In order to establish a highly efficient,convenient,and effective circular RNA(circRNA)vaccine preparation system,enhanced green fluorescent protein(EGFP)circRNA was constructed using permuted intron exon(PIE)strategy based on type Ⅰ introns.Then,circRNA circularization rates of RNA after in vitro transcription(IVT),primary circularization(IVC1),and secondary circularization(IVC2)were compared after purification.The constructed circRNA system was fur-ther applied to porcine reproductive and respiratory syndrome virus(PRRSV),and two circRNAs based on PRRSV GP5 protein were constructed and developed for in vitro expression.Results showed that circularization rates and protein expression effects of EGFP circRNA in IVC1 RNA and IVC2 RNA were similar,but both were significantly better than those of IVT RNA.Purity of EGFP circRNA reached 74％,and purities of two PRRSV GP5 protein circRNAs constructed using this preparation system were 71％and 64％,respectively.Western blot and indirect immunofluo-rescence assay(IFA)results indicated that both of the PRRSV GP5 protein circRNAs were suc-cessfully expressed.The results demonstrated that an easy-to-operate,low-cost circRNA prepara-tion system with high circularization rate was successfully constructed.Two PRRSV GP5 protein circRNA vaccines were successfully prepared using this system and expressed efficiently,which provides a reference for the development of animal circRNA vaccines and novel candidate vaccines against PRRSV.

Objective To analyze the correlation of cholecystectomy and changes in intestinal microbiota composition and function by observing functional characteristics of differential microbial communities.Methods A cross-sectional study was conducted on the patients(PC group,n=73)undergoing cholecystectomy in our hospital from 2020 to 2021.Another 56 healthy age-and gender-matched individuals(HC group)without a history of cholecystectomy were subjected and served as the control group.Fecal specimens were collected from the 2 groups.16S rRNA sequencing analysis was performed to examine the changes in composition and function of intestinal microbiota.Results There were no statistical differences between the 2 groups in baseline indicators,such as gender,age,BMI,smoking and drinking history,blood pressure,heart rate,and comorbidities,but significant difference was observed in total bilirubin(TBIL)between them(P<0.01).Alpha diversity analysis showed no significant difference in Chao1,Shannon,and Simpson indices between the 2 groups.Beta diversity analysis using the Bray-Curtis distance algorithm revealed a significant difference between the 2 groups at the class and genus levels(P<0.05).The analysis of microbiota relative abundance using LEFSE showed that Enterobacteriaceae,Lactobacillales,Citrobacter,Megasphaera,Lactobacillus,Enterococcus,Akkermansia,Streptococcus,Klebsiella,and Ruminococcus_gnavus were up-regulated in the PC group,and Lachnospiraceae,Sutterellaceae,Lachnospirales,Lachnospira,and Sutterella were down-regulated.Kyoto Encyclopedia of Genes and Genomes(KEGG)functional prediction analysis indicated that significant differences were seen between the 2 groups in metabolic pathways,including ascorbic acid(vitamin C)metabolism and aldonic acid metabolism(P<0.05),tricarboxylic acid cycle(TCA cycle)(P<0.05),glutathione metabolism(P<0.05),glutamic acid metabolism(P<0.05),secondary bile acid metabolism(P<0.05),and pentose phosphate pathway(P<0.01).Conclusion Cholecystectomy is closely associated with the structural alterations in the composition of intestinal microbiota.Variations in microbiota composition and function may induce perturbations in TCA cycle and glutathione metabolism,glutamate metabolism,secondary bile acid metabolism,and pentose phosphate pathways.

Objective To investigate the diversity and composition of gut fungi microbiota in patients with polycystic kidney disease(PKD)and its correlation with clinical indicators.Methods A total of 44 PKD patients,44 patients with non-polycystic chronic kidney disease(NPCKD)and 22 healthy controls(HC)admitted to our hospital from February 2023 to February 2024 were recruited.ITS1 DNA sequencing was applied to analyze the gut fungal composition.Bioinformatics analysis was used to compare the diversity and structural differences of fungi among the 3 groups.Pearson correlation analysis was performed to analyze the relationship between gut fungi and clinical indicators.Results There were no significant differences in baseline characteristics(gender,age,body mass index,etc.)among the 3 groups,but statistical differences were seen in terms of serum indicators(such as serum creatinine,blood urea nitrogen,uric acid,estimated glomerular filtration rate,etc.)(P<0.01).Alpha diversity analysis showed no significant difference was seen between the PKD and HC groups,but the PKD group had significant differences to the NPCKD group(P<0.01).Beta diversity analysis revealed significant differences among the 3 groups and in pairwise comparisons(P=0.001).Fungi composition analysis found that the abundance of Candida was significantly higher in the PKD group than the other 2 groups(P<0.01),while the abundances of Aspergillus and Cladosporium were significantly lower in the PKD group than the HC group(P<0.05).Linear discriminant analysis(LEFSe)indicated that Candida was significantly enriched,while Aspergillus and Cladosporium were significantly reduced in the PKD group.Correlation analysis revealed that the abundance of Cladosporium was negatively correlated with cyst diameter and immunoglobulin light chain Kappa/Lambda ratio in the PKD group(P<0.05),while the abundance of Candida was positively correlated with liver/kidney cyst diameter(P<0.01).Conclusion PKD patients exhibit characteristic changes in gut fungi diversity and composition.The abundances of Cladosporium and Candida are closely associated with clinical indicators of PKD patients.

