OBJECTIVES: To investigate the role and mechanism of Brahma-related gene 1 (<i>Brg1i>) in regulating the Wnt/β-catenin signaling pathway in a bronchopulmonary dysplasia (BPD) model. METHODS: Wild-type C57BL/6 and <i>Brg1i>^f1/f1 mice were randomly divided into four groups: wild-type control, wild-type BPD, <i>Brg1i>^f1/f1 control, and <i>Brg1i>^f1/f1 BPD (<i>ni>=5 each). Immortalized mouse pulmonary alveolar type 2 cells (imPAC2) were cultured, and <i>Brg1i> gene was knocked down using lentivirus transfection technology. Cells were divided into three groups: control, empty vector, and <i>Brg1i> knockdown. Hematoxylin and eosin staining and immunofluorescence were used to detect pathological changes in mouse lung tissue. Western blot and real-time fluorescent quantitative PCR were used to measure Brg1 protein and mRNA expression levels in mouse lung tissue. Western blot and immunofluorescence were used to detect the expression of homeodomain-containing protein homeobox (HOPX), surfactant protein C (SPC), and Wnt/β-catenin signaling pathway proteins in mouse lung tissue and imPAC2 cells. The CCK8 assay was used to assess the proliferation of imPAC2 cells, and co-immunoprecipitation was performed to verify the interaction between Brg1 and β-catenin proteins in imPAC2 cells. RESULTS: Compared to the <i>Brg1i>^f1/f1 control group and wild-type BPD group, the <i>Brg1i>^f1/f1 BPD group showed increased alveolar diameter and SPC protein expression, and decreased relative density of pulmonary vasculature and HOPX protein expression (<i>Pi><0.05). Compared to the control group, the <i>Brg1i> knockdown group showed increased cell proliferation ability, protein expression levels of SPC, Wnt5a and β-catenin, and β-catenin protein fluorescence intensity, along with decreased HOPX protein expression (<i>Pi><0.05). An interaction between Brg1 and β-catenin proteins was confirmed. CONCLUSIONS The <i>Brg1i> gene may promote the proliferation of alveolar type 2 epithelial cells by regulating the Wnt/β-catenin signaling pathway, thus influencing the occurrence and development of BPD.
Animals ; DNA Helicases/genetics* ; Transcription Factors/genetics* ; Wnt Signaling Pathway/physiology* ; Nuclear Proteins/genetics* ; Mice ; Bronchopulmonary Dysplasia/etiology* ; Mice, Inbred C57BL ; beta Catenin/physiology* ; Disease Models, Animal ; Cell Proliferation ; Lung/pathology* ; Male

SMARCA4-deficient non small cell lung cancer (SMARCA4-dNSCLC) has recently garnered increasing attention due to its high malignancy and poor prognosis. The literature suggests that in non small cell lung cancer (NSCLC), the loss of SMARCA4 frequently co-occurs with mutations in KRAS, KEAP1, and STK11 rather than in EGFR, ALK, and ROS1. Herein, we present the first documented case of SMARCA4-dNSCLC accompanied with rare mutations of EGFR exon 20 S768I and exon 18 G719X. The patient achieved partial response with afatinib for 17 months. Our case highlights the importance of EGFR mutations in the precision targeted treatment of SMARCA4-dNSCLC.
Humans ; Afatinib/therapeutic use* ; Antineoplastic Agents/therapeutic use* ; Carcinoma, Non-Small-Cell Lung/pathology* ; DNA Helicases/genetics* ; ErbB Receptors/genetics* ; Lung Neoplasms/pathology* ; Mutation ; Nuclear Proteins/genetics* ; Transcription Factors/genetics*

