A highly sensitive immuno-PCR assay based on sandwich ELISA and PCR was developed to detect the circulating antigen in trichinellosis. Antigens were purified from the muscle larvae of Trichinella spiralis, and the myeloma cells were fused with spenocytes immunized with T. spiralis antigens to product the specific monoclonal antibodies. Indirect ELISA was used to select the antibody-secreting hybrodoma cells. By this method of procedure, monoclonal antibody F4C6 against the T. spiralis ES antigen was obtained, which was used as the indicator antibody, while the rabbit polyclonal antibodies against T. spiralis were to be used as capturing antibodies. The plasmid Bluecript Ⅱ KS was amplified by PCR with biotin-labeled primer M13-20, and thus the biotin-labeled DNA was obtained. Both the second antibody and DNA labeled with biotin were to be linked with 100 ng/ml avidin. The whole procedures of assay consisted of two steps, in which the circulating antigens were captured by monoclonal antibody through sandwich ELISA in the first step, and the DNA linked by monoclonal antibody was amplified by PCR in the second step. The sensitivity of this method was compared with that of the ELISA assay. It was found that the measuring ranges to detect the circulating antigens in trichinellosis were 50 pg/L to 0.005 pg/L for the immuno-PCR assay, and 5 μg/L to 0.05 μg/L for ELISA assay, the former was quite higher than that of the latter. It is evident that this method is highly sensitive for the detection of circulating antigens in trichinellosis.

Objective To observethe efficacy of mice infected with Sparganum mansoni by using different dosages of praziquantel.Methods A total of 156 Kunming mice were divided into 2 batches.each of them wag orally infted with 5 spargana.Thirty-six mice in the first batch were equally divided into 6 groups.the mice in group 1-5 were inoculated with spargana cultured in different concentrations of praziquantel for 3 days,the group 6 served as a control.One hundred and twenty mice in the second batch were equally divided into 12 groups,each mouse was inoculated with spargana obtained from frogs or tadpoles,group 1-9 were treated by different desages of praziquantel 1 or5 weeks post infection.group 10-12 served as controls.All of the mice wore sacriftced and dissectedl or 2 weeks after the treatment.the mean number of worms recovered was cmculated and worm reduction rates were determined.Results The number of worm recovered from mice infected with spargana cultured in 10-40 μg/ml of praziquantel had no significant difference with that of the control(P>0.05).The worm reduction rate wag 16.60%while the spargana beins cultured in 50 μg/ml of praziquantel.The worm reduction rates of the mice that sacrificed 1 week or 2 weeks after being treated by the same dosage of praziquantel had no significant difference(P>0.05).When being treated with 200.400 or 800 mg/kg of praziquantel 1 week post infection,the number of worm recovered from mice infected with spargana from frogs had no significant difference with those of the control 1 and 2 weeks after the treatment(P>0.05).The worm reduction rates between the groups with the same dosage 1 week and 2 weeks post treatment had no significant difference(P>0.05).When being treated with 200 or 400 mg/kg of praziquantel 1 week post infection,the number of worm recovered from mice infected with spargana from tadpoles had no statistically difference with that of the control 1 week after treatment (P>0. 05). The worm reduction rate of mice was only 17.02% while being treated with 800 mg/kg of praziquantel. The worm reduction rates among groups with different dosages had no significant difference (P>0.05). Compared with the mice infected with spargana from frogs treated with 1 200 or 1 800 mg/kg of praziquantel 5 weeks post infection, the difference between the numbers of worm recovered from mice 1 week and 2 weeks after treatment had no statistically significance (P > 0.05), but they were significantly higher than those of the controls (P<0.05 ). The worm reduction rates among the groups with the same dosage had no significant difference (P>0.05). ConclusionsPraziquantel (10-50 μg/ml) has no evident killing effect on spargnna in vitro, but when the dosage is higher(1 800 mg/kg), it has certain efficacy for treating the mice infected with spargana by oral inoculation.

