[The effect of ubiquitin ligase Cullin3 on the proliferation, migration and invasion of triple-negative breast cancer cells and exploration of its mechanism].

Objective: To investigate the effects of ubiquitin ligase Cullin3 (CUL3) on the proliferation, migration and invasion ability of triple-negative breast cancer (TNBC) cells and its mechanism of action. Methods: Bioinformatics-based methods were used to obtain CUL3 gene and protein expression data in TNBC tissues, and to assess the expression of CUL3 in tumour tissues of TNBC patients (n=160) and in normal breast tissues (n=572), and its relationship with clinical prognosis. The effects of overexpression of CUL3 on the proliferation, migration and invasion ability of TNBC cells in vitro were detected by CCK8 cell proliferation assay, scratch assay and transwell assay; proteins that might interact with CUL3 were screened by immunoprecipitation combined with mass spectrometry analysis, and the substrate protein regulated by CUL3 was identified as Glutathione S-Transferase Pi 1 (GSTP1); the effects of overexpression of GSTP1 on the migration and invasion ability of TNBC cells were detected by scratch assay and Transwell assay, and it was explored whether overexpression of CUL3 could reverse the effects of GSTP1 on the migration and invasion ability of cells; and the effects of overexpression of GSTP1 on the migration and invasion ability of cells were detected by Western blot and IP (Immunoprecipitation) to detect the effect of CUL3 on the ubiquitination modification of GSTP1 protein, and to verify the molecular mechanism by which CUL3 regulates the expression of GSTP1 to affect TNBC migration and invasion. Results: CUL3 expression was significantly higher in TNBC (P<0.000 1), and high CUL3 expression was closely associated with poor prognosis of TNBC patients (OS, P=0.018; RFS, P=0.008); overexpression of CUL3 significantly increased the proliferation of TNBC cells (F=11.97, P=0.002 for the 231-cell group, F=51.92, P<0.001 for the 468-cell group), migration [74.7±4.0 and 128.0±6.1 perforating cells in the overexpression groups of 231 and 468 cell lines, compared with 21.0±2.7 and 70.0±6.6 in the blank control (NC) group, and the t-values of 231 and 468 cell groups were-19.24 and-11.23, with P-values<0.001] and invasive ability (48 h cell proliferation rates were 56.6%±4.4% and 51.6%±3.7% in the 231 and 468 cell line overexpression groups, compared with 40.5%±2.9% and 32.9%±4.8% in the NC group, respectively, t=-5.26, P=0.006 3 in the 231 cell group; t=-5.38 in the 468 cell group, P=0.005 8); GSTP1 expression was reduced in TNBC, and up-regulation of GSTP1 inhibited TNBC cell migration (the number of membrane-penetrating cells in the overexpression groups of 231 and 468 cell lines were 16.3±6.5 and 33.0±6.2, respectively, compared with 34.3±2.5 and 77.3±5.0 in the NC group, and t=5.44 in the 231 cell group, P=0.006; 468 cell group t=7.20, P=0.002) and invasion (48 h cell proliferation rates of 49.6%±1.7% and 36.2%±1.4% in the 231 and 468 cell line overexpression groups, compared to 59.4%±4.7% and 53.0%±1.7% in the NC group, t=3.42, P=0.027 in the 231 cell group; 468 cell group t=13.18, P<0.001), whereas up-regulation of CUL3 reversed the effects of GSTP1 on cell migration (37.0±1.0 and 67.0±5.3 membrane-penetrating cells in the overexpression groups of 231 and 468 cell lines, respectively, 231 cell group t=-3.97, P=0.017; 468 cell group t=-6.12, P=0.004), and invasion (48 h cell proliferation rates of 71.9%±3.6% and 59.4%±2.1% in the 231 and 468 cell line overexpression groups, respectively, with t-values of -9.61 and -16.01 in the 231 and 468 cell groups, respectively, P-values<0.001) inhibitory effects; and CUL3, by increasing GSTP1 ubiquitylation modification, promotes ubiquitin-proteasome system to degrade GSTP1 protein, thereby reducing the stability of GSTP1 protein. Conclusion: Overexpression of CUL3 promotes TNBC development by promoting GSTP1 ubiquitination degradation inducing cell migration and invasion.

