In order to prepare monoclonal antibodies with blocking activity against feline TNF-α,this study successfully constructed,expressed and purified the recombinant plasmid pET-28a-sTNFα based on the soluble feline TNF-α(sTNFα)gene,and further investigated the induced ex-pression.The conditions were explored and optimized to identify its biological activity;secondly,the feline TNF-α recombinant protein was used as an immunogen for mouse immunization,after cell fusion,screening of blocking active hybridoma cells and ascites preparation,the obtained mon-oclonal antibodies were tested.The results showed that the pET-28a-sTNFα plasmid was success-fully constructed and the bioactive feline TNF-α recombinant protein was expressed in E.coli sys-tem.The molecular weight was 34 kDa and the 50％inhibitory concentration was 1.22 pg/L.Three monoclonal antibodies(A6-B7-9,H5-E2-94 and C8-A10-100)with blocking activity were success-fully screened out.The results of Western blot showed that all the three mAbs could specifically bind to TNF-α with a titer of 1:512 000.When the concentration of the three mAbs was 100 mg/L,the inhibitory effect on TNF-α was the strongest.In this study,we screened antibodies that can block the activity of cat TNF-α,in order to provide novel,safe and effective candidate drugs for the treatment of TNF-α mediated diseases in cats.

Traditional Chinese medicines have demonstrated clinical efficacy in preventing and treating chemotherapy-induced peripheral neuropathic pain(CIPNP).However,their specific clinical application and mechanism of action require further in-depth study and exploration.There is thus a need to develop more accurate and clinically relevant animal models that reflect the occurrence and development of human diseases as a tool for research.This review provides an in-depth analysis and discussion of the recent establishment and detection criteria of existing rat and mouse animal models of paclitaxel-induced peripheral neuropathic pain.We also evaluate and explain the application of these models for the prevention and treatment of CIPNP in traditional Chinese medicine,thus providing a theoretical basis and reference for future experimental and mechanistic research on the subject.This research will benefit clinical practice and promotion,offering valuable insights into preventing and treating CIPNP using traditional Chinese medicines.

Small intestine

Objective:To comprehensively understand the current situation of outpatient management in tertiary medical institutions in Anhui Province under the COVID-19 epidemic, and to provide empirical reference for effective prevention and control of the epidemic.Methods:In December 2020, a stratified cluster sampling method was used to investigate and score the current situation of outpatient management in 56 tertiary medical institutions in Anhui Province. The survey content included four dimensions: appointment and triage setting and management, fever clinic setting and management, nosocomial infection prevention and control management, and medical resource storage management. The scoring results were divided into three grades, namely " good" (total score≥90 points), " acceptable" (85≤total score<90 points)and " inadequate" (total score<85 points). SPSS 21.0 software was used for data statistical analysis. Independent sample t-test was used to compare the outpatient setting and management scores of medical institutions, and the proportion of assessment grades was used chi-square inspection.Results:The outpatient setting and management of 56 tertiary medical institutions in Anhui Province were evaluated as good in 36, acceptable in 6 and inadequate in 14. The outpatient setting and management score was(81.55±24.94), including(16.53±2.66)in " appointment and triage setting and management" , (47.62±19.60) in " fever clinic setting and management" , (8.69±1.44)in " nosocomial infection prevention and control management" , and (8.75±3.02) in " medical resource storage management" . The total scores and four dimension scores of 39 general hospitals were higher than those of 17 specialized hospitals, and the differences were significant( P<0.05). Conclusions:The overall situation of outpatient epidemic prevention and control management in tertiary medical institutions in Anhui Province is good, but the construction and control of specialized hospitals need to be further strengthened.

Drug metabolism and pharmacokinetics (DMPK) are the science to study the process of drug absorption, distribution, metabolism and excretion. It is very important to evaluate the characteristics of DMPK for the early development of drugs and the later clinical precision medication. The innovative construction of DMPK models promotes the development and improvement of drug evaluation system. Based on our research results, this review summarized the latest progress and application of innovative DMPK models, focusing on the following two aspects: (1) CRISPR/Cas9 gene editing rat models, including Cyp2e1

