Study of GCN repeats of PHOX2B gene among individuals from southwest China and diagnosis of two patients with Congenital central hypoventilation syndrome / 中华医学遗传学杂志

OBJECTIVE@#To study the trinucleotide repeats of GCN (GCA, GCT, GCC, GCG) encoding Alanine in exon 3 of the PHOX2B gene among healthy individuals from southwest China and two patients with Congenital central hypoventilation syndrome (CCHS).@*METHODS@#The number and sequence of the GCN repeats of the PHOX2B gene were analyzed by capillary electrophoresis, Sanger sequencing and cloning sequencing of 518 healthy individuals and two newborns with CCHS, respectively.@*RESULTS@#Among the 1036 alleles of the 518 healthy individuals, five alleles were identified, including (GCN)7, (GCN)13, (GCN)14, (GCN)15 and (GCN)20. The frequency of the (GCN)20 allele was the highest (94.79%). And five genotypes were identified, which included (GCN)7/(GCN)20, (GCN)13/(GCN)20, (GCN)14/(GCN)20, (GCN)15/(GCN)20, (GCN)20/(GCN)20. The homozygous genotypes were all (GCN)20/(GCN)20, and the carrier rate was 89.58%. Four GCN sequences of the (GCN)20 homozygous genotypes were identified among the 464 healthy individuals. The GCN repeat numbers in the exon 3 of the PHOX2B gene showed no significant difference between the expected and observed values, and had fulfilled the,Hardy-Weinberg equilibrium. The genotypes of the two CCHS patients were (GCN)20/(GCN)25 and (GCN)20/(GCN)30, respectively.@*CONCLUSION@#It is important to determine the GCN repeats and genotypic data of the exon 3 of the PHOX2B gene among the healthy individuals. The number of GCN repeats in 518 healthy individuals was all below 20. The selection of appropriate methods can accurately detect the polyalanine repeat mutations (PARMs) of the PHOX2B gene, which is conducive to the early diagnosis, intervention and treatment of CCHS.

Genetic analysis of two children with Coffin-Siris syndrome due to variants of ARID1B gene / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic basis of two children with unexplained psychomotor developmental delay and facial dysmorphisms suggestive of Coffin-Siris syndrome (CSS).@*METHODS@#A boy and a girl suspected for CSS at the 980th Hospital of the People's Liberation Army Joint Service Support Force respectively in July 2019 and January 2021, and seven members from their families, were selected as the study subjects. Clinical data and family history of the children were collected, and detailed physical examination was carried out, in addition with laboratory and related auxiliary examinations. Potential variants and copy number variations (CNVs) were detected by whole exome sequencing (WES) and copy number variation sequencing (CNV-seq).@*RESULTS@#Child 1, an 8-month-old female, had featured microcephaly, atrial septal defect, curving of fifth finger/toe, and low limb muscle tone. Child 2 was a 2.5-year-old male with language delay, social impairment, dense hair but no curving of the fifth fingers. Genetic testing revealed that child 1 had loss of heterozygosity for exons 8 to 21 of the ARID1B gene, which was unreported previously. Family verification showed that both of her parents were of the wild type. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG) and American Society of Molecular Pathology (AMP), the variant was rated as pathogenic (PVS1+PS2+PM2-supporting). Child 2 was found to harbor a heterozygous c.4263-6 (IVS17) T>G variant of the ARID1B gene. Transcriptome sequencing confirmed that the variant can affect the normal splicing, resulting in retention of a 5 bp sequence in intron 17. Family verification showed that both of his parents were of the wild type. Based on the guidelines from the ACMG, the variant was rated as pathogenic (PS2+PM2-supporting+PP3+PS3).@*CONCLUSION@#WES and RNA-seq have confirmed the diagnosis of CSS in both children. Discovery of the novel variants has expanded the spectrum of pathogenic mutations underlying CSS, and provided a basis for the genetic counseling.

