaining a relatively stable proportion of various phyla of gut microbiota,and had significant promoting and inhibitory effects on multiple bacterial genera.Among them,it had the strongest inhibitory effect on Lactobacillus and Limosilactobacillus,and the most obvious promoting effect on Clostridium.Analyzing the data from two sets of experimental results,76 differential metabolites and 70 potential biomarkers were found.Among them,the top ten differential metabolites and their differences were xylose,acetic acid,propionic acid,and dimethylglycine with reduced production,while the production of palmitoleic acid,proline,and T-α-MCA,T-β-MCA,3-DHCA,HCA increased.The eight strongest metabolic pathways involved are Propanoate Metabolism,Butanoate Metabolism,TCA cycle,Alanine-Aspartate-Glutamate Metabolism,D-Glutamine and D-Glutamate Metabolism,Primary Bile Acid Biosynthesis,Nitrogen Metabolism,Valine-Leucine-Isoleucine Biosythesis,all of which are closely related to bile acid production.Conclusion The HJDAE promotes the production of primary bile acids by promoting the metabolism of sugars,amino acids,and fatty acids in rats.At the same time,the HJDAE causes a large number of Clostridium species to proliferate,and multiple Clostridium species metabolize primary bile acids into secondary bile acids,jointly leading to positive regulation of bile acid metabolism.

The incidence of osteoporotic thoracolumbar vertebral fracture (OTLVF) in the elderly is gradually increasing. The kyphotic deformity caused by various factors has become an important characteristic of OTLVF and has received increasing attention. Its clinical manifestations include pain, delayed nerve damage, sagittal imbalance, etc. Currently, the definition and diagnosis of OTLVF with kyphotic deformity in the elderly are still unclear. Although there are many treatment options, they are controversial. Existing guidelines or consensuses pay little attention to this type of fracture with kyphotic deformity. To this end, the Lumbar Education Working Group of the Spine Branch of the Chinese Medicine Education Association and Editorial Committee of Chinese Journal of Trauma organized the experts in the relevant fields to jointly develop Clinical guidelines for the diagnosis and treatment of osteoporotic thoracolumbar vertebral fractures with kyphotic deformity in the elderly ( version 2024), based on evidence-based medical advancements and the principles of scientificity, practicality, and advanced nature, which provided 18 recommendations to standardize the clinical diagnosis and treatment.

ObjectiveTo explore the impact of Gegen Qinliantang（GQT） on the fecal short-chain fatty acids（SCFAs） metabolism in antibiotic-associated diarrhea（AAD） through targeted metabolomics. MethodA total of 240 SD rats were randomly divided into six groups（n=40， half male and half female）， including blank group， model group， bifidobiogen group（0.15 g·kg^-1）， and GQT high-， medium-， and low-dose groups（10.08， 5.04， 2.52 g·kg^-1）， except for the blank group， clindamycin（250 mg·kg^-1） was given to all groups by gavage for modeling every day for 7 d. After successful modeling， each administered group was gavaged with the corresponding dose of the drug， and the blank and model groups were gavaged with an equal volume of normal saline solution， 1 time/d， for 14 d. At 0， 3， 7， 14 d after the drug intervention， eight rats were randomly selected from each group， respectively. Gas chromatography-time-of-flight mass spectrometry（GC-TOF-MS） was used to perform targeted metabolomic analysis of SCFAs in the feces of rats， and partial least squares-discriminant analysis（PLS-DA） was applied to compare the differences in metabolic profiles between groups at different treatment times， and to compare the changes in the contents of SCFAs in rat feces between groups. ResultPLS-DA results showed that the blank group could be clearly distinguishable from the model group， with GQT exhibiting a closer proximity to the blank group after 7 d of treatment. After further analyzing the composition of SCFAs， it was found that the proportion of acetic acid increased and the proportions of butyric acid， valeric acid， hexanoic acid and isovaleric acid decreased in the model group compared with the blank group. After the treatment with GQT， the proportions of butyric acid， isobutyric acid， valeric acid， and isovaleric acid increased， and the proportions of acetic acid， propionic acid and caproic acid decreased. Subsequent differential analysis revealed that GQT could significantly improve the content of butyric acid， and had a certain retrogressive effect on the contents of valeric acid and hexanoic acid. ConclusionThe medium dose group of GQT can improve the contents of SCFAs in AAD feces after 7 days of treatment， which may be related to the improvement of the composition ratio of SCFAs and the contents of butyric acid， valeric acid and caproic acid.

