This study aims to establish a high-performance size-exclusion chromatography (HPSEC) method for determining the content of the classical swine fever virus (CSFV) E2 protein and screen the optimal stabilizer to enhance the stability of this protein. The optimal detection conditions were determined by optimizing the composition of the mobile phase, and characteristic chromatographic peaks were identified by SDS-PAGE and Western blotting. The specificity, repeatability, precision, linearity, limit of detection (LOD), and limit of quantitation (LOQ) of the method were assessed. The method established was used to determine the content of CSFV E2 protein antigen and vaccine. Differential scanning fluorimetry (DSF) was employed to screen the buffer system, pH, and salt ion concentrations, and sugar, amino acid, and alcohol stabilizers were further screened. The results showed that using a 200 mmol/L phosphate buffer provided the best column efficiency. An antigen-specific chromatographic peak appeared at the retention time of 18 min, which was identified as the CSFV E2 protein by SDS-PAGE and Western blotting. The method exhibited high specificity for detecting the CSFV E2 protein, with no absorbance peak observed in the blank control. The relative standard deviation (RSD) of the peak area for six repeated injections of the CSFV E2 protein was 0.74%, indicating good repeatability of the method. The RSD for repeated detection of two different concentrations of CSFV E2 protein samples by different operators at different time points was less than 2%, suggesting good intermediate precision of the method. The peak area of the CSFV E2 protein was linearly related to its concentration, with the regression equation showing R² of 1.000. The LOD and LOQ of the method were 14.88 μg/mL and 29.75 μg/mL, respectively. Application of the developed method in the detection of three batches of CSFV E2 protein antigen and three batches of vaccine demonstrated results consistent with those from the bicinchoninic acid (BCA) assay, which meant that the method could accurately determine the content of CSFV E2 protein antigen and vaccine. The DSF method identified 50 mmol/L Tris-HCl at pH 8.0 as the optimal buffer, and the addition of sugar and alcohol stabilizers further improved the stability of the CSFV E2 protein. The HPSEC method established in this study is simple, fast, and exhibits good accuracy and repeatability, enabling precise measurement of the CSFV E2 protein content. It is expected to play a crucial role in the quality control of the CSFV E2 vaccine. Furthermore, the strategy for improving the CSFV E2 protein stability, identified through DSF screening, has significant implications for enhancing the stability of the CSFV E2 vaccine.
Classical Swine Fever Virus/chemistry* ; Chromatography, Gel/methods* ; Animals ; Swine ; Viral Envelope Proteins/immunology*

BACKGROUND: In China, lung cancer remains the cancer with the highest incidence and mortality rate. Among early-stage lung adenocarcinomas (LUAD), the micropapillary (MPP) component is prevalent and typically exhibits high aggressiveness, significantly correlating with early metastasis, lymphatic infiltration, and reduced five-year survival rates. Therefore, the study is to explore the similarities and differences between MPP and non-micropapillary (non-MPP) components in malignant pulmonary nodules characterized by GGOs in early-stage LUAD, identify unique mutational features of the MPP component and analyze the relationship between the ZNF469 gene, a member of the zinc-finger protein family, and the prognosis of early-stage LUAD, as well as its correlation with immune infiltration. METHODS: A total of 31 malignant pulmonary nodules of LUAD were collected and dissected into paired MPP and non-MPP components using microdissection. Whole-exome sequencing (WES) was performed on the components of early-stage malignant pulmonary nodules. Mutational signatures analysis was conducted using R packages such as maftools, Nonnegative Matrix Factorization (NMF), and Sigminer to unveil the genomic mutational characteristics unique to MPP components in invasive LUAD compared to other tumor tissues. Furthermore, we explored the expression of the ZNF469 gene in LUAD using The Cancer Genome Atlas (TCGA) database to investigate its potential association with the prognosis. We also investigated gene interaction networks and signaling pathways related to ZNF469 in LUAD using the GeneMANIA database and conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Lastly, we analyzed the correlation between ZNF469 gene expression and levels of immune cell infiltration in LUAD using the TIMER and TISIDB databases. RESULTS: MPP components exhibited a higher number of genomic variations, particularly the 13th COSMIC (Catalogue of Somatic Mutations in Cancer) mutational signature characterized by the activity of the cytidine deaminase APOBEC family, which was unique to MPP components compared to non-MPP components in tumor tissues. This suggests the potential involvement of APOBEC in the progression of MPP components in early-stage LUAD. Additionally, MPP samples with high similarity to APOBEC signature displayed a higher tumor mutational burden (TMB), indicating that these patients may be more likely to benefit from immunotherapy. The expression of ZNF469 was significantly upregulated in LUAD compared to normal tissue, and was associated with poor prognosis in LUAD patients (P<0.05). Gene interaction network analysis and GO/KEGG enrichment analysis revealed that COL6A1, COL1A1, COL1A2, TGFB2, MMP2, COL8A2 and C2CD4C interacted with ZNF469 and were mainly involved in encoding collagen proteins and participating in the constitution of extracellular matrix. ZNF469 expression was positively correlated with immune cell infiltration in LUAD (P<0.05). CONCLUSIONS The study has unveiled distinctive mutational signatures in the MPP components of early-stage invasive LUAD in the Asian population. Furthermore, we have identified that the elevated expression of mutated ZNF469 impacts the prognosis and immune infiltration in LUAD, suggesting its potential as a diagnostic and prognostic biomarker in LUAD.
Humans ; Lung Neoplasms/genetics* ; Adenocarcinoma of Lung/genetics* ; China ; Prognosis ; Transcription Factors

