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 explore the status and influencing factors of stress disorder in different periods of postpartum women.Methods:From October 2019 to April 2021, convenience sampling was used to select 332 puerperae from 8 community health service stations in Xi'an as the research object. The Posttraumatic Stress Checklist-Civilian version (PCL-C) was used to investigate the puerperae in T1 (1 to 3 months postpartum) , T2 (4 to 6 months postpartum) , T3 (7 to 12 months postpartum) .Results:Among 332 puerperae, there were no significant differences in the posttraumatic stress disorder (PTSD) positive rate, PTSD total score and symptom cluster scores in T1, T2 and T3 ( P>0.05) . From 1 to 3 months postpartum, occupation, husband participation, sleep, delivery mode, neonatal gender coincidence and initial delivery were the influencing factors of postpartum PTSD ( P<0.05) . From 4 to 6 months postpartum, pregnancy complications was the factor affecting postpartum PTSD ( P<0.05) . From 7 to 12 months, sleep was the influencing factor of postpartum PTSD ( P<0.05) . Conclusions:PTSD was relatively stable within one year in postpartum women. Targeted intervention measures should be taken according to the influencing factors of PTSD in different postpartum periods.

Objective:To investigate the effect of blocking P21 activated kinase 1 (PAK1) activity on the proliferation, differentiation, and apoptosis of acute megakaryocytic leukemia (AMKL) cell lines (CHRF and CMK) .Methods:Cell counts were used to detect the effects of PAK1 inhibitors (IPA-3 and G5555) on AMKL cell proliferation inhibition and colony formation, and flow cytometry was used to detect its effects on AMKL cell cycle. The effect of PAK1 inhibitor on the expression of cyclin D1 and apoptosis-related protein Cleaved caspase 3 was detected using Western blot, while interference with the protein expression level of PAK1 in AMKL cells was assessed using lentivirus-mediated shRNA transfection technology. Flow cytometry was used to detect the effects of knockdown of PAK1 kinase activity on the ability of polyploid DNA formation and cell apoptosis in AMKL cells.Results:PAK1 inhibitors inhibited the proliferation of AMKL cells in a dose-dependent manner and reduced the ability of cell colony formation, and the difference was statistically significant when compared with the control group ( P<0.05) . Moreover, they also reduced the percentage of AMKL cells in S phase, and Western blot detection showed that the expression levels of phosphorylated PAK1 and cyclin D1 decreased significantly. Finally, PAK1 inhibitors induced AMKL cell apoptosis by up-regulating Cleaved caspase 3 and showed different abilities to increase the content of polyploid DNA in megakaryocytes. Only high concentrations of IPA-3 and low doses of G5555 increased the number of polyploid megakaryocytes, while knockdown of PAK1 kinase activity promoted AMKL cell differentiation and increased the apoptosis rate. Conclusion:PAK1 inhibitor significantly arrests AMKL cell growth and promotes cell apoptosis. Knocking down the expression of PAK1 promotes the formation of polyploid DNA and induces AMKL cell apoptosis. The above findings indicate that inhibiting the activity of PAK1 may control AMKL effectively.

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

In the nucleus, chromatin is folded into hierarchical architecture that is tightly linked to various nuclear functions. However, the underlying molecular mechanisms that confer these archi-tectures remain incompletely understood. Here, we investigated the functional roles of H3 lysine 9 dimethylation (H3K9me2), one of the abundant histone modifications, in three-dimensional (3D) genome organization. Unlike in mouse embryonic stem cells, inhibition of methyltransferases G9a and GLP in differentiated cells eliminated H3K9me2 predominantly at A-type (active) genomic compartments, and the level of residual H3K9me2 modifications was strongly associated with B-type (inactive) genomic compartments. Furthermore, chemical inhibition of G9a/GLP in mouse hepatocytes led to decreased chromatin-nuclear lamina interactions mainly at G9a/GLP-sensitive regions, increased degree of genomic compartmentalization, and up-regulation of hundreds of genes that were associated with alterations of the 3D chromatin. Collectively, our data demonstrated essential roles of H3K9me2 in 3D genome organization.