BackgroundThe occurrence rate of dangerous behaviors in patients with severe mental disorders is higher than that of the general population. In China， there is limited research on the prediction of dangerous behaviors in community-dwelling patients with severe mental disorders， particularly in terms of predicting models using data mining techniques other than traditional methods. ObjectiveTo explore the influencing factors of dangerous behaviors in community-dwelling patients with severe mental disorders and testing whether the classification decision tree model is superior to the Logistic regression model. MethodsA total of 11 484 community-dwelling patients with severe mental disorders who had complete follow-up records from 2013 to 2022 were selected on December 2023. The data were divided into a training set （n=9 186） and a testing set （n=2 298） in an 8∶2 ratio. Logistic regression and classification decision trees were separately used to establish predictive models in the training set. Model discrimination and calibration were evaluated in the testing set. ResultsDuring the follow-up period， 1 115 cases （9.71%） exhibited dangerous behaviors. Logistic regression results showed that urban residence， poverty， guardianship， intellectual disability， history of dangerous behaviors， impaired insight and positive symptoms were risk factors for dangerous behaviors （OR=1.778， 1.459， 2.719， 1.483， 3.890， 1.423， 2.528， 2.124， P<0.01）. Being aged ≥60 years， educated， not requiring prescribed medication and having normal social functioning were protective factors for dangerous behaviors （OR=0.594， 0.824， 0.422， 0.719， P<0.05 or 0.01）. The predictive effect in the testing set showed an area under curve （AUC） of 0.729 （95% CI： 0.692~0.766）， accuracy of 70.97%， sensitivity of 59.71%， and specificity of 72.05%. The classification decision tree results showed that past dangerous situations， positive symptoms， overall social functioning score， economic status， insight， household registration， disability status and age were the influencing factors for dangerous behaviors. The predictive effect in the testing set showed an AUC of 0.721 （95% CI： 0.705~0.737）， accuracy of 68.28%， sensitivity of 64.46%， and specificity of 68.60%. ConclusionThe classification decision tree does not have a greater advantage over the logistic regression model in predicting the risk of dangerous behaviors in patients with severe mental disorders in the community. ［Funded by Chengdu Medical Research Project （number， 2020052）］

Objective To investigate changes in the intestinal flora and function in rats with antibiotic-associated diarrhea(A AD)treated with GeGen QinLian Decoction(GQD).Methods Sixty male or female SPF-grade SD rats were fed for 7 days and then divided randomly into Control(Con)and modeling groups(1∶5 ratio).Rats in the modeling group received clindamycin 250 mg/kg by gavage once a day for 7 consecutive days.After successful modeling,the rats were divided randomly into model(Mod),high-dose GQD(GQD-H,10.08 g/kg),medium-dose GQD(GQD-M,5.04 g/kg),low-dose GQD(GQD-L,2.52 g/kg),and live Bifidobacterium power(LBP,0.15 g/kg)groups(n=10 rats per group).GQD and LBP were administered by gavage,and the Con and Mod groups were given an equal volume of saline by gavage once a day.Feces were collected after 7 consecutive days of administration for macrogenomics sequencing analysis.Results α diversity and β diversity suggested that intestinal microbial diversity differed between the Mod and GQD-treated groups.GQD increased the abundance of thick-walled bacteria and decreased the abundance of Aspergillus at the phylum level,and increased the relative abundances of the intestinal mucus bacteria Blautia,Bacteroides,Thomasclavelia,and Mediterraneibacter,and decreased the relative abundances of Adlercreutzia,Muribaculum,and Escherichia at the genus level.GQD also up-regulated the amino acid,carbohydrate,and immune disease pathways.Conclusions GQD improves the abundance ratio of beneficial and pathogenic intestinal bacteria in rats with antibiotic-associated diarrhea,which in turn reduces the intestinal inflammatory response and repairs the intestinal immune system.

Objective To investigate changes in the intestinal flora and function in rats with antibiotic-associated diarrhea(A AD)treated with GeGen QinLian Decoction(GQD).Methods Sixty male or female SPF-grade SD rats were fed for 7 days and then divided randomly into Control(Con)and modeling groups(1∶5 ratio).Rats in the modeling group received clindamycin 250 mg/kg by gavage once a day for 7 consecutive days.After successful modeling,the rats were divided randomly into model(Mod),high-dose GQD(GQD-H,10.08 g/kg),medium-dose GQD(GQD-M,5.04 g/kg),low-dose GQD(GQD-L,2.52 g/kg),and live Bifidobacterium power(LBP,0.15 g/kg)groups(n=10 rats per group).GQD and LBP were administered by gavage,and the Con and Mod groups were given an equal volume of saline by gavage once a day.Feces were collected after 7 consecutive days of administration for macrogenomics sequencing analysis.Results α diversity and β diversity suggested that intestinal microbial diversity differed between the Mod and GQD-treated groups.GQD increased the abundance of thick-walled bacteria and decreased the abundance of Aspergillus at the phylum level,and increased the relative abundances of the intestinal mucus bacteria Blautia,Bacteroides,Thomasclavelia,and Mediterraneibacter,and decreased the relative abundances of Adlercreutzia,Muribaculum,and Escherichia at the genus level.GQD also up-regulated the amino acid,carbohydrate,and immune disease pathways.Conclusions GQD improves the abundance ratio of beneficial and pathogenic intestinal bacteria in rats with antibiotic-associated diarrhea,which in turn reduces the intestinal inflammatory response and repairs the intestinal immune system.