Objective:To compare the clinical effects of minimally invasive reduction through a bone tunnel combined with Jail screwing and those of posterolateral locking plating in the treatment of simple posterolateral tibial plateau fractures.Methods:A retrospective analysis was conducted of the data of 48 patients who had been operatively treated and completely followed up at Department of Orthopedics, Suqian Hospital of Nanjing Drum Tower Hospital Group for simple posterolateral tibial plateau fractures from October 2016 to October 2020. There were 26 males and 22 females, aged from 35 to 68 years. They were divided into a minimally invasive group (25 cases subjected to minimally invasive reduction through a bone tunnel combined with Jail screwing) and an incision group (23 cases subjected to posterolateral locking plating) according to their surgical methods. The operation time, incision length, intraoperative blood loss, fracture healing time, cumulative fluoroscopy time, hospital stay and posterior inclination angles of the tibial plateau and Hospital for Special Surgery (HSS) knee function scores at 1, 3, 6, 9, and 12 months after operation were compared between the 2 groups. Complications in the 2 groups of patients were recorded.Results:There was no significant difference in the preoperative general data between the 2 groups, showing comparability ( P>0.05). The 48 patients were followed up for 12 to 36 months (average 16.5 months). The minimally invasive group was significantly better than the incision group in operation time [(42.6±9.1) min versus (65.7±11.5) min], incision length [(4.0±0.4) cm versus (15.0±1.5) cm], intraoperative blood loss[(22.6±5.8) mL versus (31.5±8.8) mL], hospital stay [(7.6±1.4) d versus (11.1±2.4) d], and HSS score one month after operation [(84.8±1.9) points versus (72.9±4.1) points], but the cumulative fluoroscopy time in the incision group [(4.1±1.4) s]was significantly less than that in the minimally invasive group [(22.3±4.2) s] ( P<0.05). There were no significant differences in fracture healing time, HSS scores at 3, 6, 9, or 12 months after operation, or posterior inclination angle of the tibial plateau between the 2 groups ( P>0.05). There were no such complications as wound infection, vascular injury, internal fixation failure, nonunion or malunion of fractures in either of the 2 groups. Two cases in the incision group presented with symptoms of common peroneal nerve injury but recovered 3 months after operation. Conclusions:Although both minimally invasive reduction through a bone tunnel combined with Jail screwing and posterolateral locking plating can achieve satisfactory outcomes in the treatment of simple posterolateral tibial plateau fractures, the minimally invasive technique is preferable because it shows the advantages of a smaller incision, less bleeding, shorter operation time, a lower operation risk, quicker postoperative recovery and shorter hospital stay.

To rapidly and accurately manipulate genome such as gene deletion, insertion and site mutation, the whole genome of a very virulent strain Md5 of Marek's disease virus (MDV) was inserted into bacterial artificial chromosome (BAC) through homogeneous recombination. The recombinant DNA was electroporated into DH10B competent cells and identified by PCR and restriction fragment length polymorphism analysis. An infectious clone of Md5BAC was obtained following transfection into chicken embryo fibroblast (CEF) cells. Furthermore, a lorf10 deletion mutant was constructed by two step Red-mediated homologous recombination. To confirm the specific role of gene deletion, the lorf10 was reinserted into the original site of MDV genome to make a revertant strain. All the constructs were rescued by transfection into CEF cells, respectively. The successful packaging of recombinant viruses was confirmed by indirect immunofluorescence assay. The results of growth kinetics assay and plaques area measurement showed that the lorf10 is dispensable for MDV propagation in vitro. Overall, this study successfully constructed an infectious BAC clone of MDV and demonstrated its application in genome manipulation; the knowledge gained from our study could be further applied to other hepesviruses.
Animals ; Chick Embryo ; Chickens ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; Herpesvirus 2, Gallid/genetics* ; Marek Disease