Clinicopathological features of gastric alpha-fetoprotein-producing adenocarcinoma with SWI/SNF complex deletion / 中华病理学杂志

Objective: To investigate the clinicopathological features and treatment of gastric alpha-fetoprotein (AFP)-producing adenocarcinoma with SWI/SNF complex deletion. Methods: Four cases of gastric AFP-producing adenocarcinoma with SWI/SNF complex deletion diagnosed in Zhongshan Hospital of Fudan University from January 2021 to December 2022 were collected, and their histomorphological characteristics, immunohistochemical (IHC), in situ hybridization of Epstein-Barr virus-encoded RNA (EBER), next-generation sequencing results, clinicopathological features and treatment were summarized, and literature review was conducted. Results: Among the 4 patients, there were three males and one female. They presented with abdominal pain, belching and melena. Serum AFP was significantly elevated in three patients, and endoscopy showed ulcerative lesions. Microscopically, the tumor cells showed mainly diffuse flaky or nest-like growth and typical characteristics of hepatoid adenocarcinoma. In two cases there were adenoid growth, and the tumor cells in these areas possessed clear cytoplasm, suggesting enteroblastic differentiation. The tumor cell nuclei were pleomorphic with large nucleoli and brisk mitoses. The IHC results showed that the tumor cells expressed AFP, GPC3 and SALL4, and there was retained expression of broad-spectrum keratin (CKpan) and E-cadherin. IHC detection of SWI/SNF complex subunits, namely INI1 (SMARCB1), BRG1 (SMARCA4), BRM (SMARCA2), ARID1A protein was performed. In all four cases the hepatoid adenocarcinoma region and enteroblastic differentiation region showed SMARCA2 deletion, and one case with enteroblastic differentiation also showed ARID1A deletion. SMARCB1 and SMARCA4 deletions were not seen. All the four cases were diffusely positive for p53 protein, and the Ki-67 proliferation index was 80%-90%. There were no mismatch repair deletion detected; one cases showed HER2 was strongly positive (3+), and EBER was negative. None of the four cases had mutations in the SWI/SNF complex-related subunits detected by next-generation sequencing. Among the four patients, two underwent palliative surgery due to distant metastasis at the time of surgery, two underwent radical resection. Postoperative adjuvant chemotherapy was given to three patients. Conclusions: AFP-producing adenocarcinoma is a rare subtype of gastric cancer, which can be combined with SWI/SNF complex deletion, and the pathomorphological manifestations are different from the classical SWI/SNF complex deletion of undifferentiated carcinoma with rhabdoid phenotype.

Clinicopathological study of epithelioid and spindle cell rhabdomysarcoma with EWSR1/FUS-TFCP2 fusion / 中华病理学杂志

Objective: To investigate the clinicopathological and genetic features of epithelioid and spindle cell rhabdomysarcoma with EWSR1-TFCP2 or FUS-TFCP2 fusion. Methods: The clinical, morphological and immunohistochemical features of 14 cases of epithelioid and spindle cell rhabdomysarcoma with EWSR1-TFCP2 or FUS-TFCP2 fusion diagnosed from January 2019 to December 2022 in the Department of Pathology, Foshan Traditional Chinese Medicine Hospital, Foshan, China were retrospectively analyzed. The cases were all subject to FISH or next generation sequencing for analysis of molecular genetic features. The literature was reviewed. Results: There were 5 males and 9 females, with the age at presentation ranging from 6 to 36 years (mean, 22 years). Tumors occurred in the head and neck (9 cases), pelvic region (2 cases), bladder (one case), right humerus (one case), and the abdominal wall, humerus and pubic at the same time (one case). Presenting symptoms varied by location but often included pain or discomfort. Most of the patients showed aggressive radiographic features with soft tissue extension. The tumors had a median size of 6.6 cm (range, 2-23 cm). The tumors were poorly defined and irregularly shaped. Microscopic examination showed diffuse proliferation of spindle or epithelioid cells. While morphologically high-grade tumors displayed obvious cytological atypia, a high mitotic count and tumor necrosis, low-grade tumors grew in sheets and fascicles composed of spindle, epithelioid cells with moderate or abundant amounts of eosinophilic cytoplasm, without pronounced cytological atypia. The tumor cells expressed Desmin, MyoD1, and Myogenin, as well as ALK, EMA, and CKpan. EWSR1/FUS-TFCP2 gene fusion was detected in 14 cases with next generation sequencing and confirmed by FISH. Six cases had EWSR1-TFCP2 fusions and 8 cases showed FUS-TFCP2 fusions. Follow-up information was available in 13 patients, ranged from 5 to 37 months. At the end of follow-up period, 7 patients died of the disease. Six patients were alive:two cases had local recurrences and metastases, two cases of recurrences, one case of metastasis and one case without recurrences and metastasis. Conclusions: Epithelioid and spindle cell rhabdomysarcomas with EWSR1-TFCP2 or FUS-TFCP2 fusion show a very aggressive clinical course, and more commonly occur in the head and neck. Their genetic hallmark is the presence of EWSR1/FUS-TFCP2 fusions. Familiarity with its clinicopathological characteristics is helpful in avoiding misdiagnoses.