Sini Decoction (SNT) is a traditional formula recognized for its efficacy in warming the spleen and stomach and dispersing cold. However, elucidating the mechanism of action of SNT remains challenging due to its complex multiple components. This study utilized a synergistic approach combining two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE)-based drug affinity responsive target stability (DARTS) with label-free quantitative proteomics techniques to identify the direct and indirect protein targets of SNT in myocardial infarction. The analysis identified 590 proteins, with 30 proteins showing significant upregulation and 51 proteins showing downregulation when comparing the SNT group with the model group. Through the integration of 2D-DIGE DARTS with proteomics data and pharmacological assessments, the findings indicate that protein disulfide-isomerase A3 (PDIA3) may serve as a potential protein target through which SNT provides protective effects on myocardial cells during myocardial infarction.
Myocardial Infarction/genetics* ; Proteomics/methods* ; Drugs, Chinese Herbal/chemistry* ; Animals ; Protein Disulfide-Isomerases/genetics* ; Male ; Two-Dimensional Difference Gel Electrophoresis/methods* ; Humans ; Rats ; Rats, Sprague-Dawley ; Electrophoresis, Gel, Two-Dimensional

<i>Bacillus coagulansi> can utilize the hydrolyzed carbon source of agricultural waste to produce lactic acid <i>viai> a homofermentative pathway. However, a significant carbon source metabolic repression effect was observed when the strain metabolized mixed sugars (glucose and xylose), reducing the productivity of lactic acid. In this study, we obtained the fermentation conditions for the simultaneous utilization of the mixed sugars by <i>Bi>. <i>coagulansi> by changing the ratio of glucose to xylose in the medium. Through transcriptome sequencing, several key genes responsible for xylose utilization were identified. The critical role of xylose isomerase (XylA, EC 5.3.1.5) in the synchronous utilization of glucose/xylose in <i>Bi>. <i>coagulansi> was investigated <i>viai> qRT-PCR (quantitative real-time polymerase chain reaction). Subsequently, the heterologous expression and characterization of the XylA-encoding gene (<i>XylAi>) were conducted. It was determined that the gene encoded a protein composed of 440 amino acid residues. The secondary structure of the encoded protein was predominantly composed of α-helixes and random coils, while the higher structure of the protein was identified as a homotetramer. Then, <i>XylAi> was cloned and expressed in <i>Escherichia colii> BL21(DE3), and the recombinant protein Bc-XlyA was obtained with a molecular weight of approximately 50 kDa. The optimal pH and temperature of Bc-XylA were 8.0 and 60 ℃, respectively, and Mn²⁺, Mg²⁺, and Co²⁺ had positive effects on the activity of Bc-XlyA. The present study provides scientific data on the molecular modification of <i>Bi>. <i>coagulansi>, offering theoretical support for the efficient utilization of xylose in the strain.
Xylose/metabolism* ; Cloning, Molecular ; Bacillus coagulans/enzymology* ; Aldose-Ketose Isomerases/metabolism* ; Fermentation ; Bacterial Proteins/metabolism* ; Glucose/metabolism*

Geminiviruses cause substantial crop yield losses worldwide. The replication initiator protein (Rep) encoded by geminiviruses is indispensable for geminiviral replication. The Rep protein encoded by beet severe curly top virus (BSCTV, genus <i>Curtovirusi>, family <i>Geminiviridaei>) induces <i>VARIANT IN METHYLATION 5i> (<i>VIM5i>) expression in <i>Arabidopsisi> leaves upon BSCTV infection. VIM5 functions as a ubiquitination-related E3 ligase to promote the proteasomal degradation of methyltransferases, resulting in reduction of methylation levels in the BSCTV C2-3 promoter. However, the specific domains of Rep responsible for <i>VIM5i> induction remain poorly characterized. Although Rep proteins from several geminiviruses act as viral suppressors of RNA silencing (VSRs), whether BSCTV Rep also possesses VSR activity remains to be illustrated. In this study, we employed a transient expression system in the 16c-GFP transgenic and the wild-type <i>Nicotiana benthamianai> plants to analyze the VSR and the <i>VIM5i>-inducing activities of different truncated Rep proteins haboring distinct domains. We found that the N-terminal domain (amino acids 1-180) of Rep suppressed <i>GFPi> silencing in 16c-GFP transgenic <i>Ni>. <i>benthamianai> leaves. The minimal N-terminal fragment (amino acids 1-104) induced <i>VIM5i> expression upon co-infiltration, while C-terminal truncations lacked <i>VIM5i>-inducing activity. Our results indicate that the N-terminal domain of Rep encoded by BSCTV mediates the suppression of RNA silencing and induces <i>VIM5i> expression. Thus, our findings contribute to a better understanding of interactions between geminiviral Rep and plant hosts.
Geminiviridae/genetics* ; Nicotiana/metabolism* ; Arabidopsis/metabolism* ; RNA Interference ; Viral Proteins/metabolism* ; Arabidopsis Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Protein Domains ; Plant Diseases/virology* ; Methyltransferases/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; DNA Helicases/genetics*