Objective To investigate the significance of test of receptor activator of nuclear factor?kappa B(NF?κB)ligand(RANKL)in serum and urine for the diagnosis of osteoporosis. Methods A total of 53 patients with osteoporosis(the experimental group)and 45 healthy controls(the normal control group)were recruited in this study. The expression levels of RANKL in serum and urine was measured and compared by enzyme?linked immunosorbent assay. Results The serum and urine levels of RANKL in the experimental group were significantly higher than those in the normal control group(P<0.01). The areas under receiver operating characteristic(ROC)curve of serum and urine RANKL were 0.898 and 0.734, respectively. The combined detection of serum and urine RANKL and Ca2+reached a high sensitivity of 89.5%and a specificity of 86.1%for diagno?sis of osteoporosis. Conclusion RANKL may be closely associated with the progression of osteoporosis. Serum and urine RANKL test may be help?ful in the diagnosis of osteoporosis.

Currently, the definition, classification and Chinese nomenclature of intra-hepatic cholangiocarcinoma (ICC) are controversial. Whether ICC belongs to liver cancer or carcinoma of bile duct is debatable, and the two terms"intrahepatic cholangiocarcinoma"and"cholangiocellular carcinoma"are simultaneously used without distinction, bringing great confusions to clinical practice. Based on authoritative literatures at home and abroad, the authors give suggestions on the definition, classification and Chinese nomenclature of ICC, as well as the classification of carcinoma of bile duct, which recommend that the Chinese translation of "cholangiocarcinoma" should be "epithelial carcinoma of bile duct (cholangiocellular carcinoma)", the mass-forming type ICC should be classified as primary liver cancer, naming as"intrahepatic cholangiocarcinoma"and the periductal-infiltrating type and intraductal-growing type ICCs still be classified as carcinoma of bile duct, naming as"perihilar cholangiocarcinoma". The authors recommend to classify carcinoma of bile duct into: perihilar cholangiocarcinoma, hilar cholangiocarcinoma, and distal cholangiocarcinoma.

Objective To explore the effect and possible pharmacological mechanism of Bufei Tongbi Decoction in the treatment of systemic lupus erythematosus interstitial lung disease (SLE-ILD).Methods The effective components and related targets of Bufei Tongbi Decoction were obtained using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Uniprot database. Key genes for SLE-ILD were screened based on DrugBank,DisGeNET,GeneCards,PharmGKB,OMIM,and GEO databases. Using Cytoscape software,a drug active ingredient-target-disease relationship network diagram was constructed to obtain the effective active ingredients and possible mechanisms of action of Bufei Tongbi Decoction in the treatment of SLE-ILD. Gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to reveal related target genes and pathway functions. Taking C57BL/6 mice as normal group,MRL/lpr mice were injected with bleomycin 5mg/kg in the nasal cavity. According to the random number table method,the mice were divided into model group,Bufei Tongbi Decoction low-dose group (10.4 g/(kg·d)),Bufei Tongbi Decoction medium-dose group (20.8 g/(kg·d)),Bufei Tongbi Decoction high-dose group (41.6 g/(kg·d)) and prednisone group (3 mg/(kg·d)). The intervention lasted for 28 days. Hematein eosin and Masson staining were used to observe the pathological changes of mouse lung tissue,the expressions of transforming growth factor-β1 (TGF-β1) and collagen type Ⅲ (Col-Ⅲ) in lung tissue were detected by immunohistochemistry,and the expressions of interleukin-1β(IL-1β),interleukin-10 (IL-10) and interleukin-17 (IL-17) in serum were detected by ELISA. The mRNA expressions of matrix metallopeptidase 1(MMP-1),hypoxia inducible factor-1α(HIF-1α),retinoid-related orphan receptor γt (RORγt ) and forkhead box P3 (FOXP3) in lung tissue were analyzed by RT-PCR,the protein expressions of HIF-1α and MMP-1 in lung tissue were detected by Western blotting,and the expressions of T helper 17 cells (Th17) and regulatory T cells (Treg cells) in blood were detected by cytometry.Results A total of 163 effective ingredients,259 targets,1729 SLE-ILD disease targets,958 SLE-ILD differential genes and 40 drug-disease interaction targets were obtained by screening. GO functional enrichment and KEGG pathway enrichment showed that IL-17 signaling pathway activated by IL-1β and MMP-1,and Th17 cell differentiation activated by IL-1β and HIF-1α were the main pathways. Animal experiments showed that Bufei Tongbi Decoction could effectively improve the degree of lung interstitial lesion and reduce the expressions of TGF-β1 and Col-Ⅲ in SLE-ILD mice (P<0.01). The expressions of IL-1β,HIF-1α and IL-17 were decreased (P<0.01). Medium and high doses of Bufei Tongbi Decoction decreased the expressions of MMP-1 and RORγt mRNA (P<0.01),and increased the expressions of IL-10 and FOXP3 mRNA (P<0.01). Bufei Tongbi Decoction could reduce the proportion of Th17 cells,increase the proportion of Treg cells,downregulate the balance of Th17/Treg (P<0.05),and improve the immune disorder. Conclusion Bufei Tongbi Decoction has the characteristics of multi-target and multi-pathway in treating SLE-ILD,and its mechanism may be related to the regulation of Th17/Treg cell balance.