Mutational Signatures Analysis of Micropapillary Components and Exploration of ZNF469 Gene in Early-stage Lung Adenocarcinoma with Ground-glass Opacities / 中国肺癌杂志

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.

Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

Functional mechanisms of WRKY transcription factors in regulating plant response to abiotic stresses / 生物工程学报

WRKYs is a unique family of transcription factors (TFs) in plants, and belongs to the typical multifunctional regulator. It is involved in the regulation of multiple signaling pathways. This type of transcription factor is characterized to contain about 60 highly conservative amino acids as the WRKY domain, and usually also has the Cys2His2 or Cys2His-Cys zinc finger structure. WRKYs can directly bind to the W-box sequence ((T)(T) TGAC (C/T)) in the promoter region of the downstream target gene, and activate or inhibit the transcription of the target genes by interacting with the target protein. They may up-regulate the expression of stress-related genes through integrating signal pathways mediated by abscisic acid (ABA) and reactive oxygen species (ROS), thus playing a vital role in regulating plant response to abiotic stresses. This review summarizes the advances in research on the structure and classification, regulatory approach of WRKYs, and the molecular mechanisms of WRKYs involved in response to drought and salt stresses, and prospects future research directions, with the aim to provide a theoretical support for the genetic improvement of crop in response to abiotic stresses.

Role of post-translational modification of basic leucine zipper transcription factors in response to abiotic stresses in plants / 生物工程学报

Abiotic stresses substantially affect the growth and development of plants. Plants have evolved multiple strategies to cope with the environmental stresses, among which transcription factors play an important role in regulating the tolerance to abiotic stresses. Basic leucine zipper transcription factors (bZIP) are one of the largest gene families. The stability and activity of bZIP transcription factors could be regulated by different post-translational modifications (PTMs) in response to various intracellular or extracellular stresses. This paper introduces the structural feature and classification of bZIP transcription factors, followed by summarizing the PTMs of bZIP transcription factors, such as phosphorylation, ubiquitination and small ubiquitin-like modifier (SUMO) modification, in response to abiotic stresses. In addition, future perspectives were prospected, which may facilitate cultivating excellent stress-resistant crop varieties by regulating the PTMs of bZIP transcription factors.

MAGED4B Promotes Glioma Progression via Inactivation of the TNF-α-induced Apoptotic Pathway by Down-regulating TRIM27 Expression / 神经科学通报·英文版

MAGED4B belongs to the melanoma-associated antigen family; originally found in melanoma, it is expressed in various types of cancer, and is especially enriched in glioblastoma. However, the functional role and molecular mechanisms of MAGED4B in glioma are still unclear. In this study, we found that the MAGED4B level was higher in glioma tissue than that in non-cancer tissue, and the level was positively correlated with glioma grade, tumor diameter, Ki-67 level, and patient age. The patients with higher levels had a worse prognosis than those with lower MAGED4B levels. In glioma cells, MAGED4B overexpression promoted proliferation, invasion, and migration, as well as decreasing apoptosis and the chemosensitivity to cisplatin and temozolomide. On the contrary, MAGED4B knockdown in glioma cells inhibited proliferation, invasion, and migration, as well as increasing apoptosis and the chemosensitivity to cisplatin and temozolomide. MAGED4B knockdown also inhibited the growth of gliomas implanted into the rat brain. The interaction between MAGED4B and tripartite motif-containing 27 (TRIM27) in glioma cells was detected by co-immunoprecipitation assay, which showed that MAGED4B was co-localized with TRIM27. In addition, MAGED4B overexpression down-regulated the TRIM27 protein level, and this was blocked by carbobenzoxyl-L-leucyl-L-leucyl-L-leucine (MG132), an inhibitor of the proteasome. On the contrary, MAGED4B knockdown up-regulated the TRIM27 level. Furthermore, MAGED4B overexpression increased TRIM27 ubiquitination in the presence of MG132. Accordingly, MAGED4B down-regulated the protein levels of genes downstream of ubiquitin-specific protease 7 (USP7) involved in the tumor necrosis factor-alpha (TNF-α)-induced apoptotic pathway. These findings indicate that MAGED4B promotes glioma growth via a TRIM27/USP7/receptor-interacting serine/threonine-protein kinase 1 (RIP1)-dependent TNF-α-induced apoptotic pathway, which suggests that MAGED4B is a potential target for glioma diagnosis and treatment.