Porcine deltacoronavirus (PDCoV) is a major pathogen causing fatal diarrhea in suckling piglets, and there is currently a lack of effective vaccines and drugs to prevent and control the virus. The nonstructural protein 13 (NSP13) serves as a virus-coded helicase and is considered to be a crucial target for antiviral drugs, making it imperative to investigate the helicase activity of NSP13. In this study, the <i>NSP13i> gene of PDCoV was synthesized and integrated into the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-NSP13. NSP13 was successfully expressed in BL21 (DE3) and subsequently purified. The study also verified the helicase activity of the purified NSP13 and explored the factors that influence this activity. The results indicated that NSP13 from PDCoV was effectively expressed in the prokaryotic system and exhibited helicase activity, capable of unwinding double-stranded DNA with a tail at the 5' end. Additionally, NSP13 demonstrated an annealing function by promoting the complementary pairing of single-stranded nucleotide chains to form double strands. The helicase activity of NSP13 was affected by metal ions, but Mg²⁺concentrations in the range of 0.5-6.0 mmol/L had no significant effect on helicase activity of NSP13. When the solution pH was in the range of 4-9, there was no difference in helicase activity. ATP concentrations in the range of 0.25-6.00 mmol/L had a weak effect on helicase activity, and NSP13 concentration ≥80 nmol/L inhibited the helicase activity. We obtained the NSP13 of PDCoV and investigated its helicase activity. These findings provided a theoretical foundation for the further research on the regulatory mechanism of NSP13 in PDCoV replication and the development of anti-coronaviral drugs.
Viral Nonstructural Proteins/metabolism* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Swine ; Animals ; DNA Helicases/metabolism* ; Genetic Vectors/metabolism*

Objective: To investigate the clinicopathological features and differential diagnosis of olfactory carcinoma (OC). Methods: Twenty-one cases of sinonasal tumors, including those initially diagnosed as olfactory neuroblastoma (ONB) and those with uncertain diagnosis, were collected from the Department of Pathology, the First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) from January 2016 to August 2022, among which 3 cases were reclassified as OC. The clinicopathological features were investigated, and the remaining 18 cases were used as control. Results: Of the three OC patients, 2 were male and 1 was female, with an average age of 57 years ranging from 35 to 74 years. Microscopically, the tumor cells were arranged in solid, nested or lobulated patterns with occasional palisading around the solid nests. The stroma was highly vascular with focal neurofibrillary areas. There were prominent rosettes or pseudorosettes formation. The tumor cells were mainly ovoid to spindly with scant to moderate amount of cytoplasm, one or several small nucleoli, and fine chromatin content. Brisk mitotic figures were seen. In all 3 cases of OC, there were scanty atypical glands and some were ciliated. Immunohistochemically, at least one epithelial marker and neuroendocrine marker were diffusely expressed in the tumor. Some of the tumor cells were positive for p40 and p63, and the sustentacular cells showed the expression of S-100 protein. All cases tested were negative for NUT, CD99 and desmin, with intact expression of SMARCA4 (BRG1) and SMARCB1 (INI-1). Ki-67 proliferation index varied from 20% to 80%. Follow-up after 16-18 months showed no mortality with tumor recurrence from 1 patient after 16 months. Conclusion: OC is a rare sinonasal tumor with neuroepithelial differentiation, its histomorphology is diverse, and the combination of immunohistochemical markers is essential for appropriate diagnosis.
Humans ; Male ; Female ; Middle Aged ; Paranasal Sinus Neoplasms/chemistry* ; Biomarkers, Tumor/metabolism* ; Carcinoma/chemistry* ; Diagnosis, Differential ; S100 Proteins ; DNA Helicases/metabolism* ; Nuclear Proteins/metabolism* ; Transcription Factors/metabolism*