[Objective]To analyze the value of grey-scale reversed T1-weighted(rT1)MRI in the detection of structur-al lesions of the sacroiliac joint(SIJ)in patients with axial spondyloarthritis(ax-SpA).[Methods]Fifty-two ax-SpA pa-tients who underwent both MRI and CT in our hospital within a week from February 2020 to December 2022 were retrospec-tively included.Both sacral and iliac side of each SIJ on oblique coronal images were divided into anterior,middle and pos-terior portion.Two radiologists reviewed independently three groups of MRI including T1-weighted imaging(T1WI),rT1 and T1WI+rT1 images to evaluate the structural lesions like erosions,sclerosis and joint space changes in each of the 6 re-gions of the SIJ.One of the radiologist did the evaluation again one month later.CT images were scored for lesions by a third radiologist and served as the reference standard.Intra-class correlation coefficients(ICC)were calculated to test the inter-and intra-reader agreement for the assessment of SIJ lesions.A Friedman test was performed to compare the lesion results of MRI and CT image findings.We examined the diagnostic performance[accuracy,sensitivity(SE)and specifici-ty]of different groups of MRI in the detection of lesions by using diagnostic test.A McNemar test was used to compare the differences of three groups of MRI findings.[Results]CT showed erosions in 71 joints,sclerosis in 65 and joint space changes in 53.Good inter-and intra-reader agreements were found in three groups of MRI images for the assessment of le-sions,with the best agreement in T1WI+rT1.There were no difference between T1WI+rT1 and CT for the assessment of all lesions,nor between rT1 and CT for the assessment of erosions and joint space changes(P>0.05).T1WI+rT1 yielded better accuracy and SE than T1WI in detection of all lesions(Accuracy erosions:90.3%vs 76.9%;SE erosions:91.6%vs 76.1%;Accu-racy sclerosis:89.4%vs 80.8%;SE sclerosis:84.6%vs 73.9%;Accuracy joint space changes:86.5%vs 73.1%;SE joint space changes:84.9%vs 60.4%;P<0.05).rT1 yielded better accuracy and SE than T1WI in detection of erosions and joint space changes(Accuracy erosions:87.5%vs 76.9%;SE erosions:88.7%vs 76.1%;Accuracy joint space changes:85.6%vs 73.1%;SE joint space changes:83.0%vs 60.4%;P<0.05).[Conclusions]In the detection of SIJ structural lesions in ax-SpA,rT1 improves the diagnostic perfor-mance and T1WI+rT1 is more superior to others.