Objective To analyze the diversity and composition of gut microbiota in individuals with circadian rhythm disruption and their correlation with cognition.Methods Night shift workers and regular shift workers were subjected from our hospital during August 2022 and October 2024.The participants with circadian rhythm disorders were assigned into an experimental group(n=24),and those with normal circadian rhythms were into a control group(n=24).Their height,weight,age,gender,body mass index(BMI)and fresh fecal samples were collected,and Montreal Cognitive Assessment(MoCA)and Mini-Mental State Examination(MMSE)were used to evaluate their mental status.Metagenomics,Alpha and Beta diversity analyses,Linear Discriminant Analysis Effect Size(LEfSe),and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were employed to investigate the diversity and function characteristics of gut microbiota in the participants.Results There were no statistical differences between the 2 groups in baseline data,such as height,weight,gender,age,and BMI(P>0.05).Alpha diversity analysis indicated that no statistical differences were observed in the ACE,Chao1,Shannon,or Simpson indices between the 2 groups,while beta diversity analysis revealed significant differences(P<0.01),suggesting different structure of gut microbiota between them.In the experimental group,the abundance of Faecalibacterium prausnitzii and Agathobacter rectalis was decreased,while that of Escherichia coli and Phocaeicola vulgaratus was increased,with significant differences when compared with the control group(P<0.05).Additionally,KEGG functional analysis showed that the experimental group had obviously higher expression levels in Th17 cell differentiation and the IL-17 signaling pathway than the control group(P<0.05).Agathobacter rectalis and Faecalibacterium prausnitzii were positively correlated with MoCA score and MMSE score(P<0.05,P<0.01).Agathobacter rectalis was negatively correlated with the IL-17 signaling pathway and Th17 cell differentiation.Conclusion Individuals with circadian rhythm disorders have significant changes in the structure and function of gut microbiota when compared to those with normal circadian rhythms.Agathobacter rectalis may be involved in the regulation of the IL-17 signaling pathway and differentiation of Th17 cells,thereby possibly impacting the increases of cognitive score related to circadian rhythm disorders.

BACKGROUND:The regulatory mechanisms of acupotomy intervention for knee osteoarthritis at a transcriptome level are not well understood despite its proven clinical efficacy.OBJECTIVE:Using acupotomy therapy in a rat model of knee osteoarthritis,to conduct transcriptome sequencing and bioinformatics analysis on cartilage samples,along with validation,and to reveal the molecular regulatory mechanisms involved in this therapy for knee osteoarthritis in rats.METHODS:Forty-eight Sprague-Dawley rats were selected and divided into three groups by random number table method,acupotomy group,model group,and sham operation group,with 16 rats in each group.Osteoarthritis models were induced by medial meniscus instability in the acupotomy group and model group.After successful modeling,acupotomy group rats were treated with acupotomy once a week,for 4 weeks in total.After the intervention,cartilage samples from the rat's knee joint were stained with hematoxylin-eosin staining and safranin O-fast green staining,evaluated by Mankin scores,and underwent MicroCT scanning.Serum inflammatory factor levels were detected by Elisa.Transcriptome sequencing was performed on the remaining cartilage samples,and the data were analyzed using R software to identify differential gene expression levels among the groups.Core targets were screened through protein-protein interaction network and Cytoscape and validated using RT-qPCR.RESULTS AND CONCLUSION:Compared with the sham operation group,rats in the model group had rough and uneven articular cartilage surfaces,narrowed joint spaces,destroyed articular surface structures,elevated Mankin scores,and significant increases in serum levels of interleukin-6,tumor necrosis factor-α,and matrix metalloproteinase 13(P<0.05).Compared with the model group,rats in the acupotomy group had smoother articular cartilage surfaces,wider joint spaces,slightly irregular articular surfaces,lower Mankin scores,and significantly lower serum levels of interleukin-6,tumor necrosis factor-α,and matrix metalloproteinase-13(P<0.05).Gene ontology and Kyoto genome and genome encyclopedia analyses involved proteolytic metabolism,autophagy,mitogen-activated protein kinase,nuclear factor kB,and Wnt signaling pathways.Protein-protein interaction network and Cytoscape screened for four key genes,including ataxia-telangiectasia mutations,Myb SWIRM and MPN domain protein 1,heat shock protein 90α1,and NIPBL cohesion-loading factor.The mRNA expression of ataxia-telangiectasia mutations,Myb SWIRM and MPN domain protein 1,heat shock protein 90α1,and NIPBL cohesion-loading factor in the articular cartilage of rats in the model group was lower than that of the sham operation group(P<0.05),while the mRNA expression of ataxia-telangiectasia mutations,Myb SWIRM and MPN domain protein 1,heat shock protein 90α1,and NIPBL cohesion-loading factor in the articular cartilage of rats in the acupotomy group was higher than that in the model group(P<0.05).To conclude,acupotomy treatment of knee osteoarthritis in rats may act on signaling pathways such as MAPK,nuclear factor kB,and Wnt to promote cartilage repair,and is closely related to the expression of genes associated with ataxia-telangiectasia mutations,Myb SWIRM and MPN domain protein 1,heat shock protein 90α1,and NIPBL cohesion-loading factor.