Objective:To identify the pathogenic gene of a pedigree with symphalangism and to prove the pathogenicity of this locus in vitro.Methods:The clinical data of patients’families were collected at Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, peripheral blood was collected and genomic DNA was extracted and NOG, FGF9, GDF5 exon regions were amplified by PCR, and the exon gene mutations were detected by first-generation sequencing technique. The structure of noggin-GDF5 protein complex was simulated in silicon. COS-7 cells were transfected with 5 μg empty plasmid, wild type plasmid and V202G mutant plasmid in vitro. Each group of plasmids was transfected into 3 well cells. The experiment was repeated for 3 times, and the expression of noggin protein was detected by Western blotting. C2C12 cells were also transfected with the above plasmids in vitro for osteogenic differentiation. By applying alkaline phosphatase staining and quantitative assay. Relative expression level of osteoblast-related genes Col1α1, ALP and Runx2 were detected by qRT-PCR. Each group of plasmids was transfected into 3 well cells, and the experiment was repeated for 3 times. All statistical analysis were performed by Prism 6 software. The result were shown as mean±standard deviation, and the comparison between groups was done by unpaired t-test. Data were considered statistically significant when P value is less than 0.05. Results:Both the proband and his mother suffered from symphalangism. The result of Sanger sequencing showed that there was a heterozygous missense mutation of NOG gene (p.V202G) in all patients in this pedigree. No disease-related mutations were detected in FGF9 and GDF5. Computer three-dimensional mechanism simulation showed that the site was located at the α helix. The result of Western blotting showed that the expression of mutant protein was significantly lower than that of wild type. Osteogenic differentiation in vitro showed that the inhibitory effect of V202G mutant protein on osteogenic differentiation decreased. The quantitative result of alkaline phosphatase staining showed that the alkaline phosphatase activity in the vector group was (12.3±0.8) U/L, and the alkaline phosphatase activity in the wild type plasmid group was (2.6±0.3) U/L, which was significantly lower than that in the vector group ( t=11.550, P<0.001). The alkaline phosphatase activity in the mutant plasmid group was (10.8±0.3) U/L. There was no significant difference between the mutant group and the vector group ( t=1.830, P=0.141). The mRNA expression level of osteogenesis-related genes was consistent with the above result . Compared with vector group, the expression of osteogenesis-related genes in wild-type noggin group decreased significantly ALP、 Col1α1 and Runx2 ( t=5.987, 4.498, 4.170; P=0.004, 0.011, 0.014). There was no significant difference between mutant plasmid group and blank vector group in ALP、 Col1α1 and Runx2 ( t=0.433, 0.177, 1.159; P=0.688, 0.868, 0.311). Conclusions:NOG gene c. 605T < G p. V202G is a novel mutation in symphalangism, which is located in the α helix of noggin protein, leading to the decrease of the expression of noggin protein and the manifestation of ankylosis.

Objective:To identify the pathogenic gene of a pedigree with symphalangism and to prove the pathogenicity of this locus in vitro.Methods:The clinical data of patients’families were collected at Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, peripheral blood was collected and genomic DNA was extracted and NOG, FGF9, GDF5 exon regions were amplified by PCR, and the exon gene mutations were detected by first-generation sequencing technique. The structure of noggin-GDF5 protein complex was simulated in silicon. COS-7 cells were transfected with 5 μg empty plasmid, wild type plasmid and V202G mutant plasmid in vitro. Each group of plasmids was transfected into 3 well cells. The experiment was repeated for 3 times, and the expression of noggin protein was detected by Western blotting. C2C12 cells were also transfected with the above plasmids in vitro for osteogenic differentiation. By applying alkaline phosphatase staining and quantitative assay. Relative expression level of osteoblast-related genes Col1α1, ALP and Runx2 were detected by qRT-PCR. Each group of plasmids was transfected into 3 well cells, and the experiment was repeated for 3 times. All statistical analysis were performed by Prism 6 software. The result were shown as mean±standard deviation, and the comparison between groups was done by unpaired t-test. Data were considered statistically significant when P value is less than 0.05. Results:Both the proband and his mother suffered from symphalangism. The result of Sanger sequencing showed that there was a heterozygous missense mutation of NOG gene (p.V202G) in all patients in this pedigree. No disease-related mutations were detected in FGF9 and GDF5. Computer three-dimensional mechanism simulation showed that the site was located at the α helix. The result of Western blotting showed that the expression of mutant protein was significantly lower than that of wild type. Osteogenic differentiation in vitro showed that the inhibitory effect of V202G mutant protein on osteogenic differentiation decreased. The quantitative result of alkaline phosphatase staining showed that the alkaline phosphatase activity in the vector group was (12.3±0.8) U/L, and the alkaline phosphatase activity in the wild type plasmid group was (2.6±0.3) U/L, which was significantly lower than that in the vector group ( t=11.550, P<0.001). The alkaline phosphatase activity in the mutant plasmid group was (10.8±0.3) U/L. There was no significant difference between the mutant group and the vector group ( t=1.830, P=0.141). The mRNA expression level of osteogenesis-related genes was consistent with the above result . Compared with vector group, the expression of osteogenesis-related genes in wild-type noggin group decreased significantly ALP、 Col1α1 and Runx2 ( t=5.987, 4.498, 4.170; P=0.004, 0.011, 0.014). There was no significant difference between mutant plasmid group and blank vector group in ALP、 Col1α1 and Runx2 ( t=0.433, 0.177, 1.159; P=0.688, 0.868, 0.311). Conclusions:NOG gene c. 605T < G p. V202G is a novel mutation in symphalangism, which is located in the α helix of noggin protein, leading to the decrease of the expression of noggin protein and the manifestation of ankylosis.

