Objective: To explore the therapeutic mechanism of puerarin in treating rheumatoid arthritis (RA) rats based on the serine/tyrosine protein kinase B (AKT)-phosphorylated forkhead box protein O1 (FOXO1)-interleukin-9 (AKT-FOXO1-IL-9) signaling pathway. Methods : 36 rats were randomly divided into a blank group , a model group , a positive control group , and low , medium , and high dose groups of puerarin. Except for the blank group , the other groups were induced with type Ⅱ collagen to establish a RA rat model. After successful modeling , different doses of puerarin and methotrexate were given to treat the rats. The body mass and toe thickness of the rats were measured , and biochemical indicators of rat blood rheology were detected. X-ray was used to observe changes in rat joint morphology. Safranin green staining were used to observe the pathology of rat joint tissue. ELISA was used to detect the levels of IL-9 and rheumatoid factors in rat serum , and Western blot was used to detect changes in levels of AKT and FOXO1 . 36 rats were randomly divided into a blank group , a model group , a positive control group , and low , medium , and high dose groups of puerarin. Except for the blank group , the other groups were induced with type Ⅱ collagen to establish a RA rat model. After successful modeling , different doses of puerarin and methotrexate were given to treat the rats. The body mass and toe thickness of the rats were measured , and biochemical indicators of rat blood rheology were detected. X-ray was used to observe changes in rat joint morphology. Safranin green staining were used to observe the pathology of rat joint tissue. ELISA was used to detect the levels of IL-9 and rheumatoid factors in rat serum , and Western blot was used to detect changes in levels of AKT and FOXO1 . Results: Compared with the blank group , the model group had the lowest toe thickness , and X-ray images showed more obvious segmental stenosis and more severe marginal bone invasion ; scaly like changes appeared at the edges of joints stained with safranin green , accompanied by the exudation of inflammatory cells and increased proliferation and secretion of chondrocytes ; the expression levels of inflammatory factors IL-9 and rheumatoid factors were the highest , and the expression levels of AKT and FOXO1 proteins were the highest (P < 0. 05) . Compared with the model group , the toe thickness of rats treated with different doses of puerarin decreased ; X-ray images showed that the puerarin treatment group of rats showed improvement in plantar joint stenosis and marginal bone invasion ; the results of safranin green staining showed that after treatment with different doses of puerarin , the infiltration of inflammatory cells decreased , and the expression levels of inflammatory factor IL-9 , rheumatoid factors , AKT , and FOXO1 proteins decreased significantly ( P < 0. 05 ) , with the high-dose puerarin group showing the most significant difference. Compared with the high-dose puerarin group , the positive control group showed a significant decrease in the above results and statistical differences (P < 0. 05) . Conclusion Puerarin has a good therapeutic effect on rats with RA by inhibiting the AKT-FOXO1-IL-9 pathway. The high-dose puerarin group (60 mg/kg) has the best therapeutic effect and the results show a dose-response relationship.

This study constructed a porcine reproductive and respiratory syndrome virus(PRRSV)NADC30-like strain GP3 recombinant adenovirus vector vaccine through in vitro homologous re-combination to explore its immunological efficacy evaluation at the mouse level.Using type 5 ade-novirus as a vector,a recombinant adenovirus expressing PRRSV GP3 protein was prepared and i-dentified in vitro by fluorescence observation,PCR,and Western blot analysis.Immunize mice with recombinant adenovirus and detect humoral and cellular immune responses induced by recombi-nant adenovirus using indirect ELISA and ELISpot methods.The recombinant adenovirus rAdGP3 was identified by enzyme digestion,PCR,fluorescence and Western blot,indicating that the recom-binant adenovirus rAdGP3 was successfully constructed and packaged.After immunizing mice,spe-cific antibodies and neutralizing antibodies were produced,indicating that the recombinant adenovi-rus could elicit strong humoral immunity.ELISpot and lymphocyte proliferation assays showed that the recombinant adenovirus vaccine could stimulate the secretion of IFN-γ-specific T lymphocytes and induce the proliferation of lymphocytes,indicating that the recombinant adenovi-rus could enhance the level of cellular immune response.In this study,rAdGP3 recombinant adeno-virus was successfully constructed and had good immunogenicity at the mouse level,which provid-ed a reference for the development of novel PRRSV vaccines.

