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This paper presents a finite element analysis (FEA) of and reliability study on concrete-filled steel tube (CFST) members under the combined effects of corrosion and compressive loading. First, a stochastic-based FE model is established through the proposed secondary development program based on ABAQUS 2021 software. The model could account for the uncertainties of material, geometric, and corrosion effect on CFST members. The reliability of the built model was validated through experimental data of corroded CFST members under compression loading. Subsequently, the compressive performance of CFST under a combination of corrosion and loading was further investigated by numerical parameter analysis. A total of 1800 models were created to clarify the coupling mechanism among the core concrete strength, the steel tube strength, the steel ratio, and the maximum strength of the CFST member. Three theoretical formulas presented in classical design standards were used to calculate the axial compressive strength of the corroded CFST, and the uncertainty parameters μkp and δkp were also obtained for the discussed design formulas. Finally, the First Order and Second Moment (FOSM) method was employed to estimate the reliability indices β across different standards. The calculations revealed that the reliability indices β according to European standard ranges from 2.93 to 5.52, with some results falling below the target reliability index βT of 3.65. In addition, the multi-parameter coupling effects on reliability index β were investigated, and the main influencing factors were obtained. By leveraging the reliability analysis, reasonable design requirements can be proposed for CFST members under the coupling effects of corrosion and external load, which provides a design basis for the CFST member.

AIM:To explore the effect of genetic factors on the pathogenesis of keratoconus and its genetic model.METHODS: Genetic epidemiological methods were used to investigate the prevalence of keratoconus in 280 first-degree relatives of 100 patients with keratoconus who attended Henan Eye Hospital between July 2020 and April 2023. The heritability was estimated by Falconer regression method. The general genetic model was calculated using Penrose method, and the genetic model was confirmed by Falconer formula, Edwards approximation formula and the projection formula of San-Duo Jiang's threshold model theory.RESULTS: The results showed that there were 16(5.714%)first-degree relatives of keratoconus probands suffering from keratoconus, and the heritability of keratoconus was(86.100±7.400)%. The S/q score calculated by the Penrose method was 35.348, which was near to 1/(q)1/2, suggesting that the genetic model of keratoconus might be polygenic inheritance. The expected prevalence in first-degree relatives of keratoconus patients by Falconer formula, Edwards approximation formula and the projection formula of San-Duo Jiang's threshold model theory were 5.900%, 7.714% and 5.700%, respectively, which showed no significant differences from the actual prevalence(5.714%), suggesting that keratoconus was a polygenetic disease.CONCLUSION:Genetic factors might play an important role in the pathogenesis of keratoconus, and keratoconus is a polygenetic disease.

Abstract Background As one of the most common malignancies, esophageal cancer has two subtypes, squamous cell carcinoma and adenocarcinoma, arising from distinct cells-of-origin. Distinguishing cell-type-specific molecular features from cancer-specific characteristics is challenging. Results We analyze whole-genome bisulfite sequencing data on 45 esophageal tumor and nonmalignant samples from both subtypes. We develop a novel sequence-aware method to identify large partially methylated domains (PMDs), revealing profound heterogeneity at both methylation level and genomic distribution of PMDs across tumor samples. We identify subtype-specific PMDs that are associated with repressive transcription, chromatin B compartments and high somatic mutation rate. While genomic locations of these PMDs are pre-established in normal cells, the degree of loss is significantly higher in tumors. We find that cell-type-specific deposition of H3K36me2 may underlie genomic distribution of PMDs. At a smaller genomic scale, both cell-type- and cancer-specific differentially methylated regions (DMRs) are identified for each subtype. Using binding motif analysis within these DMRs, we show that a cell-type-specific transcription factor HNF4A maintains the binding sites that it generates in normal cells, while establishing new binding sites cooperatively with novel partners such as FOSL1 in esophageal adenocarcinoma. Finally, leveraging pan-tissue single-cell and pan-cancer epigenomic datasets, we demonstrate that a substantial fraction of cell-type-specific PMDs and DMRs identified here in esophageal cancer are actually markers that co-occur in other cancers originating from related cell types. Conclusions These findings advance our understanding of DNA methylation dynamics at various genomic scales in normal and malignant states, providing novel mechanistic insights into cell-type- and cancer-specific epigenetic regulations.