To explore the interaction between ASFV capsid protein M1249L and host from the host cellular perspective,M1249L was selected for constructing the bait plasmid(pGBKT7-M1249L)to screen the bone marrow-derived macrophages(BMDMs)cDNA library.After again co-transform and sequence alignment,20 candidate interacting host proteins were screened,such as IL-1β,CTSB and DNAJA3.And then,co-immunoprecipitation assay was performed to verify the interaction be-tween M1249L and host proteins.GO ontology(GO)and KEGG pathway enrichment analyses re-vealed that biological regulation,cellular communication and response to stimulus and others were enriched in biological processes.And these host proteins could share some pathways,including toll-like receptor signaling pathway and Nod-like receptor signaling pathway.Therefore,the results provides the theoretical basis for further research on the mechanism of ASFV M1249L in viral in-fection and immune regulation.

In order to establish a highly efficient,convenient,and effective circular RNA(circRNA)vaccine preparation system,enhanced green fluorescent protein(EGFP)circRNA was constructed using permuted intron exon(PIE)strategy based on type Ⅰ introns.Then,circRNA circularization rates of RNA after in vitro transcription(IVT),primary circularization(IVC1),and secondary circularization(IVC2)were compared after purification.The constructed circRNA system was fur-ther applied to porcine reproductive and respiratory syndrome virus(PRRSV),and two circRNAs based on PRRSV GP5 protein were constructed and developed for in vitro expression.Results showed that circularization rates and protein expression effects of EGFP circRNA in IVC1 RNA and IVC2 RNA were similar,but both were significantly better than those of IVT RNA.Purity of EGFP circRNA reached 74％,and purities of two PRRSV GP5 protein circRNAs constructed using this preparation system were 71％and 64％,respectively.Western blot and indirect immunofluo-rescence assay(IFA)results indicated that both of the PRRSV GP5 protein circRNAs were suc-cessfully expressed.The results demonstrated that an easy-to-operate,low-cost circRNA prepara-tion system with high circularization rate was successfully constructed.Two PRRSV GP5 protein circRNA vaccines were successfully prepared using this system and expressed efficiently,which provides a reference for the development of animal circRNA vaccines and novel candidate vaccines against PRRSV.

To explore the interaction between ASFV capsid protein M1249L and host from the host cellular perspective,M1249L was selected for constructing the bait plasmid(pGBKT7-M1249L)to screen the bone marrow-derived macrophages(BMDMs)cDNA library.After again co-transform and sequence alignment,20 candidate interacting host proteins were screened,such as IL-1β,CTSB and DNAJA3.And then,co-immunoprecipitation assay was performed to verify the interaction be-tween M1249L and host proteins.GO ontology(GO)and KEGG pathway enrichment analyses re-vealed that biological regulation,cellular communication and response to stimulus and others were enriched in biological processes.And these host proteins could share some pathways,including toll-like receptor signaling pathway and Nod-like receptor signaling pathway.Therefore,the results provides the theoretical basis for further research on the mechanism of ASFV M1249L in viral in-fection and immune regulation.