Objective Few studies are reported on the construction of a finite element model of human complex knee joint using multimodality CT and MRI images.In this study, we developed a finite element model of the knee joint for total knee arthroplasty (TKA) using matched and fused CT and MRI data, hoping to provide a useful tool for the simulation study of knee joint biomechanics of TKA.Methods The CT and MRI image data about an intact knee of a 26-year-old male volunteer were imported into the Mimics software for the establishment of 3D models of bony and soft-tissue structures.A complete knee model was developed following the registration and fusion of the constructed 3D models based on the external landmarks.After the simulated implantation of TKA components, a finite element model of the TKA knee was constructed with the Hypermesh software.Then the finite element model was analyzed following the definition of its material behavior, boundary conditions and loading.Results The finite element model of the TKA knee, which was composed of bones, ligaments, components, polyethylene insert and bone cement, was developed from CT-MRI image registration and fusion and maintained its important spatial relationship among different structures in the TKA knee.The results obtained from the finite element analysis showed the characteristics of stress distribution in the TKA knee.Conclusion The finite element model of the knee joint for TKA can be established by matching and fusing CT and MRI image data, which can be employed as a useful tool for the study of knee joint biomechanics of TKA.

The cDNA fragment of JEV prME gene was cloned into the baculovirus shuttle vector (bacmid) to construct a recombinant baculovirus vector, defined as AcBac-prME. Then the recombinant baculovirus Ac-prME was obtained by transfecting Sf9 cells with AcBac-prME. Western blot analysis and immunofluorescence results indicated that both prM and E proteins were efficiently expressed in Sf9 cells. Electron microscopy suggested that prME was assembled into JEV-VLPs. To further evaluate the potential of JEV-VLPs as vaccine, the mice were immunized with JEV-VLPs and then challenged with lethal JEV. The results of mice survival and pathological changes demonstrated that the JEV-VLPs performed complete protection against JEV-P3 strain and relieved pathological changes in the mice brain significant. This study suggest that JEV-VLPs would be a potential vaccine for Japanese encephalitis virus.
Animals ; Antibodies, Viral ; immunology ; Encephalitis Virus, Japanese ; genetics ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Humans ; Japanese Encephalitis Vaccines ; administration & dosage ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Sf9 Cells ; Vaccination ; Vaccines, Virus-Like Particle ; administration & dosage ; genetics ; immunology ; Viral Envelope Proteins ; administration & dosage ; genetics ; immunology

ObjectiveTo evaluate the treatment efifcacy of the treatment promotion of standardized management for chil-dren with asthma.MethodsMedical records of 150 children with asthma were reviewed and divided into management group or control group according to whether standardized management was accepted. Comprehensive asthma education for asthma pa-tients and their parents including asthma associated basic knowledge education, health education as well as follow-ups at deifned intervals was conducted in 78 cases. In the meantime, standardized asthma therapies were performed. Control group involved 72 cases who did not receive asthma education managements and only accepted regular clinical therapies. After 1-year observational follow-up, , clinical efifcacy of children with asthma, changes of knowledge-attitude-practice of parents, and compliance of med-ication were compared between the two groups.ResultsAfter promotion of standardized managements treatment, asthma con-trol rates in the management group were signiifcantly higher than that of the control group(χ2=54.68,P<0.01); In addition, the rate of asthma attacks, emergency visits as well as hospitalizations were obviously reduced in the management group than control group (both withP<0.01). Knowledge associated with asthma, therapy and management executions as well as knowledge-atti-tude-practice of parents also demonstrated apparent elevations in the management group (P<0.01); At the same time, management group has illustrated superior medication compliance over the control group (χ2=66.27,P<0.01).ConclusionPromotion of standardized treatment management among children with asthma can help to achieve effective control by raising levels of knowl-edge-attitude-practice of the parents as well as the patient’s compliance to the treatment.