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Additional file 1: Fig. S1. Normalized LINP1 expression levels in a variety of tumors analyzed in TCGA database. The original data is from The Cancer Genome Atlas Program (TCGA) database and the diagram shows the expression levels of LINP1 in different types of cancer analyzed by Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/ ). Fig. S2. LINP1 promotes cell proliferation, migration and invasiveness in HSC-1 cells. (A) LINP1 expression was detected after LINP1 depletion in HSC-1 cells. Measurements of cell proliferation by CCK-8 assay (B), colony formation assay (C), transwell migration assay (D) and Matrigel invasiveness measurement (E) were performed in HSC-1 cells treated with siRNAs targeting LINP1. (F) Apoptosis assay by Annexin V/PI double staining were performed in HSC-1cells treated with siRNAs targeting LINP1. Scale bars, 500 mm (C), 100 μm (D, E). (G) qPCR validations of key gene expression in endoplasmic reticulum signaling including GRP78, XBP1, DDIT3 and DR5 after LINP1 depletion in HSC-1 cells. (H) Protein levels of GRP78, XBP1, eIF2α, p-eIF2α, DDIT3, DR5, and cleavages of Caspase-8, Caspase-7 and Caspase-3 were detected by Western blot after LINP1 knockdown in HSC-1 cells. GAPDH was using as loading control. Each experiment was performed in at least triplicate and results are presented as mean ± s.d. One-way ANOVA and Dunnett’s multiple comparison test were used to analyze the data (*P < 0.05, **P < 0.01, ***P < 0.001). Fig. S3. LINP1 promotes cell proliferation, migration and invasiveness in A431 cells. (A, F) LINP1 expression was detected after LINP1 depletion or overexpression in A431 cells. Measurements of cell proliferation by CCK-8 assay (B, G), colony formation assay (C, H), transwell migration assay and Matrigel invasiveness measurement (D, E, I, J) were performed after LINP1 knockdown or overexpression in A431 cells. Scale bars, 500 mm (C, H), 100 μm (D, E, I, J). Each experiment was performed in at least triplicate and results are presented as mean ± s.d. One-way ANOVA and Dunnett’s multiple comparison test were used to analyze the data (*P < 0.05, **P < 0.01, ***P < 0.001). Fig. S4. LINP1 functions as oncogene to repress UPR-mediated cell apoptosis in A431 cells. (A) The protein of GRP78, XBP1, eIF2α, p-eIF2α, ATF4 and DDIT3 were detected by Western blot in A431 cells after knockdown of LINP1 or overexpression of LINP1. (B, C) The expression of LINP1, DDIT3, DR5, XBP1 and GRP78 was detected by qRT-PCR in A431 cells after knockdown of LINP1 or overexpression of LINP1. (D) The levels of DR5 and cleaved Caspase-3, cleaved Caspase-7, cleaved Caspase-8 were detected by Western blot in A431 cells after knockdown of LINP1 or overexpression of LINP1. Statistical data of qRT-PCR represent the average of three independent experiments ± s.d. (E, F) Apoptosis assay by Annexin V/PI double staining were performed in LINP1-knockdown or LINP1-overexpression A431 cells. One-way ANOVA and Dunnett’s multiple comparison test were used to analyze the data (*P < 0.05, **P < 0.01, ***P < 0.001). Fig. S5. Peptides of eIF2α binding to LINP1 identified by HPLC-MS. (A) The protein mix precipitated by in vitro-transcribed Biotin-labelled LINP1 RNA was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) to identify the amino acid sequences of eIF2α peptides. (B) The ability of eIF2α to interact with LINP1 is predicted by lncPro ( http://bioinfo.bjmu.edu.cn/lncpro/ ). (C) Predicted secondary structure of LINP1 by RNAfold ( http://rna.tbi.univie.ac.at//cgi-bin/RNAWebSuite/RNAfold.cgi ). Fig. S6. Knockdown of DDIT3 promotes the proliferation, migration but represses apoptosis in cSCC cells. (A) DDIT3 expression was detected after depletion of DDIT3 by siNC and siDDIT3 in cSCC cells. (B-E) Depletion of DDIT3 repressed cell apoptosis and enhanced cell proliferation, migration in cSCC cells by CCK-8 assay, colony formation assay, transwell migration assay and Annexin V/PI double staining measurement. Scale bars, 500 mm (C), 100 μm (D). (F) DR5, cleaved forms of Caspase-8, Caspase-7 and Caspase-3 proteins were detected in cSCC cells by Western blot after DDIT3 knockdown, GAPDH was using as loading control. Each experiment was performed in at least triplicate and results are presented as mean ± s.d. One-way ANOVA and Dunnett’s multiple comparison test were used to analyze the data (*P < 0.05, **P < 0.01, ***P < 0.001). Fig. S7–S9. (A431, HSC-1 and HaCaT cell lines authentication and quality check).