[Colorectal adenocarcinoma with enteroblastic differentiation: a clinicopathological analysis of eight cases].

Objective: To investigate the clinicopathological features of colorectal adenocarcinoma with enteroblastic differentiation (CAED). Methods: Eight cases of CAED diagnosed at the Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, China from January 2017 to August 2023 were collected. The histopathological, immunohistochemical, molecular and prognostic features of 8 CAED cases were analyzed. The relevant studies were also reviewed. Results: Among the eight patients, there were six males and two females, with an average age of 58 years (range: 29-77 years, median age: 61.5 years). Preoperative serum alpha-fetoprotein levels were elevated in five patients (14.0-286.6 µg/L). Four tumors were located in the colon, and four tumors in the rectum. Two patients were clinically staged as advanced stage (stage Ⅳ), and distant metastasis occurred at the initial diagnosis (one case had liver metastasis, and the other had lung, bone and multiple lymph nodes metastases). Six patients were clinically staged as locally-advanced stage (Stage Ⅱ-Ⅲ). Three of them developed distant metastases after surgery (one case had liver metastasis, one case had lung metastasis, and one case had peritoneal metastasis). Additionally, two patients died at 9 months and 24 months after surgery, respectively. The tumors were composed of various proportions of adenocarcinoma components with enteroblastic differentiation (30%-100%) and classical tubular adenocarcinoma components. The component with enteroblastic differentiation exhibited morphology similar to embryonic intestinal epithelium: cuboidal or columnar tumor cells arranged in tubular, papillary, cribriform, or solid nest patterns, with clear cytoplasm. Immunohistochemical studies showed that tumor cells expressed at least one oncofetal protein (SALL4, Glypican-3, and AFP). In addition, focal squamous differentiation was observed in 3 cases (3/8). Compared to the primary tumor, both CAED and squamous differentiation components were increased in the metastatic tumors. Based on the sequencing results of KRAS, NRAS and BRAF of the primary and/or metastatic tumors, 5 cases were wild-type, while KRAS exon 2 (G13D) mutations were identified in 2 cases. Conclusions: CAED is a rare colorectal malignancy with a dismal prognosis. Accurate pathological diagnosis is prognostically valuable. The histological features of enteroblastic differentiation, elevated serum AFP levels, and the expression of oncofetal proteins play an important role in the tumor diagnosis.

[Gastric SWI/SNF-complex deficient undifferentiated/rhabdoid carcinoma: a clinicopathological study].

Objective: To investigate the clinicopathological features, immunohistochemical characteristics, differential diagnosis and prognosis of gastric SWI/SNF-complex deficient undifferentiated/rhabdoid carcinomas. Methods: Two cases of gastric SWI/SNF-complex deficient undifferentiated/rhabdoid carcinoma were collected at Fudan University Shanghai Cancer Center, Shanghai, China from 2017 to 2018. The clinicopathological characteristics were analyzed. Hematoxylin and eosin, and immunohistochemical stains were performed, and the relevant literatures were reviewed. Results: The two patients were both male, aged 60 and 74 years, respectively. Their symptoms were both abdominal pain. The tumor arose in the esophagogastric junction in case 1, and the cardia to the fundus and the posterior wall of the upper part of gastric body in case 2. Both tumors were present as an ulcerative mass. The patients died of tumor 11 months and 8 months after surgery, respectively. Histologically, the tumor cells arranged in sheets, nests, cords or trabecular patterns, and pseudoavleolar structure. The tumor cells were epithelioid with uniform morphology, while the tumors showed scant stroma and massive necrosis. Variable rhabdoid cells and multinucleated giant cells were seen in both cases. SMARCA4 encoding protein BRG1 was undetectable in both tumors, while SMARCB1 encoding protein INI1 was detected. The tumor cells were diffusely positive for vimentin and negative for epithelial marker (CKpan), gastrointestinal stromal tumor markers (CD117 and DOG1), myogenic markers (desmin and myogenin), melanoma markers (S-100 protein, SOX10 and HMB45), and lymphohematopoietic markers (LCA and CD20). Conclusions: Gastric SWI/SNF-complex deficient undifferentiated/rhabdoid carcinoma is a rare and highly aggressive tumor with poor prognosis. The detection of subunits protein expression of SWI/SNF complex is important for diagnosis of the tumor.