Doublecortin-like kinase 1 activates Hippo pathway to promote migration, invasion and proliferation of pancreatic cancer cells / 中华肿瘤杂志

Objective: To explore the mechanism of Doublecortin-like kinase 1 (DCLK1) in promoting cell migration, invasion and proliferation in pancreatic cancer. Methods: The correlation between DCLK1 and Hippo pathway was analyzed using TCGA and GTEx databases and confirmed by fluorescence staining of pancreatic cancer tissue microarrays. At the cellular level, immunofluorescence staining of cell crawls and western blot assays were performed to clarify whether DCLK1 regulates yes associated protein1 (YAP1), a downstream effector of the Hippo pathway. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to analyze the expressions of YAP1 binding transcription factor TEA-DNA binding proteins (TEAD) and downstream malignant behavior-promoting molecules CYR61, EDN1, AREG, and CTGF. Transwell test of the DCLK1-overexpressing cells treated with the Hippo pathway inhibitor Verteporfin was used to examine whether the malignant behavior-promoting ability was blocked. Analysis of changes in the proliferation index of experimental cells used real-time label-free cells. Results: TCGA combined with GTEx data analysis showed that the expressions of DCLK1 and YAP1 molecules in pancreatic cancer tissues were significantly higher than those in adjacent tissues (P<0.05). Moreover, DCLK1was positively correlated with the expressions of many effectors in the Hippo pathway, including LATS1 (r=0.53, P<0.001), LATS2 (r=0.34, P<0.001), MOB1B (r=0.40, P<0.001). In addition, the tissue microarray of pancreatic cancer patients was stained with multicolor fluorescence, indicated that the high expression of DCLK1 in pancreatic cancer patients was accompanied by the up-regulated expression of YAP1. The expression of DCLK1 in pancreatic cancer cell lines was analyzed by the CCLE database. The results showed that the expression of DCLK1 in AsPC-1 and PANC-1 cells was low. Thus, we overexpressed DCLK1 in AsPC-1 and PANC-1 cell lines and found that DCLK1 overexpression in pancreatic cancer cell lines promoted YAP1 expression and accessible to the nucleus. In addition, DCLK1 up-regulated the expression of YAP1 binding transcription factor TEAD and increased the mRNA expression levels of downstream malignant behavior-promoting molecules. Finally, Verteporfin, an inhibitor of the Hippo pathway, could antagonize the cell's malignant behavior-promoting ability mediated by high expression of DCLK1. We found that the number of migrated cells with DCLK1 overexpressing AsPC-1 group was 68.33±7.09, which was significantly higher than 22.00±4.58 of DCLK1 overexpressing cells treated with Verteporfin (P<0.05). Similarly, the migration number of PANC-1 cells overexpressing DCLK1 was 65.66±8.73, which was significantly higher than 37.00±6.00 of the control group and 32.33±9.61 of Hippo pathway inhibitor-treated group (P<0.05). Meanwhile, the number of invasive cells in the DCLK1-overexpressed group was significantly higher than that in the DCLK1 wild-type group cells, while the Verteporfin-treated DCLK1-overexpressed cells showed a significant decrease. In addition, we monitored the cell proliferation index using the real-time cellular analysis (RTCA) assay, and the proliferation index of DCLK1-overexpressed AsPC-1 cells was 0.66±0.04, which was significantly higher than 0.38±0.01 of DCLK1 wild-type AsPC-1 cells (P<0.05) as well as 0.05±0.03 of DCLK1-overexpressed AsPC1 cells treated with Verteporfin (P<0.05). PANC-1 cells showed the same pattern, with a proliferation index of 0.77±0.04 for DCLK1-overexpressed PANC-1 cells, significantly higher than DCLK1-overexpressed PANC1 cells after Verteporfin treatment (0.14±0.05, P<0.05). Conclusion: The expression of DCLK1 is remarkably associated with the Hippo pathway, it promotes the migration, invasion, and proliferation of pancreatic cancer cells by activating the Hippo pathway.