Objective: To study the differences in clinical characteristics, risk factors, and complications across age-groups among the inpatients with the coronavirus disease 2019 (COVID-19).Methods: In this population-based retrospective study, we included all the positive hospitalized patients with COVID-19 at Wuhan City from December 29, 2019 to April 15, 2020, during the first pandemic wave. Multivariate logistic regression analyses were used to explore the risk factors for death from COVID-19. Canonical correlation analysis (CCA) was performed to study the associations between comorbidities and complications.Results: There are 36,358 patients in the final cohort, of whom 2,492 (6.85%) died. Greater age (odds ration [OR] = 1.061 [95% CI 1.057–1.065], p < 0.001), male gender (OR = 1.726 [95% CI 1.582–1.885], p < 0.001), alcohol consumption (OR = 1.558 [95% CI 1.355–1.786], p < 0.001), smoking (OR = 1.326 [95% CI 1.055–1.652], p = 0.014), hypertension (OR = 1.175 [95% CI 1.067–1.293], p = 0.001), diabetes (OR = 1.258 [95% CI 1.118–1.413], p < 0.001), cancer (OR = 1.86 [95% CI 1.507–2.279], p < 0.001), chronic kidney disease (CKD) (OR = 1.745 [95% CI 1.427–2.12], p < 0.001), and intracerebral hemorrhage (ICH) (OR = 1.96 [95% CI 1.323–2.846], p = 0.001) were independent risk factors for death from COVID-19. Patients aged 40–80 years make up the majority of the whole patients, and them had similar risk factors with the whole patients. For patients aged OR = 17.112 [95% CI 6.264–39.73], p < 0.001) and ICH (OR = 31.538 [95% CI 5.213–158.787], p < 0.001) were significantly associated with higher odds of death. For patients aged >80 years, only age (OR = 1.033 [95% CI 1.008–1.059], p = 0.01) and male gender (OR = 1.585 [95% CI 1.301–1.933], p < 0.001) were associated with higher odds of death. The incidence of most complications increases with age, but arrhythmias, gastrointestinal bleeding, and sepsis were more common in younger deceased patients with COVID-19, with only arrhythmia reaching statistical difference (p = 0.039). We found a relatively poor correlation between preexisting risk factors and complications.Conclusions: Coronavirus disease 2019 are disproportionally affected by age for its clinical manifestations, risk factors, complications, and outcomes. Prior complications have little effect on the incidence of extrapulmonary complications.

Xingyuan Liu,1 Chuang Feng,2 Guohua Wei,1 Wencong Kong,1 Hai Meng,3 Yaqin Du,3 Jingyuan Li4 1Pathology Department, College of Basic Medical Sciences, Jinzhou Medical University, Jinzhou, Liaoning 121001, People’s Republic of China; 2Science and Technology Department, Jinzhou Medical University, Jinzhou, Liaoning 121001, People’s Republic of China; 3Clinicopathological Center, First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning 121001, People’s Republic of China; 4Faculty of Pharmaceutical Sciences, First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning 121001, People’s Republic of ChinaCorrespondence: Jingyuan LiFaculty of Pharmaceutical Sciences, First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning Province 121001, People’s Republic of ChinaTel/ Fax +86-416-4145198Email lijingyuan1024@yeah.netBackground: Epithelial-to-mesenchymal transition (EMT) has been considered a latent mediator of diverse biological processes in cancer. However, the mechanisms involved in high glucose-associated EMT in lung adenocarcinoma (LAD) have not been fully clarified. In this study, we aimed to investigate whether mitofusin1 (MFN1) is involved in the EMT of LAD cells induced by glucose and to identify the molecular mechanism involved in this process.MaterialsandMethods: The expression of specific proteins was analysed by Western blotting, immunohistochemistry, co-immunoprecipitation and immunofluorescence analysis. The proliferation, migration and invasion of cells were assessed by Cell Counting Kit-8, bromodeoxyuridine incorporation, wound-healing and transwell assays. Lung tissues of adjacent normal regions and lung tissues from patients with LAD and LAD combined with diabetes mellitus were collected to determine the expression and significance of MFN1.Results: Here, we showed that the expression of MFN1 was increased in LAD tissues compared with adjacent normal tissues and expression was even higher in lung tissues from patients with LAD combined with diabetes. In the lung cancer cell line A549, increased cell proliferation, invasion and EMT induced by high glucose were inhibited by MFN1 silencing. Mechanistic studies demonstrated that inhibiting autophagy reversed the abnormal EMT triggered by high glucose conditions. In addition, our data provide novel evidence demonstrating that PTEN-induced kinase (Pink) is a potential regulator involved in MFN1-mediated cell autophagy, which eventually leads to high glucose-induced proliferation, invasion and EMT of A549 cells.Conclusion: Taken together, our data show that MFN1 interacts with Pink to induce the autophagic process and that the abnormal occurrence of autophagy ultimately contributes to glucose-induced pathological EMT in LAD.Keywords: lung adenocarcinoma, glucose, mitofusin1, epithelial-to-mesenchymal transition, autophagy

A novel series of indole-2-carbohydrazide derivatives were synthesized, characterized, and evaluated for their antiproliferative activities against two cancer cell lines, HCT116 and SW480, and a normal human fetal lung fibroblast cell line, MRC-5. Among this series, compound 24 f displayed potent cytotoxic activities in vitro against HCT116 and SW480 cell lines with GI50 values of 8.1 and 7.9 μm, respectively, and was inactive against MRC-5 cells. The newly synthesized compounds were also evaluated for anti-angiogenesis capabilities by chick chorioallantoic membrane, human umbilical vein endothelial cell (HUVEC) migration, and endothelial microtubule formation assays. Moreover, the effects of 24 f on the vascular endothelial growth factor receptor-2 and the signaling pathway in HUVECs indicated that this compound inhibits VEGFR-2 and its downstream related proteins. These results indicate that compound 24 f, as well as the other derivatives, are promising inhibitors of angiogenesis.