Objective:To detect gene mutations in a pedigree with dominant dystrophic epidermolysis bullosa (DDEB) .Methods:A 20-year-old male proband presented with repeated blisters, ulceration, pigmentation, scars on the limbs, and deformation of the nails/toenails after birth. There were 5 patients in the 3-generation family, and they all presented with typical skin lesions. Peripheral blood samples were obtained from 14 members of the pedigree (including the 5 patients) and 100 unrelated healthy controls. Whole-exome sequencing was performed in the proband to identify relevant mutation sites, which were then confirmed in the family by Sanger sequencing.Results:Genetic testing indicated that the proband and the other 4 patients all carried a missense mutation (c.7885G>A) in exon 107 of the COL7A1 gene, resulting in the substitution of glycine by arginine at amino acid position 2629 (p.G2629R). The mutation was identified neither in the 9 healthy relatives nor in the 100 unrelated healthy controls. The mutation co-segregated with DDEB in the family, and was not included in databases such as Pubmed, HGMD or ClinVar, suggesting it was a novel missense mutation. The amino acid encoded by this mutation may alter the structure of type Ⅶ collagen, thereby affecting its function.Conclusion:A novel missense mutation was identified in exon 107 of the COL7A1 gene in the family with DDEB, expanding the spectrum of mutations in the COL7A1 gene.

Objective To investigate the protective effect of 5-hydroxy-6,7-dimethoxyflavone(5-HDF),a compound extracted from Elsholtzia blanda Benth.,against lung injury induced by H1N1 influenza virus and explore its possible mechanism of action.Methods 5-HDF was extracted from Elsholtzia blanda Benth.using ethanol reflux extraction and silica gel chromatography and characterized using NMR and MS analyses.In an A549 cell model of H1N1 influenza virus infection(MOI=0.1),the cytotoxicity of 5-HDF was assessed using MTT assay,and its effect on TRAIL and IL-8 expressions was examined using flow cytometry;Western blotting was used to detect the expression levels of inflammatory,apoptosis,and ferroptosis-related proteins.In a mouse model of H1N1 influenza virus infection established by nasal instillation of 50 μL H1N1 virus at the median lethal dose,the effects of 30 and 60 mg/kg 5-HDF by gavage on body weight,lung index,gross lung anatomy and lung histopathology were observed.Results 5-HDF exhibited no significant cytotoxicity in A549 cells within the concentration range of 0-200 μg/mL.In H1N1-infected A549 cells,treatment with 5-HDF effectively inhibited the activation of phospho-p38 MAPK and phospho-NF-κB p65,lowered the expressions of IL-8,enhanced the expression of anti-ferroptosis proteins(SLC7A11 and GPX4),and inhibited the expressions of apoptosis markers PARP and caspase-3 and the apoptotic factor TRAIL.In H1N1-infected mice,treatment with 5-HDF for 7 days significantly suppressed body weight loss and increment of lung index and obviously alleviated lung tissue pathologies.Conclusion 5-HDF offers protection against H1N1 influenza virus infection in mice possibly by suppressing H1N1-induced ferroptosis,inflammatory responses,and apoptosis via upregulating SLC7A11 and GPX4,inhibiting the activation of phospho-NF-κB p65 and phospho-p38 MAPK,and decreasing the expression of cleaved caspase3 and cleaved PARP.