[Clinicopathological features and outcome of gastroenteropancreatic high-grade (WHO G3) neuroendocrine tumors: a study of 60 cases].

Objective: To investigate the clinicopathological features and outcome of gastroenteropancreatic high-grade neuroendocrine tumors. Methods: A total of 60 gastroenteropancreatic high-grade neuroendocrine tumors were collected from January 1st, 2013 to December 31th, 2018 at Fudan University Shanghai Cancer Center, with available pathology databases and clinic follow-up information. At the same time, 157 cases of gastrointestinal pancreatic neuroendocrine neoplasm (NEN) diagnosed at the hospital in 2018 were collected and the incidence of NEN at all grades was compared. Results: There were 32 males and 28 females, aged 13-80 years (mean 54 years). Pancreas primary was the most common (48%, 29/60). Nodal metastatic rate was 9/16 and distant metastatic rate was 41%(18/44). Liver was the most common site of metastasis. Among all the gastroenteropancreatic neuroendocrine neoplasms diagnosed in the hospital in 2018, the incidence of high-grade neuroendocrine tumors was the lowest (7%, 11/157). High-grade neuroendocrine tumors had typical pathologic features of well-differentiated/moderate neuroendocrine tumors, but with significant differences in mitotic rates. By immunohistochemical staining, most of the tumors expressed neuroendocrine markers and somatostatin receptor 2 was positive in 60% (12/20) of the cases. The average Ki-67 index was 30%-40%, and there was significant difference between cases (18%-80%). The overall survival of high-grade neuroendocrine tumors was 43 months, and the disease-free survival was 12 months. Conclusions: High-grade neuroendocrine tumor is a rare group of neuroendocrine tumors, with unique clinicopathological features and good prognosis. Pathological classification and grading of gastroenteropancreatic neuroendocrine neoplasms can help clinicians to select appropriate treatment and accurately evaluate prognosis.

Numerical investigation of airflow, heat transfer and particle deposition for oral breathing in a realistic human upper airway model.

Inhalation injury from exposure to fire smoke is one of the causes of burn-related death. In this study, a realistic three-dimensional human upper airway model was built from magnetic resonance imaging (MRI) scanned images, including the nasal, oral, pharynx, larynx, trachea and part of the first generation of the tracheobronchial tree, as well as a tissue region from the pharynx to the upper bronchi. The Transition Shear Stress Transport (SST-transition) turbulence model, Pennes bioheat transfer equation, convective boundary conditions and a Lagrangian frame were applied and verified with experimental measurements to simulate the airflow fields, temperature distributions and particle deposition in the human airway model. The effects of flow rate, inhalation temperature and particle diameter were studied. It showed that the oral cavity is more likely to be affected by the inlet air conditions. The mucosa in the oral, pharynx and larynx are more likely to cause the thermal injury. The inspiration flow rate significantly influences the airflow fields, temperature distributions and particle deposition fraction interior of the human upper airway model, especially in the pharynx-larynx region. The rising flow rate, inhalation air temperature and particle diameter all contribute to boosting the total deposition fraction in the model. The heated particles with a higher temperature are more likely to be deposited in the oral cavity and the influence of the inlet temperature has a minor influence in the case of a bigger particle diameter.

Prognostic significance of Versican expression in gastric adenocarcinoma.

