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 immunohistochemical phenotypes of gastric SMARCA4-deficient undifferentiated carcinoma, and to discuss the daily diagnostics of this entity and analyze its prognosis. Methods: The cases of gastric SMARCA4-deficient undifferentiated carcinoma diagnosed at the Department of Pathology, Peking University Cancer Hospital, China from January 2010 to August 2022 were collected. The histological sections were reviewed, the immunohistochemical results and clinicopathological features were analyzed, and relevant literature was reviewed. Results: Pure foci of undifferentiated carcinoma were seen in 7 cases, and 1 case was accompanied by a moderately differentiated tubular adenocarcinoma component. Undifferentiated carcinoma foci showed similar sheet-like or solid diffuse growth pattern, medium-sized tumor cells characterized by 1-2 nucleoli, and abundant cytoplasm and rhabdoid appearance. The average patient age was 65±8 years. Six patients were male and 2 were female. Immunohistochemical staining showed that undifferentiated carcinoma of all 8 tumors were negative for SMARCA4 (BRG1). Among 7 patients who underwent SMARCA2 (BRM) and SMARCB1 (INI1) staining, 4 cases showed loss of BRM expression, 2 cases showed weakly positive staining, and 1 case was diffusely positive, but all 7 cases were diffusely strong positive for INI1. The neuroendocrine marker, synaptophysin, was weakly positive in 5 cases, while CgA and CD56 were negative in 8 cases. Ki-67 index was more than 70%. Two cases were mismatch repair deficient and showed the loss of MLH1/PMS2 expression, while 1 case showed only MSH2 loss. PD-L1 staining showed that combined positive score (CPS)≥1 in 4 cases (CPS ranging from 1 to 55) and CPS<1 in the other 3 cases. Four patients had clinical stage Ⅳ disease. Two of them died within 3 months after diagnosis. Conclusions: Gastric SMARCA4-deficient undifferentiated carcinoma/rhabdoid carcinoma is a rare group of highly malignant tumors with a poor prognosis. Loss of the core subunit of SWI/SNF complex may be associated with the development of dedifferentiated histological pattern and aggressive tumor progression, which may be more frequently accompanied with mismatch repair deficiency.
Male ; Female ; Humans ; Carcinoma/pathology* ; Adenocarcinoma ; Colorectal Neoplasms ; Cell Differentiation ; Stomach Neoplasms ; Biomarkers, Tumor ; DNA Helicases ; Nuclear Proteins ; Transcription Factors

Objective: To investigate and elucidate the clinicopathological and prognostic characteristics of SMARCA4-deficient non-small cell lung cancer. Methods: The clinicopathological and prognostic data were collected in 127 patients with SMARCA4-deficient non-small cell lung cancer diagnosed in Shanghai Pulmonary Hospital, Shanghai, China from January 2020 to March 2022. The variation and expression of biomarkers related to treatment were retrospectively reviewed. Results: One hundred and twenty-seven patients were eligible for enrollment. Among them 120 patients (94.5%) were male and 7 cases (5.5%) were female, while the average age was 63 years (range 42-80 years). There were 41 cases (32.3%) of stage Ⅰ cancer, 23 cases (18.1%) of stage Ⅱ, 31 cases (24.4%) of stage Ⅲ and 32 cases (25.2%) of stage Ⅳ. SMARCA4 expression detected by immunohistochemistry was completely absent in 117 cases (92.1%) and partially absent in 10 cases (7.9%). PD-L1 immunohistochemical analyses were performed on 107 cases. PD-L1 was negative, weakly positive and strongly positive in 49.5% (53/107), 26.2% (28/107) and 24.3% (26/107) of the cases, respectively. Twenty-one cases showed gene alterations (21/104, 20.2%). The KRAS gene alternation (<i>ni>=10) was most common. Mutant-type SMARCA4-deficient non-small cell lung cancer was more commonly detected in females, and was associated with positive lymph nodes and advanced clinical stage (<i>Pi><0.01). Univariate survival analysis showed that advanced clinical stage was a poor prognosis factor, and vascular invasion was a poor predictor of progression-free survival in patients with surgical resection. Conclusions: SMARCA4-deficient non-small cell lung cancer is a rare tumor with poor prognosis, and often occurs in elderly male patients. However, SMARCA4-deficient non-small cell lung cancers with gene mutations are often seen in female patients. Vascular invasion is a prognostic factor for disease progression or recurrence in patients with resectable tumor. Early detection and access to treatment are important for improving patient survivals.
Humans ; Male ; Female ; Aged ; Adult ; Middle Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung/pathology* ; B7-H1 Antigen/metabolism* ; Lung Neoplasms/pathology* ; Retrospective Studies ; China ; Prognosis ; Biomarkers, Tumor/analysis* ; DNA Helicases/genetics* ; Nuclear Proteins/genetics* ; Transcription Factors/genetics*

BACKGROUND: The SMARCA4 mutation has been shown to account for at least 10% of non-small cell lung cancer (NSCLC). In the present, conventional radiotherapy and targeted therapy are difficult to improve outcomes due to the highly aggressive and refractory nature of SMARCA4-deficient NSCLC (SMARCA4-DNSCLC) and the absence of sensitive site mutations for targeted drug therapy, and chemotherapy combined with or without immunotherapy is the main treatment. Effective SMARCA4-DNSCLC therapeutic options, however, are still debatable. Our study aimed to investigate the efficacy and prognosis of programmed cell death 1 (PD-1) immune checkpoint inhibitors (ICIs) in combination with chemotherapy and chemotherapy in patients with stage III-IV SMARCA4-DNSCLC. METHODS: 46 patients with stage III-IV SMARCA4-DNSCLC were divided into two groups based on their treatment regimen: the chemotherapy group and the PD-1 ICIs plus chemotherapy group, and their clinical data were retrospectively analyzed. Efficacy assessment and survival analysis were performed in both groups, and the influencing factors for prognosis were explored for patients with SMARCA4-DNSCLC. RESULTS: Male smokers are more likely to develop SMARCA4-DNSCLC. There was no significant difference in the objective response rate (76.5% vs 69.0%, P=0.836) between chemotherapy and the PD-1 ICIs plus chemotherapy or the disease control rate (100.0% vs 89.7%, P=0.286). The one-year overall survival rate in the group with PD-1 ICIs plus chemotherapy was 62.7%, and that of the chemotherapy group was 46.0%. The difference in median progression-free survival (PFS) between the PD-1 ICIs plus chemotherapy group and the chemotherapy group was statistically significant (9.3 mon vs 6.1 mon, P=0.048). The results of Cox regression analysis showed that treatment regimen and smoking history were independent influencing factors of PFS in patients with stage III-IV SMARCA4-DNSCLC, and family history was an individual influencing factor of overall survival in patients with stage III-IV SMARCA4-DNSCLC. CONCLUSIONS Treatment regimen may be a prognostic factor for patients with SMARCA4-DNSCLC, and patients with PD-1 ICIs plus chemotherapy may have a better prognosis.
Humans ; Male ; Carcinoma, Non-Small-Cell Lung/genetics* ; Lung Neoplasms/genetics* ; Immune Checkpoint Inhibitors/therapeutic use* ; Programmed Cell Death 1 Receptor/genetics* ; Retrospective Studies ; Antineoplastic Agents, Immunological/therapeutic use* ; Prognosis ; DNA Helicases/genetics* ; Nuclear Proteins/genetics* ; Transcription Factors/genetics*

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