We explored clinicopathological features and treatment strategies for thoracic <i>SMARCA4i>-deficient undifferentiated tumor (<i>SMARCA4i>-UT). Thoracic <i>SMARCA4i>-UT is a new entity recently acknowledged in the 2021 edition of World Health Organization Classification of Thoracic Tumors, and doctors are relatively unfamiliar with its diagnosis, treatment, and prognosis. Taking a case of <i>SMARCA4i>-UT treated in Peking University First Hospital as an example, this multi-disciplinary discussion covered several hot issues on diagnosing and treating thoracic <i>SMARCA4i>-UT, including histological features, immu- nohistochemical and molecular phenotype, immune checkpoint inhibitor (ICI) therapy, and pathological assessment of neoadjuvant therapy response. The patient was an older man with a long history of smoking and was admitted due to a rapidly progressing solid tumor in the lower lobe of the right lung. Histologically, tumor cells were epithelioid, undifferentiated, diffusely positive for CD34, and partially positive for SALL4.The expression of BRG1 protein encoded by <i>SMARCA4i> gene was lost in all of tumor cells, and next-generation sequencing(NGS)confirmed <i>SMARCA4i> gene mutation (c.2196T>G, p.Y732Ter). The pathological diagnosis reached as thoracic <i>SMARCA4i>-UT, and the preoperative TNM stage was T1N2M0 (ⅢA). Tumor proportion score (TPS) detected by immunohistochemistry of programmed cell death 1-ligand 1 (PD-L1, clone SP263) was 2%. Tumor mutation burden (TMB) detected by NGS of 1 021 genes was 16. 3/Mb. Microsatellite detection showed the tumor was microsatellite stable (MSS). Neo-adjuvant therapy was implemented with the combined regimen of chemotherapy and ICI. Right lower lobectomy was performed through thoracoscopy after the two weeks' neoadjuvant. The pathologic assessment of lung tumor specimens after neoadjuvant therapy revealed a complete pathological response (CPR). The post-neoadjuvant tumor TNM stage was ypT0N0M0. Then, five cycles of adjuvant therapy were completed. Until October 2022, neither tumor recurrence nor metastasis was detected, and minimal residual disease (MRD) detection was negative. At present, it is believed that if BRG1 immunohistochemical staining is negative, regardless of whether <i>SMARCA4i> gene mutation is detected, it should be classified as <i>SMARCA4i>-deficient tumors. <i>SMARCA4i>-deficient tumors include a variety of carcinomas and sarcomas. The essential criteria for diagnosing <i>SMARCA4i>-UT includes loss of BRG1 expression, speci-fic histological morphology, and exclude other common thoracic malignant tumors with <i>SMARCA4i>-deficiency, such as squamous cell carcinoma, adenocarcinoma and large cell carcinoma. <i>SMARCA4i>-UT is a very aggressive malignant tumor with a poor prognosis. It has almost no targeted therapy mutations, and little response to chemotherapy, but ICI is currently the only effective drug. The successful diagnosis and treatment for this case of <i>SMARCA4i>-UT should enlighten significance for various kinds of <i>SMARCA4i>-deficient tumors.
Humans ; Immune Checkpoint Inhibitors ; Neoplasm Recurrence, Local ; Lung Neoplasms/genetics* ; Thoracic Neoplasms/pathology* ; Adenocarcinoma ; DNA Helicases ; Nuclear Proteins ; Transcription Factors