Clinicopathological characteristics of gastric SMARCA4-deficient undifferentiated/rhabdoid carcinoma / 中华病理学杂志

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.

Clinicopathological features and prognosis of SMARCA4-deficient non-small cell lung carcinoma: an analysis of 127 cases / 中华病理学杂志

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 (n=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 (P<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.

Thoracic SMARCA4-deficient undifferentiated tumor-pathological diagnosis and combined immune checkpoint inhibitor treatment / 北京大学学报(医学版)

We explored clinicopathological features and treatment strategies for thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). Thoracic SMARCA4-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 SMARCA4-UT treated in Peking University First Hospital as an example, this multi-disciplinary discussion covered several hot issues on diagnosing and treating thoracic SMARCA4-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 SMARCA4 gene was lost in all of tumor cells, and next-generation sequencing(NGS)confirmed SMARCA4 gene mutation (c.2196T>G, p.Y732Ter). The pathological diagnosis reached as thoracic SMARCA4-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 SMARCA4 gene mutation is detected, it should be classified as SMARCA4-deficient tumors. SMARCA4-deficient tumors include a variety of carcinomas and sarcomas. The essential criteria for diagnosing SMARCA4-UT includes loss of BRG1 expression, speci-fic histological morphology, and exclude other common thoracic malignant tumors with SMARCA4-deficiency, such as squamous cell carcinoma, adenocarcinoma and large cell carcinoma. SMARCA4-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 SMARCA4-UT should enlighten significance for various kinds of SMARCA4-deficient tumors.

Tra2β Enhances Cell Proliferation by Inducing the Expression of Transcription Factor SP1 in Cervical Cancer / 生物医学与环境科学（英文）

OBJECTIVE@#In this study, the role and potential mechanism of transformer 2β (Tra2β) in cervical cancer were explored.@*METHODS@#The transcriptional data of Tra2β in patients with cervical cancer from Gene Expression Profiling Interactive Analysis (GEPIA) and cBioPortal databases were investigated. The functions of Tra2β were evaluated by using Western blot, MTT, colony formation, Transwell assays, and nude mouse tumor formation experiments. Target genes regulated by Tra2β were studied by RNA-seq. Subsequently, representative genes were selected for RT-qPCR, confocal immunofluorescence, Western blot, and rescue experiments to verify their regulatory relationship.@*RESULTS@#The dysregulation of Tra2β in cervical cancer samples was observed. Tra2β overexpression in Siha and Hela cells enhanced cell viability and proliferation, whereas Tra2β knockdown showed the opposite effect. Alteration of Tra2β expression did not affect cell migration and invasion. Furthermore, tumor xenograft models verified that Tra2β promoted cervical cancer growth. Mechanically, Tra2β positively regulated the mRNA and protein level of SP1, which was critical for the proliferative capability of Tra2β.@*CONCLUSION@#This study demonstrated the important role of the Tra2β/SP1 axis in the progression of cervical cancer in vitro and in vivo, which provides a comprehensive understanding of the pathogenesis of cervical cancer.

Advances of structure and mechanisms of bromodomain-containing protein 4 and its related research in tumors / 生物工程学报

The bromodomain and extraterminal domain (Bet) family are the regulators of the epigenome and also the pivotal driving factors for the expression of tumor related genes that tumor cells depend on for survival and proliferation. Bromodomain-containing protein 4 (Brd4) is a member of the Bet protein family. Generally, Brd4 identifies acetylated histones and binds to the promoter or enhancer region of target genes to initiate and maintain expression of tumor related genes. Brd4 is closely related to the regulation of multiple transcription factors and chromatin modification and is involved in DNA damage repair and maintenance of telomere function, thus maintaining the survival of tumor cells. This review summarizes the structure and function of Brd4 protein and the application of its inhibitors in tumor research.

Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans / 生物工程学报

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC80 values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

Development and optimization of a cell screening system for farnesoid X receptor agonist / 生物工程学报

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.