Gastric cancer (GC) is the leading malignancy in the digestive system. Versican is a ubiquitous component of the extracellular matrix and has a role in tumor progression. We aim to examine the expression of Versican in GC and the relationship between Versican levels and patient survival. We detected the mRNA expression of Versican in tumorous pairs and adjacent normal tissues (ANTs) of 78 GC patients by quantitative real-time polymerase chain reaction. The protein expression of Versican in 101 cases of matched GC and ANT, as well as in 27 intraepithelial neoplastic (IN) samples, was evaluated by immunohistochemistry. We analyzed the correlation between Versican levels and clinical outcomes. Finally, we performed CCK-8 cell counting assay and transwell assay in GC cell lines. Versican mRNA expression was significantly greater in tumor tissues (P<0.001) than in ANT. Versican was majorly expressed in the stroma surrounding tumor epithelium and minorly some areas of tumor epithelium. The Versican expression level was higher in GC than in ANT (P=0.004), but no significant difference was observed between ANT and IN (P=0.517). The Versican mRNA and protein levels were consistent in GC. High Versican mRNA and protein expression correlated with greater tumor invasion depth (P=0.030, P=0.027). Univariate and multivariate analysis revealed that patients with high Versican mRNA expression exhibited poor disease-specific survival (P<0.001). In vitro experiments showed that Versican overexpression promoted cell proliferation and invasion. Our data indicate that Versican may be a novel prognostic indicator in GC and may be a potential target for clinical diagnosis.

First Report of Stem Rot of Konjac Caused by Phytophthora nicotianae in China.

Konjac (Amorphophallus) is an important economic crop widely used in health products and biomaterials in Asia (2). A serious foliage disease of Konjac was observed in Fuyuan County, Yunnan Province, China, in July 2012. The symptoms began with leaf color changes from light green to yellow, followed by discoloration on the stem base, plant wilting, bulb rotting, and ultimately plant death. Symptomatic tissues were cut into small pieces, surface-sterilized, and cultured on 20% V8 juice agar at 28°C. Five days after incubation, white fluffy colonies with the typical sporangium of Phytophthora sp. were observed from root and stem pieces. Isolates were identified as P. nicotianae based on morphological characteristics and DNA analysis. The growth rate of the colonies was 16 mm/d at 28°C. Sporangia were pyriform, ovoid to spherical, and papillate, and the dimensions of the 80 sporangia measured ranged from 23.7 to 60.4 × 19.4 to 45.7 µm (avg. 42.4 × 31.5 µm) with length-to-breadth ratios of 1.19 to 1.44 (avg. 1.34). The chlamydospores were spherical with a smooth surface, and their dimensions ranged from 20.3 to 47.3 × 18.9 to 45.9 µm (avg. 32.7 × 30.4 µm) (3). DNA was extracted from one colony containing spores and hyphae of the isolated P. nicotianae, and the nuclear ribosomal DNA internal transcribed spacer (ITS) region was amplified with primers ITS6 and ITS4 (4). The obtained 854-bp amplicon was purified and sequenced. NCBI BLAST retrieved a 100% identity with P. nicotianae (GenBank Accession No. KJ506732). A pathogenicity test of the isolated P. nicotianae was conducted in a greenhouse. After 7 days in a humidity-controlled greenhouse, all 10 inoculated plants showed similar symptoms as observed initially in the field, while control plants were symptomless. P. nicotianae was re-isolated from the inoculated stems, thus successfully completing Koch's postulates (1). To our knowledge, this is the first report of P. nicotianae as a pathogen of Konjac in China. References: (1) B. Alvarez-Rodriguez et al. Plant Dis. 97:1257, 2013. (2) H. Ban, et al. Plant Cell Rep. 28:1847, 2009. (3) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. APS Press, St. Paul, MN, 1996. (4) J. M. French et al. Plant Dis. 95:1028, 2011.

[Treatment of huge hemangioma with intervention-embolism and circumferential suture maneuver].

OBJECTIVE: To demonstrate the effectiveness of operative resection for patients with huge hemangioma. METHODS: Eight cases were adopted in this study, including 5 males and 3 females. Among them, 5 cases with hemangioma on the face and neck, 1 case on the back, 1 case on the buttock, and 1 case on the leg. The maximal size of the hemangioma was 31.2 cm x 9.1 cm and the minimal size was 27.3 cm x 6.0 cm. Before operation, angiography was taken to find out the size of tumor. During operation, intervention-embolism and circumferential suture were carried out to control bleeding so that resection of tumor was practicable. RESULTS: There had been successful result in all the cases with huge hemangioma. No recurrence were found with following up 8 months to 4 years(averaged 13 months). CONCLUSION: Angiography of the hemangioma is important in providing the informations for the operation design. The intervention-embolism and circumferential suture can control and reduce the bleeding in operation. Combine of these techniques is essential for the treatment of huge hemangioma.