Objective To investigate the protective effect of 5-hydroxy-6,7-dimethoxyflavone(5-HDF),a compound extracted from Elsholtzia blanda Benth.,against lung injury induced by H1N1 influenza virus and explore its possible mechanism of action.Methods 5-HDF was extracted from Elsholtzia blanda Benth.using ethanol reflux extraction and silica gel chromatography and characterized using NMR and MS analyses.In an A549 cell model of H1N1 influenza virus infection(MOI=0.1),the cytotoxicity of 5-HDF was assessed using MTT assay,and its effect on TRAIL and IL-8 expressions was examined using flow cytometry;Western blotting was used to detect the expression levels of inflammatory,apoptosis,and ferroptosis-related proteins.In a mouse model of H1N1 influenza virus infection established by nasal instillation of 50 μL H1N1 virus at the median lethal dose,the effects of 30 and 60 mg/kg 5-HDF by gavage on body weight,lung index,gross lung anatomy and lung histopathology were observed.Results 5-HDF exhibited no significant cytotoxicity in A549 cells within the concentration range of 0-200 μg/mL.In H1N1-infected A549 cells,treatment with 5-HDF effectively inhibited the activation of phospho-p38 MAPK and phospho-NF-κB p65,lowered the expressions of IL-8,enhanced the expression of anti-ferroptosis proteins(SLC7A11 and GPX4),and inhibited the expressions of apoptosis markers PARP and caspase-3 and the apoptotic factor TRAIL.In H1N1-infected mice,treatment with 5-HDF for 7 days significantly suppressed body weight loss and increment of lung index and obviously alleviated lung tissue pathologies.Conclusion 5-HDF offers protection against H1N1 influenza virus infection in mice possibly by suppressing H1N1-induced ferroptosis,inflammatory responses,and apoptosis via upregulating SLC7A11 and GPX4,inhibiting the activation of phospho-NF-κB p65 and phospho-p38 MAPK,and decreasing the expression of cleaved caspase3 and cleaved PARP.

Zirconia crown has been widely used in the field of prosthodontics.Traditional zirconia exhibits excellent mechanical properties but lacks translucency.The introduction of transparent zirconia significantly enhances its aesthetic performance.In clinical applications,factors affecting the aesthetic results of full zirconia crown should be comprehen-sively considered,and the most suitable restoration should be chosen.Additionally,clinicians need to design appropriate tooth preparation dimensions and methods based on an individual patient's actual situation.During the clinical bonding process of zirconia,proper surface treatment of the tooth and restoration is essential.The selection of suitable adhesives is crucial for achieving optimal bonding strength and aesthetics.

Objective This study aimed to explore the expression trends of innate immune cells and immune-checkpoint molecules validated by data calculation in the process of oral mucosal carcinogenesis,as well as to explore methods of suppressing oral mucosal carcinogenesis based on immunotherapy by predicting their interactions.Me-thods 1)The cancer genome atlas(TCGA)database comprehensively scores immune cells and immune-checkpoint molecules in the process of oral mucosal carcinogenesis and screens out intrinsic immune cells and immune-check-point molecules that interfere with tumor immune escape.2)Clinical patient blood routine data were collected for the statistical analysis of peripheral blood immune cells during the progression of oral mucosal carcinogenesis.Immune cells in peripheral blood that may affect the progression of oral mucosal carcinogenesis were screened.3)Immunohis-tochemical staining was performed on intrinsic immune cells and immune-checkpoint molecules validated based on da-ta calculation in various stages of oral mucosal carcinogenesis.4)Special staining was used to identify innate immune cells in various stages of oral mucosal carcinogenesis based on data-calculation verification.5)Survival analysis was conducted on intrinsic immune cells and immune-checkpoint molecules validated based on data calculation during the process of oral mucosal carcinogenesis.The association of intrinsic immune cells and immune-checkpoint molecules with the prognosis of oral squamous cell carcinoma was verified.Results The expression of monocytes and neutro-phils increased during the process of oral mucosal carcinogenesis.The expression of eosinophils showed a single peak trend of up and down.The expression of mast cells decreased.In the process of oral mucosal carcinogenesis,the ex-pression of the immune-checkpoint molecules cytotoxic T-lymphocyte-associated protein 4(CTLA4)and programmed cell death-ligand(PD-L1)increased.The expression trends of monocytes,neutrophils,and eosinophils were positively correlated with those of CTLA4 and PD-L1 immune-checkpoint molecules.The expression trend of mast cells was negatively correlated with the expression of CTLA4 and PD-L1.Monocytes,neutrophils,and eosinophils may pro-mote tumor immune escape mediated by CTLA4 and/or PD-L1,thereby accelerating the progression of oral mucosal carcinogenesis.Mast cells may inhibit tumor immune escape mediated by CTLA4 and/or PD-L1,delaying the progres-sion of oral mucosal carcinogenesis.Conclusion Therefore,interference with specific immune cells in innate immu-nity can regulate the expression of CTLA4 and/or PD-L1 to a certain extent,inhibit tumor immune escape,and delay the progression of oral mucosal carcinogenesis.