OBJECTIVE: To explore the driving gene of hepatocellular carcinoma (HCC) occurrence and progression and its potential as new therapeutic target of HCC. METHODS: The transcriptome and genomic data of 858 HCC tissues and 493 adjacent tissues were obtained from TCGA, GEO, and ICGC databases. Gene Set Enrichment Analysis (GSEA) identified EHHADH (encoding enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase) as the hub gene in the significantly enriched differential pathways in HCC. The downregulation of EHHADH expression at the transcriptome level was found to correlate with TP53 mutation based on analysis of the TCGA- HCC dataset, and the mechanism by which TP53 mutation caused EHHADH downregulation was explored through correlation analysis. Analysis of the data from the Metascape database suggested that EHHADH was strongly correlated with the ferroptosis signaling pathway in HCC progression, and to verify this result, immunohistochemical staining was used to examine EHHADH expression in 30 HCC tissues and paired adjacent tissues. RESULTS: All the 3 HCC datasets showed signficnatly lowered EHHADH expression in HCC tissues as compared with the adjacent tissues (<i>Pi> < 0.05) with a close correlation with the degree of hepatocyte de-differentiation (<i>Pi> < 0.01). The somatic landscape of HCC cohort in TCGA dataset showed that HCC patients had the highest genomic TP53 mutation rate. The transcriptomic level of PPARGC1A, the upstream gene of EHHADH, was significantly downregulated in HCC patients with TP53 mutation as compared with those without the mutation (<i>Pi> < 0.05), and was significantly correlated with EHHADH expression level. GO and KEGG enrichment analyses showed that EHHADH expression was significantly correlated with abnormal fatty acid metabolism in HCC. The immunohistochemical results showd that the expression level of EHHADH in HCC tissues was down-regulated, and its expression level was related to the degree of hepatocytes de-differentiation and the process of ferroptosis. CONCLUSION TP53 mutations may induce abnormal expression of PPARGC1A to cause downregulation of EHHADH expression in HCC. The low expression of EHHADH is closely associated with aggravation of de-differentiation and ferroptosis escape in HCC tissues, suggesting the potential of EHHADH as a therapeutic target for HCC.
Humans ; Carcinoma, Hepatocellular/genetics* ; Transcriptome ; Liver Neoplasms/genetics* ; Gene Expression Profiling ; Fatty Acids ; Peroxisomal Bifunctional Enzyme

The cranial neural crest plays a fundamental role in orofacial development and morphogenesis. Accordingly, mutations with impact on the cranial neural crest and its development lead to orofacial malformations such as cleft lip and palate. As a pluripotent and dynamic cell population, the cranial neural crest undergoes vast transcriptional and epigenomic alterations throughout the formation of facial structures pointing to an essential role of factors regulating chromatin state or transcription levels. Using CRISPR/Cas9-guided genome editing and conditional mutagenesis in the mouse, we here show that inactivation of Kat5 or Ep400 as the two essential enzymatic subunits of the Tip60/Ep400 chromatin remodeling complex severely affects carbohydrate and amino acid metabolism in cranial neural crest cells. The resulting decrease in protein synthesis, proliferation and survival leads to a drastic reduction of cranial neural crest cells early in fetal development and a loss of most facial structures in the absence of either protein. Following heterozygous loss of Kat5 in neural crest cells palatogenesis was impaired. These findings point to a decisive role of the Tip60/Ep400 chromatin remodeling complex in facial morphogenesis and lead us to conclude that the orofacial clefting observed in patients with heterozygous KAT5 missense mutations is at least in part due to disturbances in the cranial neural crest.
Animals ; Mice ; Chromatin Assembly and Disassembly ; Cleft Lip/genetics* ; Cleft Palate/genetics* ; DNA Helicases/metabolism* ; DNA-Binding Proteins ; Neural Crest/metabolism* ; Skull ; Transcription Factors/metabolism*