Medical simulation-based assessments have emerged as a vital method for evaluating the clinical competence of healthcare professionals. These assessments provide examinees with opportunities to practice clinical skills in a controlled environment that mirrors real-life medical scenarios. However, assessment organizers must implement specific measures to ensure that these assessments yield valid and reliable results. The reliability of simulation-based assessments is a crucial component of quality assurance. This article delineates twelve practical recommendations to enhance the reliability of medical simulation-based assessments across the three key dimensions of raters, simulation environment, and assessment tools. These recommendations include ① selecting raters with relevant professional backgrounds and extensive experience in simulation-based teaching and assessment; ②providing raters with standardized training for the assessment; ③conducting mock ratings and piloting to reach consensus among raters; ④preventing excessive work hours or cognitively overload while rating; ⑤providing standardized personnel with adequate training and re-training; ⑥implementing immediate and long-term quality assurance measures for standardized patient role performance; ⑦maintaining consistent operation of simulation equipment; ⑧guaranteeing the simulation provides adequate functional fidelity for the examinees; ⑨clearly defining each element and item in assessment tools while controlling their quantity; ⑩ensuring consistency when multiple assessment tools are used; 11using assessment tools with evidence of construct validity when possible; and 12establishing systems to ensure consistency in data (scoring) collection while safeguarding data security. These strategies aim to assist medical educators in developing structured and reliable simulation-based assessment schemes.

This study constructed a porcine reproductive and respiratory syndrome virus(PRRSV)NADC30-like strain GP3 recombinant adenovirus vector vaccine through in vitro homologous re-combination to explore its immunological efficacy evaluation at the mouse level.Using type 5 ade-novirus as a vector,a recombinant adenovirus expressing PRRSV GP3 protein was prepared and i-dentified in vitro by fluorescence observation,PCR,and Western blot analysis.Immunize mice with recombinant adenovirus and detect humoral and cellular immune responses induced by recombi-nant adenovirus using indirect ELISA and ELISpot methods.The recombinant adenovirus rAdGP3 was identified by enzyme digestion,PCR,fluorescence and Western blot,indicating that the recom-binant adenovirus rAdGP3 was successfully constructed and packaged.After immunizing mice,spe-cific antibodies and neutralizing antibodies were produced,indicating that the recombinant adenovi-rus could elicit strong humoral immunity.ELISpot and lymphocyte proliferation assays showed that the recombinant adenovirus vaccine could stimulate the secretion of IFN-γ-specific T lymphocytes and induce the proliferation of lymphocytes,indicating that the recombinant adenovi-rus could enhance the level of cellular immune response.In this study,rAdGP3 recombinant adeno-virus was successfully constructed and had good immunogenicity at the mouse level,which provid-ed a reference for the development of novel PRRSV vaccines.

Medical simulation-based assessments have emerged as a vital method for evaluating the clinical competence of healthcare professionals. These assessments provide examinees with opportunities to practice clinical skills in a controlled environment that mirrors real-life medical scenarios. However, assessment organizers must implement specific measures to ensure that these assessments yield valid and reliable results. The reliability of simulation-based assessments is a crucial component of quality assurance. This article delineates twelve practical recommendations to enhance the reliability of medical simulation-based assessments across the three key dimensions of raters, simulation environment, and assessment tools. These recommendations include ① selecting raters with relevant professional backgrounds and extensive experience in simulation-based teaching and assessment; ②providing raters with standardized training for the assessment; ③conducting mock ratings and piloting to reach consensus among raters; ④preventing excessive work hours or cognitively overload while rating; ⑤providing standardized personnel with adequate training and re-training; ⑥implementing immediate and long-term quality assurance measures for standardized patient role performance; ⑦maintaining consistent operation of simulation equipment; ⑧guaranteeing the simulation provides adequate functional fidelity for the examinees; ⑨clearly defining each element and item in assessment tools while controlling their quantity; ⑩ensuring consistency when multiple assessment tools are used; 11using assessment tools with evidence of construct validity when possible; and 12establishing systems to ensure consistency in data (scoring) collection while safeguarding data security. These strategies aim to assist medical educators in developing structured and reliable simulation-based assessment schemes.