Objective Using network pharmacology to explore the mechanism of action of tripterygium wilfordii-white peony in the treatment of rheumatoid arthritis(RA)and validating it using an adjuvant arthritis rat model.Methods Obtain the effective ingredients of tripterygium wilfordii and white peony through TCMSP database and predict the action targets;Obtain the disease targets of RA using DisGeNET and GeneCards online database;Draw the network of"tripterygium wilfordii-white peony-active ingredient-RA"and screen the core effective components using the software Cytoscape 3.9.1;Construct the protein-protein interaction(PPI)network using String database and screen the core targets using the CytoNCA plug-in,using the Metascape database for gene enrichment analysis of common targets,and using Auto Dock Tools and Pymol software for molecular docking of core active ingredients and targets.Enzyme linked immunosorbent assay(ELISA)detection of serum tumor necrosis factor-α(TNF-α),interleukin(IL)-4,IL-6,and IL-17 levels in adjuvant arthritis rats,Western blot was used to detect the expression of phosphorylated phosphatidylinositol 3-hydroxy kinase(p-PI3K)and phosphorylated AKT protein(p-AKT),validate the results of network pharmacology analysis.Results There were a total of 61 effective ingredients in the treatment of RA with tripterygium wilfordii-white peony,with 116 targets and 191 signaling pathways involved.Animal experiments have shown that compared with the model group rats,the serum cytokine TNF-α,IL-6 and IL-17 of rats in tripterygium wilfordii-white peony group and tripterygium wilfordii extract group were significantly decreased(P<0.05),IL-4 significantly increased(P<0.05);The expression of p-PI3K and p-AKT in synovial tissue was significantly reduced(P<0.05);Compared with rats in tripterygium wilfordii extract group,the TNF-α,IL-6,IL-17,p-PI3K and p-AKT of rats in tripterygium wilfordii-white peony group were significantly reduced(P<0.05),while IL-4 was significantly increased(P<0.05).Conclusion Tripterygium wilfordii-white peony can inhibit the overexpression of phosphatidylinositol 3-hydroxy kinase(PI3K)/protein kinase B(AKT)signaling pathway,regulate cytokine release,exert anti-inflammatory effects,and ultimately treat RA through multiple active ingredients and targets.

OBJECTIVEA novel method of Nano-Immunomagnetic Separation (Nano-IMS) plus Real-time PCR was established for detecting Vibrio cholerae.

METHODSThe Nano-Immunomagnetic Beads were created by using the monoclonal antibody of Vibrio cholerae, which was named Nano-IMB-Vc. Nano-IMB-Vc has specific adsorption of Vibrio cholerae, combined with Real-time PCR technology, a method for rapid detection of Vibrio cholerae was established. The capture specificity of Nano-IMB-Vc was tested by using 15 bacteria strains. The specificity of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria strains. The sensitivity of Nano-IMS plus Real-time PCR were tested in pure culture and in artificial samples and compared with NMKL No.156.

RESULTSThe capture ratio of Nano-IMB-Vc was reached 70.2% at the level of 10(3) CFU/ml. In pure culture, the sensitivity of Nano-IMS plus Real-time PCR was reached at 5.4×10(2) CFU/ml. The specific of Real-time PCR method was tested by using 102 targets and 101 non-targets bacteria. The results showed that 102 strains of Vibrio cholerae test results were all positive, and the rest of the 101 strains of non-target bacteria test results were negative. No cross-reaction was founded. Add 1 CFU vibrio cholerae per 25 g sample, it could be detect with Nano-IMS plus Real-time PCR method after 8 hours enrichment.

CONCLUSIONSThe Nano-IMS plus Real-time PCR method of Vibrio cholerae established in this study has good specificity and sensitivity, which could be applied to the rapid detection of Vibrio cholerae.