Objective To investigate the effects of Huanglian Jiedu Decoction Aqueous Extract(HJDAE)on the gut microbiota structure and colon metabolites of rats using 16S rRNA technology and non targeted metabolomics technology.Methods SD rats were continuously gavaged with HJDAE for 7 days and then Euthanized under anesthesia to extract the colon contents of the rats,,the contents of the rats'colon were taken,and 16S rRNA high-throughput sequencing was performed on the gut microbiota of the rats'colon segments using the Illumina Miseq platform.The differential microbiota,differential metabolites,and related pathways were analyzed in combination with the fecal non targeted metabonomics method.Results The results showed that HJDAE could affect the structure of gut microbiota in rats while maintaining a relatively stable proportion of various phyla of gut microbiota,and had significant promoting and inhibitory effects on multiple bacterial genera.Among them,it had the strongest inhibitory effect on Lactobacillus and Limosilactobacillus,and the most obvious promoting effect on Clostridium.Analyzing the data from two sets of experimental results,76 differential metabolites and 70 potential biomarkers were found.Among them,the top ten differential metabolites and their differences were xylose,acetic acid,propionic acid,and dimethylglycine with reduced production,while the production of palmitoleic acid,proline,and T-α-MCA,T-β-MCA,3-DHCA,HCA increased.The eight strongest metabolic pathways involved are Propanoate Metabolism,Butanoate Metabolism,TCA cycle,Alanine-Aspartate-Glutamate Metabolism,D-Glutamine and D-Glutamate Metabolism,Primary Bile Acid Biosynthesis,Nitrogen Metabolism,Valine-Leucine-Isoleucine Biosythesis,all of which are closely related to bile acid production.Conclusion The HJDAE promotes the production of primary bile acids by promoting the metabolism of sugars,amino acids,and fatty acids in rats.At the same time,the HJDAE causes a large number of Clostridium species to proliferate,and multiple Clostridium species metabolize primary bile acids into secondary bile acids,jointly leading to positive regulation of bile acid metabolism.

ObjectiveTo investigate the mechanism of Gegen Qinliantang（GQT） on the intestinal flora of antibiotic-associated diarrhea（AAD） by 16S rRNA sequencing and network pharmacology. MethodSixty SD rats were randomly divided into six groups（n=10）， including blank group， model group， GQT high-， medium- and low-dose groups（10.08， 5.04， 2.52 g·kg^-1） as well as Lizhu Changle group（0.15 g·kg^-1）， except for the blank group， each group was given clindamycin（250 mg·kg^-1） by gavage once a day for 7 consecutive days. After successful modeling， the blank group and the model group were given equal volumes of normal saline by gavage. The other groups were given corresponding doses of drugs by gavage for 14 days. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） was used to screen the active components and targets of GQT， GeneCards， Online Mendelian Inheritance in Man（OMIM） database， Pharmacogenetics and Pharmacogenomics Knowledge Base（PharmGKB）， DrugBank and DisGeNET were used to search for AAD disease targets. The drug-disease common targets were obtained by R software. STRING was applied to analyze the target protein-protein interaction， and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis was performed. Then hematoxylin-eosin（HE） staining was used to observe the pathological changes of the colon， and 16S rRNA sequencing of AAD colon content flora structure further verified the results of network pharmacology. ResultThrough network pharmacology， it was found that 238 active components were screened from GQT and acted on 276 component targets， among which quercetin， puerarin， wogonin and apigenin were the main core components of GQT， 1 097 AAD disease targets and 127 drug-disease intersection targets. The protein-protein interaction network mainly included core targets such as protein kinase B1（Akt1）， interleukin（IL）-6 and IL-1β， which were mainly enriched in the IL-17 signaling pathway. It was verified through animal experiments that compared with the blank group， the colon structure of the model group was seriously abnormal， the intestinal epithelial columnar cells were damaged， the goblet cells were reduced， and a large number of inflammatory cells were infiltrated. Compared with the model group， the colon structure of the GQT high-dose group improved， but there were still abnormalities， the colon structure of GQT medium- and low- dose groups and Lizhu Changle group improved significantly and reached the normal level. GQT could improve the structural diversity of AAD intestinal flora. At the phylum level， the abundance of Firmicutes was increased and the abundance of Bacteroidetes was decreased. At the genus level， the abundance of Lactobacillus was increased， and the abundances of Prevotella and Bacteroides were decreased. Among them， Lactococcus could be used as a biomarker for AAD treatment with GQT， and the prediction of functional metabolism of intestinal flora revealed that GQT could promote acetate and lactate metabolic pathways in the intestine. ConclusionGQT may activate IL-17 signaling pathway by acting on the targets of Akt1 and IL-6 through key components such as quercetin and wogonin， and improve the abundance of Lactococcus in the intestinal tract as well as acetate and lactate metabolic pathways， so as to play a role in repairing the intestinal barrier for the treatment of AAD.