Objective:To explore the effect and molecular mechanism of capsaicin receptor(TRPV1) on neuronal autophagy and depression-like behavior in mice.Methods:Using the method of random number table, 87 C57 male mice were divided into Sham operation group (Sham group), cerebral ischemia/reperfusion group (I/R group) and capsazepine(CPZ) preconditioning cerebral ischemia/reperfusion group (I/R+ CPZ group), with 28 mice in each group due to 3 incompatible.Mice in the I/R group were subjected to right middle cerebral artery occlusion (MCAO) to establish a cerebral ischemia-reperfusion model.Mice in the I/R+ CPZ group were injected with CPZ in the lateral ventricle prior to moulding.Mice in the Sham group had only wire plugs inserted and no arterial embolization was performed.The mNSS score was used to evaluate the degree of neurological deficits.The depression-like behaviour of mice was detected by the tail suspension test and forced swimming test.The infarct volume was observed by TTC staining.The pathological changes in the amygdala were observed by HE staining, and the expression of Beclin-1, LC3, p62 and p-PI3K, p-AKT and p-mTOR proteins were detected by Western blot.Statistical analysis was performed using SPSS 23.0 software.The t-test was used for comparison between two groups and one-way ANOVA was used for comparison of multiple group. Results:The neurological deficit score in I/R+ CPZ group (9.77±2.32) was significantly lower than that in I/R group (12.85±2.73) ( t=3.10, P<0.01). Compared with I/R group, the tail suspension immobility time of I/R+ CPZ Group ((93.28±50.69)s, (143.80±35.61) s; t=2.94, P<0.01) and the forced swimming immobility time ((139.50±13.33)s, (175.30±19.78)s; t=2.94, P<0.01) were significantly reduced.The results of TTC staining showed that the cerebral infarct volume in I/R+ CPZ group was significantly lower than that in I/R group ((19.30±5.19)%, (33.60±3.90)%; t=5.40, P<0.01). HE staining showed that the number of cells in the amygdala region of mice in the I/R+ CPZ group increased compared with that in the I/R group, with tighter arrangement and reduced deep staining of nuclear fixation.Western blot showed that compared with I/R group, the expression levels of autophagy related proteins Beclin-1( t=2.94, P<0.05) and LC3 ( t=3.16, P<0.05) in amygdala of I/R+ CPZ group were down-regulated, while the expression levels of p62( t=3.60, P<0.05), p-PI3K ( t=7.79, P<0.01), p-AKT ( t=4.15, P<0.01) and p-mTOR ( t=6.15, P<0.01) were up-regulated. Conclusion:Cerebral ischemia/reperfusion activates neuronal autophagy, and CPZ may regulate the PI3K-AKT-mTOR pathway, thus inhibits excessive activation of autophagy, thereby acting as a neuroprotective agent and improving post-stroke depression-like behaviour.

Objective To study the common etiological components and bedside diagnostic skills of isolated transient vertigo (ITV).Methods We performed a single-center observational study that had consecutively recruited 424 middle-aged and elderly patients presenting with vertigo episodes lasted less than three minutes in the Neurology Department of Shanghai Tongji Hospital from January 2018 to January 2020.The clinical characteristics were collected and analyzed according to the established format and vertigo causes were analyzed.Binary Logistic regression analysis was used to determine some clinical parameters to benign paroxysmal positional vertigo (BPPV) and vascular ITV (vITV).Results In all 424 patients,BPPV accounted for 65.09% (n=276),vITV for 20.51% (n=87).Fifty-two cases (18.84%) of BPPV were diagnosed as posterior circulation ischemia (PCI) and 12 cases (14.63%) of vITV were suspected as BPPV.Logistic Regression analysis showed that onset time of vertigo<30 s,vertigo provoked by lying down or turning over in the supine position and typical nystagmus by positional maneuver were the independent predicted factors of BPPV (P<0.001).The higher risk of vITV was found in association with intolerance of head movement,trunk balance disturbance,spontaneous nystagmus and intolerance by positional maneuver (P<0.001).There was no independent correlation between vascular risk factors and vITV (P>0.05).Conclusion BPPV and vITV are the most common etiologies of ITV in the middle-aged and elderly patients.The etiology of ITV can be quickly and effectively distinguished by some specific clinical features and target bedside examinations.