Food Microbiology ; methods ; Immunomagnetic Separation ; methods ; Nanotechnology ; Real-Time Polymerase Chain Reaction ; methods ; Seafood ; analysis ; microbiology ; Vibrio cholerae ; genetics ; isolation & purification ; Vibrio parahaemolyticus ; genetics ; isolation & purification

Objective:To analyze the inflammatory mechanism and potential intervention drugs related to angiotensin converting enzyme 2 (ACE2) inhibitory mutations in order to provide reference for the treatment of corona virus disease 2019 (COVID-19).Methods:The data of lung adenocarcinoma with ACE2 mutations were screened from the cancer genome atlas (TCGA) database. The data were analyzed by R program language edgeR package and cluster Profiler package, gene ontology (GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Using String online analysis website for protein-protein interaction (PPI) network analysis, screening out the core genes, and finally using the Epigenomic Precision Medicine Prediction Platform (EpiMed) for multi-group association analysis of key genes, and drug candidates prediction.Results:A total of 1 005 differential genes were obtained, of which 91 were up-regulated and 914 down-regulated. A total of 71 GO were enriched, including 45 items related to biological processes, 16 items related to cell components, and 10 items related to molecular function. A total of 13 KEGG pathways were enriched, mainly in inflammatory pathways, various viral infectious diseases, transcriptional regulation, drug metabolism and protein digestion and absorption pathways. The differentially expressed genes were introduced into String online analysis website for PPI network analysis, a total of 252 proteins were obtained, and 10 core genes were H2A clustered histone 16(HIST1H2AL), H3 clustered histone 2 (HIST1H3B), H3 clustered histone 7 (HIST1H3F), H3 clustered histone 11 (HIST1H3I), H3 clustered histone 3 (HIST1H3C), H2B clustered histone 3 (HIST1H2BB), H2B clustered histone 6 (HIST1H2BI), H4 clustered histone 2 (HIST1H4B), H1-4 linker histone (HIST1H1E), H2A clustered histone 4 (HIST1H2AB). Interferon-α, resveratrol, celecoxib, heartleaf houttuynia herb, weeping forsythia capsule, dexamethasone, Chinese pulsatilla root, tumor necrosis factor-α inhibitors, liquorice root and famciclovir might be drugs for the treatment of ACE2 mutation-related inflammation.Conclusions:Inflammation associated with ACE2 inhibitory mutations is similar to the pathogenesis of COVID-19, which could lead to disease by promoting the activation of inflammatory pathways such as mitogen-activated protein kinase (MAPK), the Janus kinase signal transducer and activator of transcription (JAK/STAT), mammalian target of rapamycin (mTOR). Celecoxib, interferon and resveratrol may have the potential therapeutic effects on COVID-19.

Objective:To analyze the mechanism and potential intervention drugs of acute lung injury caused by severe acute respiratory syndrome coronavirus (SARS-CoV) infection in order to provide reference for the treatment of COVID-19.Methods:Gene Expression Omnibus (GEO) announcement database was used to screen coronavirus transcriptome data, and R language package was used for differential expression analysis and Kyoto Gene and Genome Encyclopedia (KEGG) and Gene Ontology (GO) enrichment analysis. A protein-protein interaction (PPI) network analysis was carried out by using STRING online analysis website, and the key genes were screened. Then the Epigenomic Precision Medicine Prediction Platform (EpiMed) was used to analyze the association of key genes and predict potential therapeutic drugs.Results:Based on the whole genome expression profile data of SARS-CoV, a total of 3 606 differential genes were screened, including 2 148 up-regulated and 1 458 down-regulated. GO enrichment is mainly related to viral infection, leukocyte migration and adhesion, acute inflammation and collagen secretion. KEGG enrichment is mainly related to signal transduction, acute inflammation, immune response and so on. Ten key genes related to lung injury, such as PTPRC, TIMP1, ICAM1 and IL1B, were screened by protein interaction network analysis. EpiMed platform predicted that pulsatilla chinensis, polygonum cuspidatum, tumor necrosis factor-α inhibitors, famciclovir, fluvastatin and other drugs have potential therapeutic effects.Conclusions:SARS-CoV infection can cause lung injury by activating a series of inflammation-related molecules. Drugs that may be effective in the treatment of coronavirus infections, including pulsatilla chinensis, polygonum cuspidatum, tumor necrosis factor-α inhibitors, famciclovir and fluvastatin.