Objective:To study the differences in the bone marrow histopathology between acquired aplastic anemia (AAA) in children and refractory cytopenia of childhood (RCC) to facilitate their diagnoses and differential diagnosis.Methods:The clinical data and bone marrow biopsies of the RCC and AAA cases diagnosed from January 2008 to December 2018 in Xinhua Hospital, Shanghai Jiaotong University School of Medicine and Shanghai Children′s Medical Center affiliated to Shanghai Jiaotong University School of Medicine were analyzed.Results:A total of 71 AAA and 79 RCC cases were analyzed. There were 52 males and 19 females, age ranged 1.0-15.0 years (median, 8.9 years) in the AAA group, and 53 males and 26 females, age ranged 0.5-16.0 years (median, 5.0 years) in the RCC group. All the biopsy specimens of AAA patients had severe hypocellularity; the cellularity of 88.7% (63/71) specimens was under 5.0%, and 11.3%(8/71) was 5%-24%. None of the AAA specimens showed any dysplastic change. All the biopsy specimens of RCC patients had hypocellularity, including 94.9%(75/79) of the specimens with a cellularity of 5%-50%. All of the RCC specimens showed a patchy distribution of hematopoiesis. A dysplastic change of erythroid cells and micromegakaryocytes was found in 40.5% (32/79) and in 60.8% (48/79) of the RCC cases, respectively.Conclusions:The degree of hypocellularity, the distribution pattern of hematopoiesis, the cell composition and localization of erythroid cell clusters and the appearance of micromegaryocytes could help the diagnosis and differential diagnosis of AAA and RCC.

Objective To examine the efficacy and safety of 125Iseed implantation for treating neuroblastoma (NB) in animal models.Methods A total of 45 nude mice models of neuroblastoma were constructed and divided into the 125Igroup.control group.and blank group at 15 mice per group.The long and short diameters of the tumor were measured every 3 days.and the tumor inhibition rate was calculated every 9 days.Apoptotic and proliferative protein expression levels in tumor tissue and peritumoral tissue.as well as endocrine markers and bone marrow of the nude mice.were analyzed.The independent sample t test was used to compare the mean scores.and ANOVA was used for comparison between multiple groups.Results Tumor volume inhibition rate was significantly higher in the 125Igroup than in the control group and blank group on days 9.18.and 27(all P<0.05).Caspase-3 expression in tumor tissues was significantly higher in the 125Igroup than in the control group and blank group (all P<0.05).whereas proliferating cell nuclear antigen (PCNA) expression was significantly lower in the 125Igroup than in the control group and blank group (all P<0.05).There was no significant difference in Caspase-3 and PCNA expression between the control group and blank group (all P>0.05).In addition.no significant difference in the expression of Caspase-3 and PCNA in peritumoral tissue was observed between the 125Igroup.control group.and blank group (all P>0.05).Cell apoptosis in tumor tissue was significantly lower in the blank group and control group than in the 125Igroup (all P<0.05).while there was no significant difference between the blank group and the control group (P>0.05).There was no significant difference in endocrine markers between the three groups (P>0.05).There was no significant bone marrow suppression in the 125Igroup.and this observation was similar to those in the control group and blank group (all P>0.05).Conclusions 125Iseeds have significant toxicity to NB.125Iseed implantation is safe in nude mice with NB within the therapeutic doses.

Objective To explore the clinicopathological characters of T2 gastric cancer and the value of MSCT in the preop‐erative TNM staging of T2 gastric cancer .Methods A total of 93 patients with T2 gastric cancer were included in our study and un‐derwent preoperative MSCT staging ,who were confirmed by pathologic results .Then the results were compared with those of path‐ologic TNM staging .Also the clinicopathological features of the T2 gastric cancer were analyzed .Results There were no statistical‐ly significant differences in the clinicopathological characters among T2a and T2b patients (P>0 .05) .Comparing with pathologic TNM stage ,the T staging accuracy of MSCT was 91 .40% (85/93) ,the N staging accuracies of CT was 66 .67% (62/93) ,in which , 68 .18% (30/44) ,65 .00% (26/40) ,60 .00% (3/5) and 75 .00% (3/4) were for pN0 ,pN1 ,pN2 and pN3 .And the TNM staging ac‐curacies of CT was 67 .74% (63/93) ,in which ,68 .18% (30/44) ,64 .10% (25/39) ,60 .00% (3/5) and 100% (5/5) were for stageⅠ ,Ⅱ ,Ⅲ and Ⅳ .Conclusion There are no significant different on clinicopathology features among T2a and T2b patients .MSCT can clearly determine the preoperative TNM staging of T2 gastric cancer .