BACKGROUND:The regulatory mechanisms of acupotomy intervention for knee osteoarthritis at a transcriptome level are not well understood despite its proven clinical efficacy.OBJECTIVE:Using acupotomy therapy in a rat model of knee osteoarthritis,to conduct transcriptome sequencing and bioinformatics analysis on cartilage samples,along with validation,and to reveal the molecular regulatory mechanisms involved in this therapy for knee osteoarthritis in rats.METHODS:Forty-eight Sprague-Dawley rats were selected and divided into three groups by random number table method,acupotomy group,model group,and sham operation group,with 16 rats in each group.Osteoarthritis models were induced by medial meniscus instability in the acupotomy group and model group.After successful modeling,acupotomy group rats were treated with acupotomy once a week,for 4 weeks in total.After the intervention,cartilage samples from the rat's knee joint were stained with hematoxylin-eosin staining and safranin O-fast green staining,evaluated by Mankin scores,and underwent MicroCT scanning.Serum inflammatory factor levels were detected by Elisa.Transcriptome sequencing was performed on the remaining cartilage samples,and the data were analyzed using R software to identify differential gene expression levels among the groups.Core targets were screened through protein-protein interaction network and Cytoscape and validated using RT-qPCR.RESULTS AND CONCLUSION:Compared with the sham operation group,rats in the model group had rough and uneven articular cartilage surfaces,narrowed joint spaces,destroyed articular surface structures,elevated Mankin scores,and significant increases in serum levels of interleukin-6,tumor necrosis factor-α,and matrix metalloproteinase 13(P<0.05).Compared with the model group,rats in the acupotomy group had smoother articular cartilage surfaces,wider joint spaces,slightly irregular articular surfaces,lower Mankin scores,and significantly lower serum levels of interleukin-6,tumor necrosis factor-α,and matrix metalloproteinase-13(P<0.05).Gene ontology and Kyoto genome and genome encyclopedia analyses involved proteolytic metabolism,autophagy,mitogen-activated protein kinase,nuclear factor kB,and Wnt signaling pathways.Protein-protein interaction network and Cytoscape screened for four key genes,including ataxia-telangiectasia mutations,Myb SWIRM and MPN domain protein 1,heat shock protein 90α1,and NIPBL cohesion-loading factor.The mRNA expression of ataxia-telangiectasia mutations,Myb SWIRM and MPN domain protein 1,heat shock protein 90α1,and NIPBL cohesion-loading factor in the articular cartilage of rats in the model group was lower than that of the sham operation group(P<0.05),while the mRNA expression of ataxia-telangiectasia mutations,Myb SWIRM and MPN domain protein 1,heat shock protein 90α1,and NIPBL cohesion-loading factor in the articular cartilage of rats in the acupotomy group was higher than that in the model group(P<0.05).To conclude,acupotomy treatment of knee osteoarthritis in rats may act on signaling pathways such as MAPK,nuclear factor kB,and Wnt to promote cartilage repair,and is closely related to the expression of genes associated with ataxia-telangiectasia mutations,Myb SWIRM and MPN domain protein 1,heat shock protein 90α1,and NIPBL cohesion-loading factor.

Objective:To investigate the safety of early antiplatelet therapy for non-cardioembolic mild stroke patients with thrombocytopenia.Methods:Data of acute ischemic stroke patients with baseline National Institutes of Health Stroke Scale(NIHSS)score≤3 and a platelet count<100×109/L were obtained from a multicenter register.Those who required anticoagulation or had other contraindications to antiplatelet therapy were excluded.Short-term safety outcomes were in-hospital bleeding events,while the long-term safety outcome was a 1-year all-cause death.The short-term neurological outcomes were evaluated by modified Rankin scale(mRS)score at discharge.Results:A total of 1868 non-cardioembolic mild stroke patients with thrombocytopenia were enrolled.Multivariate regression analyses showed that mono-antiplatelet therapy significantly increased the proportion of mRS score of 0-1 at discharge(OR=1.657,95%CI:1.253-2.192,P<0.01)and did not increase the risk of intracranial hemorrhage(OR=2.359,95%CI:0.301-18.503,P>0.05),compared with those without antiplatelet therapy.However,dual-antiplatelet therapy did not bring more neurological benefits(OR=0.923,95%CI:0.690-1.234,P>0.05),but increased the risk of gastrointestinal bleeding(OR= 2.837,95%CI:1.311-6.136,P<0.01)compared with those with mono-antiplatelet therapy.For patients with platelet counts≤75×109/L and>90×109/L,antiplatelet therapy significantly improved neurological functional outcomes(both P<0.05).For those with platelet counts(>75-90)×109/L,antiplatelet therapy resulted in a significant improvement of 1-year survival(P<0.05).For patients even with concurrent coagulation abnormalities,mono-antiplatelet therapy did not increase the risk of various types of bleeding(all P>0.05)but improved neurological functional outcomes(all P<0.01).There was no significant difference in the occurrence of bleeding events,1-year all-cause mortality risk,and neurological functional outcomes between aspirin and clopidogrel(all P>0.05).Conclusions:For non-cardioembolic mild stroke patients with thrombocytopenia,antiplatelet therapy remains a reasonable choice.Mono-antiplatelet therapy has the same efficiency as dual-antiplatelet therapy in neurological outcome improvement with lower risk of gastrointestinal bleeding.