In order to explore the role of BspE protein in Brucella infection,yeast two-hybrid tech-nique was used to screen host cell proteins that interact with BspE protein.The constructed BspE recombinant plasmid pGBKT7-BspE was used as bait plasmid to hybridize with the RAW264.7-cD-NA library of mouse mononuclear macrophages by yeast two-hybridization technique.The positive clones were extracted by plasmid,sequenced and co-immunoprecipitation to determine the host cell proteins that could interact with BspE.The subcellular localization of BspE proteins was analyzed by confocal laser microscopy.The physical and chemical properties,protein structure and function of BspE interacting proteins were analyzed by bioinformatics.The siRNA for one of the BspE inter-acting proteins was synthesized,the expression of its gene was silenced in HEK293T cells,and the silenced cells was infected with Brucella M5-90 and the number of intracellular bacteria was coun-ted.The results showed that the decoy plasmid pGBKT7-BspE was successfully constructed,and the plasmid could express BspE protein in yeast.Eight positive clones were obtained from the host cell genome library by yeast two-hybridization.The positive clones were identified as RBM27 and PCBP1 by sequencing,backcross and co-immunoprecipitation.Bioinformatics was used to predict the cell location,protein structure and amino acid composition of RBM27 and PCBP1.After siRNA interference,the expression level of PCBP1 was significantly decreased and the amount of M5-90 in the cell was increased.Brucellosis secreted protein BspE interacts with host proteins RBM27 and PCBPl,and PCBP1 negatively regulates the proliferation of Brucellosis.

Objective:To explore the change of medical students' self-regulated learning from traditional medicine to clinical practice in emergency department, and to provide direction for optimizing the curriculum system and exploring new education and teaching methods.Methods:Through questionnaires and in-depth interviews, the self-regulated learning scale was applied, and the 6-level Likert scale was used for scoring. A total of 118 medical students, including 8-year undergraduates majoring in clinical medicine of Peking Union Medical College, were evaluated in the traditional medicine course stage (traditional group), emergency clinical practice stage (clinical group), and emergency clinical practice stage of the "4 + 4" reform pilot class ("4 + 4" pilot class group) of clinical medicine. The learning situation and related influencing factors were self-regulated. SPSS 23.0 was used for one-way analysis of variance.Results:Among the eight-year medical students of Peking Union Medical College, there were 48 males (40.7%) and 70 females (59.3%). The three groups of 8-year traditional medicine course stage, 8-year emergency clinical practice stage and "4 + 4" pilot class of emergency clinical practice stage were analyzed. The results showed that the total score of self-regulated learning ability in the clinical group was significantly lower than that in the traditional group [(326.2±31.9) vs. (347.7±40.2) points]. The subscales of self-regulated learning ability were analyzed respectively. In the learning motivation subscale, the score of external goal orientation of the clinical group is significantly lower than that of the traditional group [(8.9±2.3) vs. (10.0±2.9) points] and the score of the "4 + 4" pilot class group is significantly higher than that of the clinical group [(11.0±3.5) vs. (8.9±2.3) points]. In the learning strategy subscale, there was no significant difference among the three groups. In the resource management subscale, the scores of time and study environment [(6.5±1.1) vs. (7.5±1.9) points], learning management [(37.7±4.0) vs. (40.3±3.0) points] and help-seeking [(32.7±5.3) vs. (37.5±9.5) points] of the clinical group decreased significantly compared with those of the traditional group, while the scores of learning management [(40.2±7.3) vs. (37.7±4.0) points] and help-seeking [(38.7±7.6) vs. (32.7±5.3) points] of the "4+4" pilot class group increased significantly compared with those of the clinical group.Conclusion:The self-regulated learning of clinical medical students has changed significantly during the transition from traditional medical class to emergency clinical practice. The decrease of external goal orientation and resource management may be the important reason for the decline of the self-regulated learning ability of 8-year undergraduate medical students in the clinical environment during emergency practice.