As an important part of tumor microenvironment, neutrophils are poorly understood due to their spatiotemporal heterogeneity in tumorigenesis. Here we defined, at single-cell resolution, CD44-CXCR2- neutrophils as tumor-specific neutrophils (tsNeus) in both mouse and human gastric cancer (GC). We uncovered a Hippo regulon in neutrophils with unique YAP signature genes (e.g., ICAM1, CD14, EGR1) distinct from those identified in epithelial and/or cancer cells. Importantly, knockout of YAP/TAZ in neutrophils impaired their differentiation into CD54+ tsNeus and reduced their antitumor activity, leading to accelerated GC progression. Moreover, the relative amounts of CD54+ tsNeus were found to be negatively associated with GC progression and positively associated with patient survival. Interestingly, GC patients receiving neoadjuvant chemotherapy had increased numbers of CD54+ tsNeus. Furthermore, pharmacologically enhancing YAP activity selectively activated neutrophils to suppress refractory GC, with no significant inflammation-related side effects. Thus, our work characterized tumor-specific neutrophils in GC and revealed an essential role of YAP/TAZ-CD54 axis in tsNeus, opening a new possibility to develop neutrophil-based antitumor therapeutics.
Humans ; Animals ; Mice ; Adaptor Proteins, Signal Transducing/metabolism* ; Transcription Factors/metabolism* ; Stomach Neoplasms/pathology* ; Neutrophils/pathology* ; Signal Transduction/genetics* ; YAP-Signaling Proteins ; Tumor Microenvironment ; Hyaluronan Receptors/genetics*

Objective: To study the effect of Musashi-2 ( MSI2 ) overexpression on the imbalance of proliferation and apoptosis of human ovarian granulosa cell line (KGN) induced by dihydrotestosterone (DHT) ． Methods: The gene expression profiles of human ovarian granulosa cells ( GCs) in primary culture were statistically analyzed to screen the differentially expressed genes．pcDNA3. 1-MSI2 eukaryotic expression plasmid was constructed and transiently transfected into the KGN cells，and the overexpression effect of MSI2 was detected by Quantitative Real-time PCR (RT-qPCR) and Western blot．After overexpressing MSI2 in DHT induced KGN cells，MTT colorimetry and Edu staining were used to detect the proliferation of cells in each group，and flow cytometry ( FCM) was further used to detect the apoptosis of cells in each group. Results: The mRNA expression level of MSI2 gradually decreased during the primary culture of human ovarian GCs．And pcDNA3. 1-MSI2 was successfully constructed and transfected into KGN cells to improve the mRNA and protein expression levels of MSI2．Then MTT，EdU and FCM results showed that compared with the blank group，DHT induction could significantly reduce the proliferation rate and increase the apoptosis rate of KGN cells (P ＜0. 05) ．However，after MSI2 overexpression，the proliferation rate of KGN cells increased and the apoptosis rate decreased (P ＜0. 05) ，which were close to the blank group. Conclusion Overexpression of MSI2 can effectively alleviate the imbalance of proliferation and apoptosis of KGN cells induced by DHT，indicating that MSI2 plays an important role in GCs growth and follicle development.