Genome-wide identification and characterization of the WOX gene family in Brassica juncea / 生物工程学报

The WUSCHEL related-homeobox (WOX) family is one of the plant-specific transcription factor families, playing important roles in plant growth and development. In this study, 51 WOX gene family members were identified from the genome data of Brassica juncea by searching and screening with HUMMER, Smart and other software. Their protein molecular weight, amino acids numbers, and isoelectric point were analyzed by using Expasy online software. Furthermore, bioinformatics software was used to systematically analyze the evolutionary relationship, conservative region, and gene structure of the WOX gene family. The mustard WOX gene family was divided into three subfamilies: ancient clade, intermediate clade, and WUS clade/modern clade. Structural analysis showed that the type, organization form and gene structure of the conservative domain of WOX transcription factor family members in the same subfamily were highly consistent, while there was a certain diversity among different subfamilies. 51 WOX genes are distributed unevenly on 18 chromosomes of mustard. Most of the promoters of these genes contain cis acting elements related to light, hormone and abiotic stress. Using transcriptome data and real-time fluorescence quantitative PCR (qRT-PCR) analysis, it was found that the expression of mustard WOX gene was spatio-temporal specific, among which BjuWOX25, BjuWOX33, and BjuWOX49 might play an important role in the development of silique, and BjuWOX10, BjuWOX32, and BjuWOX11, BjuWOX23 respectively might play an important role in the response to drought and high temperature stresses. The above results may facilitate the functional study of mustard WOX gene family.

Molecular characterization and transcriptional analysis of VrWOX genes in mungbean Vigna radiate (L.) Wilczek / 生物工程学报

WUSCHEL-related homebox (WOX) gene family is a type of plant specific transcription factor, and belongs to the homeobox (HB) transcription factor superfamily. WOX genes play an important role in plant development, such as stem cell regulation and reproductive progress, and have been identified in many plant species. However, the information of mungbean VrWOX genes is limited. In this study, we identified 42 VrWOX genes in mungbean genome using Arabidopsis AtWOX genes as BLAST queries. VrWOX genes are unevenly distributed on 11 mungbean chromosomes, and chromosome 7 contains the most VrWOX genes. VrWOX genes are classified into three subgroups, the ancient group, the intermediate group and the modern/WUSCHEL group, which contains 19, 12 and 11 VrWOX members, respectively. Intraspecific synteny analysis revealed 12 VrWOX duplicated gene pairs in mungbean. Mungbean and Arabidopsis thaliana have 15 orthologous genes, and mungbean and Phaseolus vulgaris have 22 orthologous genes, respectively. The gene structure and conserved motif are different among VrWOX genes, indicating their functional diversity. The promoter regions of VrWOX genes contain different number and type of cis-acting elements, and VrWOX genes show distinct expression levels in eight mungbean tissues. Our study investigated the bioinformation and expression profiles of VrWOX genes, and provided essential information for further functional characterization of VrWOX genes.

Identification of CjSPL gene family in Ziyang Xiangcheng rootstock and expression pattern analysis / 生物工程学报

Squamosa promoter binding protein-like (SPL) family is a group of important transcription factors involved in the regulation of plant growth and development and the response to environmental stress, but there are few studies in perennial fruit trees such as citrus. In this study, Ziyang Xiangcheng (Citrus junos Sib.ex Tanaka), an important rootstock of Citrus, was used as the material for analysis. Based on plantTFDB transcription factor database and sweet orange genome database, 15 SPL family members were genome-widely identified and cloned from Ziyang Xiangcheng, and named CjSPL1-CjSPL15. Sequence analysis showed that the open reading frame (ORF) length of CjSPLs ranged from 393 bp to 2 865 bp, encoding 130-954 amino acids. Phylogenetic tree divided 15 CjSPLs into 9 subfamilies. Gene structure and conserved domain analysis predicted 20 different conserved motifs and SBP basic domains. Analysis of cis-acting promoter elements predicted 20 different promoter elements, including those related to plant growth and development, abiotic stress and secondary metabolites. The expression patterns of CjSPLs under drought, salt and low temperature stresses were analyzed by real-time fluorescence quantitative PCR (qRT-PCR), and many CjSPLs were significantly up-regulated after stress treatment. This study provides a reference for further study on the function of SPL family transcription factors in citrus and other fruit trees.