Objective:To investigate the expressions of tissue inhibitor of matrix metalloproteinase-1 (TIMP1) and fibronectin 1 (FN1) in pregnancy associated breast cancer (PABC) and their correlations with expression of E-cadherin (E-cad).Methods:The clinicopathological data of 55 PABC patients in Binzhou People's Hospital Affiliated to Shandong First Medical University from January 2011 to December 2020 were retrospectively analyzed. Immunohistochemistry was used to detect expressions of TIMP1, FN1 and E-cad in cancer tissues and corresponding paracancerous tissues (>3 cm from the edge of the tumor foci). The expressions of TIMP1 and FN1 proteins in fresh intraoperative frozen cancer tissues and paracancerous tissues of 10 PABC patients were detected by Western blotting. The correlations of TIMP1 and FN1 expressions with clinicopathological characteristics of patients were analyzed by χ2 test, the correlation of TIMP1 and FN1 expressions with E-cad expression was analyzed by Spearman method, and the correlation of TIMP1 and FN1 expressions with survival was analyzed by Kaplan-Meier method. Results:The positive rates of TIMP1 and FN1 in PABC tissues were 72.7% (40/55) and 58.2% (32/55), and 25.5% (14/55) and 18.2% (10/55) in paracancerous tissues, and the differences were statistically significant ( χ2 values were 24.59 and 18.64, both P < 0.001). The results of Western blotting showed that the relative expressions of TIMP1 and FN1 proteins in the fresh cancer tissues of 10 PABC patients was higher than those in the corresponding paracancerous tissues (1.60±0.76 vs. 0.62±0.29, 1.31±0.62 vs. 0.44±0.15), and the differences were statistically significant ( t values were 5.92 and 4.86, both P < 0.001). The expressions of TIMP1 and FN1 in PABC tissues were correlated with estrogen receptor expression, Ki-67 positivity index, TNM stage and lymph node metastasis (all P < 0.05). The expressions of TIMP1 and FN1 were negatively correlated with expression of E-cad in PABC ( r values were -0.471 and -0.432, both P < 0.001). Five cases were lost to follow-up, and the remaining 50 cases had a median follow-up time of 43 months (12-90 months). Among the 50 cases, 36 cases were TMP1-positive and 29 cases were FN1-positive. The overall survival of TIMP1-negative group and FN1-negative group were better than those of the corresponding positive group ( χ2 values were 4.49 and 6.06, both P < 0.05); the median overall survival time of TIMP1-positive group and FN1-positive group were 51 months (95% CI 37-65 months) and 43 months (95% CI 32-53 months), while that of TIMP1-negative group and FN1-negative group were 89 months (95% CI 84-93 months) and 87 months (95% CI 85-92 months). Conclusions:TIMP1 and FN1 expressions are elevated in PABC tissues and negatively correlated with E-cad expression, TIMP1 and FN1 may be involved in PABC invasion through epithelial-mesenchymal transition and affect the prognosis of patients.