Objective: To construct a mouse derived pcDNA3. 1 ⁃3 × Flag⁃c⁃NUP85 expression plasmid and observe its effect on expression of inflammation factors in LPS⁃induced RAW264. 7 cells , as well as on the proliferation and apoptosis of RAW264. 7 cells. Methods: The NUP85 gene was amplified by PCR to construct pcDNA3. 1 ⁃3 × Flag-c⁃NUP85 eukaryotic expression plasmid. The pcDNA3. 1 ⁃3 × Flag⁃c vector was divided with enzymes. The purified PCR product was ligated with the vector, and the ligated product was transformed into bacterial competent cells. After identification by enzyme digestion , sequencing and analysis were performed. Then , it was transfected into RAW264. 7 cells , and the blank plasmid without NUP85 gene was set as the control group. The effect on cell proliferation and apoptosis were detected by CCK⁃8 assay and flow cytometry , and the expression of inflammatory cytokines such as tumor necrosis factor⁃α (TNF⁃α ) and interleukin⁃6 (IL⁃6) in LPS⁃induced RAW264. 7 cells was detected by Western blot and ELISA. Results: Enzyme digestion identification and Western blot results showed that pcDNA3. 1 ⁃3 × Flag⁃c⁃NUP85 eukaryotic expression plasmid was successfully constructed and expressed. The results of CCK⁃8 assay showed that the cell survival rate of NUP85 overexpression group was significantly lower than that of control group after 24 h[(0. 55 ± 0. 03) vs (0. 67 ± 0. 05) , F = 30. 98 , P < 0. 05 ] . The results of flow cytometry showed that the cell apoptosis rate of NUP85 overexpression group was higher than that of control group[( 15. 78 ±1. 05)% vs ( 13. 40 ± 0. 47)% , F = 75. 38 , P < 0. 05] . The results of Western blot and ELISA showed that after transfection of pcDNA3. 1 ⁃3 × Flag⁃c⁃NUP85 , the expression of TNF⁃α and IL⁃6 in RAW264. 7 cells were higher than those in the control group ,with statistical significance (P < 0. 05) . Conclusion NUP85 can inhibit the proliferation and promote apoptosis in LPS⁃stimulated RAW264. 7 cells , and NUP85 can promote the expression of inflammatory cytokines IL⁃6 and TNF⁃α in LPS⁃stimulated RAW264. 7 cells.

Acidosis, regardless of hypoxia involvement, is recognized as a chronic and harsh tumor microenvironment (TME) that educates malignant cells to thrive and metastasize. Although overwhelming evidence supports an acidic environment as a driver or ubiquitous hallmark of cancer progression, the unrevealed core mechanisms underlying the direct effect of acidification on tumorigenesis have hindered the discovery of novel therapeutic targets and clinical therapy. Here, chemical-induced and transgenic mouse models for colon, liver and lung cancer were established, respectively. miR-7 and TGF-β2 expressions were examined in clinical tissues (n = 184). RNA-seq, miRNA-seq, proteomics, biosynthesis analyses and functional studies were performed to validate the mechanisms involved in the acidic TME-induced lung cancer metastasis. Our data show that lung cancer is sensitive to the increased acidification of TME, and acidic TME-induced lung cancer metastasis via inhibition of miR-7-5p. TGF-β2 is a direct target of miR-7-5p. The reduced expression of miR-7-5p subsequently increases the expression of TGF-β2 which enhances the metastatic potential of the lung cancer. Indeed, overexpression of miR-7-5p reduces the acidic pH-enhanced lung cancer metastasis. Furthermore, the human lung tumor samples also show a reduced miR-7-5p expression but an elevated level of activated TGF-β2; the expressions of both miR-7-5p and TGF-β2 are correlated with patients' survival. We are the first to identify the role of the miR-7/TGF-β2 axis in acidic pH-enhanced lung cancer metastasis. Our study not only delineates how acidification directly affects tumorigenesis, but also suggests miR-7 is a novel reliable biomarker for acidic TME and a novel therapeutic target for non-small cell lung cancer (NSCLC) treatment. Our study opens an avenue to explore the pH-sensitive subcellular components as novel therapeutic targets for cancer treatment.

Objective To study the irradiation dose of organs at risk (OAR) in involved field radiation and extended field radiation in patients with thoracic esophageal cancer who received intensity modulated radiotherapy (IMRT). Methods A total of 40 patients with thoracic esophageal cancer were treated with IMRT. The involved field, extended field, and OAR were outlined to generate IMRT plans. The conformity index (CI) and homogeneity index (HI) of planning target volume (PTV) and the irradiation parameters of OAR were evaluated for the two plans. Paired t-test was used for comparison of irradiation parameters. Results The PTV of both plans received the prescribed dose. There were no significant differences in CI and HI of PTV between the two groups (P = 0.317, 0.130). There were significant differences in average lung dose, lung V₅, lung V₂₀, lung V₃₀, spinal cord D_mean, heart D_mean, heart D_max, heart V₃₀, heart V₄₀, and heart V₆₀ between the two groups (P < 0.01). Conclusion Compared with the extended field, the involved field can reduce the irradiation dose of ORA in patients with thoracic esophageal cancer, thus reducing the risk of radiation.