Objective To investigate the efficacy and safety of dipeptidyl peptidase 4(DDP-4)inhibitor in the treatment of diabetes mellitus.Methods Eighty-six cases patients with poor glycemic control in type 2 diabetes mellitus in Shunde First People's Hospital of Foshan from May 2015 to May 2016 were selected as the research objects and divided into two groups according to random number table method,each group with 43 cases.The control group with acarbose(50 mg/times,3 times/d,with the same as the 3 meals,sustained medication for 12 weeks)to control blood sugar,while the observation group used DPP-4 inhibitors sitagliptin(100 mg/times,1 times/d,sustained medication for 12 weeks),compared the blood glucose,blood lipid indexes and adverse reactions of two groups before and after treatment.Results After treatment,FPG,2 hPG,HbAlc level of observation group were respectively(6.71±0.65)mmol/L,(8.10±0.17)mmol/L,(7.12±0.41)％,significantly lower than control group((7.86±0.72)mmol/L,(9.20±0.65)mmol/L,(7.51±0.52)％,the differences were statistically significant(P＜0.01).After treatment,there were no significant differences in terms of TC,TG,HDL-C,LDL-C between two groups(P＞0.05).Treatment for 12 weeks,the blood glucose compliance rate of observation group was 88.37％(38/43),the control group was 67.44％(29/43),the observation group was significantly higher than the control group(P=0.019).After treatment,the HOMA-IR value of the observation group was 4.42±0.17,significantly lower than the control group(4.91±0.24),HOMA-beta value was 88.20±6.31,significantly higher than that of the control group(80.21±5.67),the differences were significant(P＜0.01).Conclusion DDP-4 inhibitor in the treatment of type 2 diabetes,can effectively reduce the level of blood glucose and glycosylated hemoglobin,and have no significant adverse reactions,is effective and safe hypoglycemic drugs.

Background and objective Brain metastasis was frequent in non-small cell lung cancer (NSCLC) patients with poor prognosis. Geiftinib was an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor which has been used in the treatment of NSCLC. Our study was to evaluate the effcacy and toxicities of geiftinib in lung adenocarcinoma patients with brain metastases. Methods We retrospectively reviewed clinical records of 32 lung adenocarcinoma patients with brain metastases, who had received geiftinib 250 mg Qd until disease progression or intolerable toxicities. Results hTe median overall survival (mOS) and median progression-free survival (mPFS) were 24.7 months and 11.2 months, respectively. Response rate (RR) and disease control rate (DCR) were 62.5%and 93.8%, respectively. hTe mOS and mPFS of geiftinib-naive patients were 35.6 months and 11.3 months, respectively, and RR and DCR were 75.0%and 100.0%, respectively. hTe mOS and mPFS of geiftinib treatment patients were 18.6 months and 6.7 months, respectively, and RR and DCR were 50.0%and 83.3%, respectively. hTe mOS and mPFS of patients with sensitive EGFR mutation were 24.8 months and 10.8 months, respectively, and RR and DCR were 75.0%and 100.0%, respectively. hTe mOS and mPFS of patients with unknown EGFR status were 35.6 months and 12.3 months, respectively, and RR and DCR were 53.3%and 86.7%, respectively. Treatment was well tolerated and no severe toxicities were observed. Common toxicities include:rash in 15 patients (46.9%), diarrhea in 7 cases (21.9%) and oral ulcer in 1 case (3.1%). Conclusion Geiftinib was highly effective and well tolerated in lung adenocarcinoma patients with brain metastases, and could be recommended as a treatment choice for this population.

Background and objective Lung cancer in young patients (less or equal to 45 years) is relatively rare. We explored the effcacy and survival of Geiftinib for young patients with unknown epidermal growth factor receptor (EGFR) gene mutation of advanced lung adenocarcinoma. Methods hTe clinical data of 55 young patients with unknown EGFR gene mutation in advanced lung adenocarcinoma referred to the Cancer Hospital&Institute, Chinese Academy of Medical Sciences from Jan 2006 through Dec 2010 were analyzed retrospectively. Results Of 55 young patients enrolled, the me-dian age was 41 years. hTe objective response rate and disease control rate were 43.6%and 90.9%, respectively. hTe median progression-free survival (PFS) was 9.0 months. Among the factors analyzed, brain metastasis had signiifcant effect on PFS (P=0.017). hTe median overall survival (OS) was 24.0 months. hTe independent prognostic factors to signiifcantly improve OS included non-smoking history (P=0.028) and receiving other anti-cancer treatment atfer Geiftinib therapy (P<0.001). Conclusion hTe median PFS and OS of the young patients with Unknown EGFR gene mutation in advanced lung adeno-carcinoma were similar with general population.