ObjectiveTo screen and validate key enzyme genes affecting the polysaccharide content in different Polygonatum species and perform in-depth amino acid sequence analysis by transcriptomic analysis of P. zanlanscianense， P. kingianum， and P. cyrtonema rhizomes to enrich the transcriptome data of Polygonatum plants and provide references for polysaccharide biosynthesis mechanism and genetic improvement. MethodThe Polygonatum transcriptome was sequenced and analyzed using the Illumina NovaSeq high-throughput sequencing platform， and the differences in the transcriptomes of the three Polygonatum species were compared and according to the annotations of Nr， Gene Ontology （GO）， and Kyoto Encyclopedia of Genes and Genomes （KEGG） databases. The key enzymes in the polysaccharide metabolism pathway were screened， and the expression of key enzyme genes was clustered and correlated with the polysaccharide content. Finally， Real-time polymerase chain reaction （Real-time PCR） was performed to validate the eight key enzyme genes， and the key genes of polysaccharide biosynthesis were further screened for homologous gene sequence analysis in combination with sequencing results， followed by constructing phylogenetic trees， predicting motifs， conserved structural domains， protein sequence isoelectric points， and molecular weights， and constructing 3D protein structures by using homology modeling method. ResultThe annotation of the Nr database revealed that three Polygonatum species had the highest gene homology with Asparagus officinalis. GO database annotation results showed that three Polygonatum species differed significantly in binding， catalytic activity， metabolic processes， and cellular components， while the KEGG pathway annotation results indicated that three Polygonatum species differed significantly in the starch and sucrose metabolic pathway and galactose metabolic pathway. According to clustering analysis， correlation analysis， Real-time PCR， expression profiles， and structural and functional predictions of amino acid sequences， the key enzyme significantly affecting the polysaccharide content in different Polygonatum species was inferred to be β-fructofuranosidase （sacA）. ConclusionSacA may be the main influencing factor for the difference in polysaccharide content of Polygonatum， and is also an important reason why Polygonatum polysaccharides are mainly fructans.

Objective:To compare clinical characteristics and results on parameters for muscle measurement in elderly sarcopenia patients of different age groups.Methods:This was a cross-sectional study, and 404 outpatients aged 60 years and over were enrolled and received a long-term follow-up.According to the World Health Organization classification of the elderly, the subjects were divided into a youngest-old group, a middle old group and an oldest-old group; and according to the diagnostic criteria of the Asian Working Group for Sarcopenia in 2019, elderly people of all ages were divided into a sarcopenia group and a non-sarcopenia group.General clinical data were recorded, and the upper arm circumference, leg circumference, skeletal muscle index, grip strength and walking speed, the Short Physical Performance Battery and timed up and go were conducted.At the same time, the body composition was measured by multifrequency bioelectrical impedance analysis.Results:The subjects had a mean age of(85.24 ± 8.23)years, including 90(22.28%)youngest-old, 165(40.84%)middle old and 149(36.88%)oldest-old.123 patients(30.45%)were diagnosed with sarcopenia, with a prevalence of 27.78%(25)in the youngest-old group, 28.48%(47)in the middle old group and 34.23%(51)in the oldest-old group.Compared with those without sarcopenia, the basal metabolic rate in sarcopenia patients of all age groups decreased significantly, the levels of hemoglobin and albumin in youngest-oldsarcopenia patients decreased, the waist circumference and body mass index in middle old and oldest-old sarcopenia patients decreased, and the body fat rate in middle old sarcopenia patients increased.In the subjects with sarcopenia, the proportion of men in the oldest-old group was higher than in the other two groups.Compared with the youngest-old group, the levels of alanine aminotransferase and prealbumin decreased, serum creatinine increased, and skeletal muscle index values did not change in the middle old and longest-old groups, but handgrip strength(27.7±5.9 vs.23.2±6.9 vs.21.4±5.8, F=5.81, P=0.004), gait speed(0.8±0.2 vs.0.7±0.2 vs.0.5±0.2, F=11.88, P=0.000)and calf circumference(33.4±3.3 vs.15.9± 8.4 vs.31.5±3.1 vs.30.9±3.3, F=3.58, P=0.031)significantly decreased, and time up and go values(10.0±2.1 vs.15.9±8.4 vs.20.8±12.8, F=6.98, P=0.001)increased in the middle old and longest-old groups.Partial correlation analysis showed that age had a significant negative correlation with handgrip( r=-0.374, P=0.001), daily gait speed( r=-0.441, P=0.000), and calf circumference( r=-0.223, P=0.017), but a significant positive correlation with timed up and go( r=0.319, P=0.009). Conclusions:Compared with youngest-old sarcopenia patients, middle old patients show significant decline in muscle function, muscle strength and muscle mass in the lower limb.With increasing age, the impact of changes in muscle function, muscle strength and regional muscle mass on adverse events should receive heightened attention.