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 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.

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.

In order to further promote the reform of medical service price and strengthen the macro management of medical service price, the author constructed a calculation method for price adjustment space of medical service. This study was centered on two parameters of the historical base and the growth coefficient, to calculate the total amountof price adjustment. The historical base used the total revenue of medical services in the region from the previous year, and the growth coefficient was measured by two methods, the comprehensive indicator method and the consumer price index (CPI) reference method.Taking Suzhou, a national pilot city, as an example, the historical base in 2022 was 18 754 million yuan. By using the comprehensive indicator method, the annual growth coefficient was calculated to be 2.38%, and adjustment space of medical service price was 446.35 million yuan; According to the CPI reference method, the growth coefficient of Suzhou was 2.10%, and adjustment space of medical service price was 393.83 million yuan.The two methods for calculating the total amount each have their own advantages and disadvantages, and need to be further optimized and improved by drawing on the practical experience of reform in each pilot city.

Objective:To design a set of evaluation index system for the medical service price reform mechanism, so as to provide reference for the evaluation of deepening the medical service price reform mechanism.Methods:On the basis of searching literature of medical service prices reformfrom August 2021 to March 2023, policy logic analysis and expert consultation were used to construct evaluation indexes for the medical service price reform mechanism, set evaluation index thresholds, and assign scores to index thresholds in different grades.Results:The evaluationindex system of medical service price reform mechanism included 5 level-1 indexes, 12 level-2 indexes and 35 level-3 indexes. After scoring, the scores of 5 level-1 indexes were 12, 24, 35, 21, and 8, respectively.Based on the percentile scoring method, the evaluation criteria were categorized into five grades: premium, excellent, good, average and failure.Conclusions:This study constructed a set of evaluation index system for the mechanism of medical service price reform, which had strong scientific and operability. However, due to the fact that medical service price reform in the new era is still in its initial stage, the index system need to be further optimized drawing on the practical experience of reforms in pilot cities.

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 investigate the omics mechanism of SARS-related immune injury and predict targeted therapeutic drugs through clinical bioinformatics analysis of the transcriptome data of SARS virus in order to provide reference for clinical treatment of COVID-19.Methods:The transcriptome data of SARA virus were collected from the Gene Expression Oibus (GEO) and used to screen differential genes. Enrichment analysis and protein interaction analysis were performed to investigate the mechanism of immune damage associated with SARS. A platform of epigenetics in precision medicine (EpiMed) was established to predict potential therapeutic drugs.Results:The mechanism of SARS-related immune injury was complex, involving affecting the function of immune cells through signaling pathways such as Toll-like receptors, increasing cytokines in plasma through Th17 signaling pathway and inducing autoimmune responses after autoantibodies were generated by molecules such as IL-6, NF-κB, and TNF. Drugs such as Chuanqiong and Etanercept might have therapeutic effects on SARS-related immune damage.Conclusions:SARS virus could cause abnormal expression of many immune-related molecules and signaling pathways. Drugs such as Chuanqiong and Etanercept might have therapeutic effects on SARS-related immune damage. This study might provide reference for clinical treatment of COVID-19.