Abstract Background Increasing evidence indicates that the tumor microenvironment (TME) is a crucial determinant of cancer progression. However, the clinical and pathobiological significance of stromal signatures in the TME, as a complex dynamic entity, is still unclear in esophageal squamous cell carcinoma (ESCC). Methods Herein, we used single-cell transcriptome sequencing data, imaging mass cytometry (IMC) and multiplex immunofluorescence staining to characterize the stromal signatures in ESCC and evaluate their prognostic values in this aggressive disease. An automated quantitative pathology imaging system determined the locations of the lamina propria, stroma, and invasive front. Subsequently, IMC spatial analyses further uncovered spatial interaction and distribution. Additionally, bioinformatics analysis was performed to explore the TME remodeling mechanism in ESCC. To define a new molecular prognostic model, we calculated the risk score of each patient based on their TME signatures and pTNM stages. Results We demonstrate that the presence of fibroblasts at the tumor invasive front was associated with the invasive depth and poor prognosis. Furthermore, the amount of α-smooth muscle actin (α-SMA)+ fibroblasts at the tumor invasive front positively correlated with the number of macrophages (MØs), but negatively correlated with that of tumor-infiltrating granzyme B+ immune cells, and CD4+ and CD8+ T cells. Spatial analyses uncovered a significant spatial interaction between α-SMA+ fibroblasts and CD163+ MØs in the TME, which resulted in spatially exclusive interactions to anti-tumor immune cells. We further validated the laminin and collagen signaling network contributions to TME remodeling. Moreover, compared with pTNM staging, a molecular prognostic model, based on expression of α-SMA+ fibroblasts at the invasive front, and CD163+ MØs, showed higher accuracy in predicting survival or recurrence in ESCC patients. Regression analysis confirmed this model is an independent predictor for survival, which also identifies a high-risk group of ESCC patients that can benefit from adjuvant therapy. Conclusions Our newly defined biomarker signature may serve as a complement for current clinical risk stratification approaches and provide potential therapeutic targets for reversing the fibroblast-mediated immunosuppressive microenvironment.

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the world’s leading causes of death, and its primary clinical therapy relies on surgical resection, chemotherapy, radiotherapy, and chemoradiotherapy. Although the genomic features and clinical significance of ESCC have been identified, the outcomes of targeted therapies are still unsatisfactory. Here, we demonstrate that mitogen-activated protein kinase (MAPK) signaling is highly activated and associated with poor prognosis in patients with ESCC. Mitogen-activated protein kinase kinase (MEK) inhibitors efficiently blocked the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in ESCC, while signal transducer and activator of transcription 3 (STAT3) signaling was rapidly activated. Combined STAT3 inhibition prevented the emergence of resistance and enhanced MEK inhibitor-induced cell cycle arrest and senescence in vitro and in vivo. Mechanistic studies revealed that the suppressor of cytokine signaling 3 (SOCS3) was downregulated, resulting in an increase in STAT3 phosphorylation in MEK-inhibited cells. Furthermore, chromatin immunoprecipitation showed that ELK1, which was activated by MEK/ERK signaling, induced SOCS3 transcription. These data suggest that the development of combined MEK and STAT3 inhibition could be a useful strategy in ESCC targeted therapy.

AIM: To investigate the characteristics of corneal stiffness parameters at the first applanation(SP-A1)in keratoconus patients among different age groups and severity classification groups. METHODS: A cross-sectional study. Three hundred and forty-five eyes of 247 consecutive keratoconus patients diagnosed in Henan Eye Hospital were enrolled during September 2018 to December 2020. The mean age was(24.51±6.38)years. According to age, the subjects were divided into ≤20 years group, 21-30 years group and ≥31 years group. The severity of keratoconus was divided into AK1, AK2, AK3 and AK4 by Amsler krumeich(AK)classification method. The visual acuity, corneal flat curvature(K1), corneal steep curvature(K2), average corneal curvature(Km)and the thinnest corneal thickness(TCT)were measured. The SP-A1, intraocular pressure(IOP), deformation amplitude max(DA Max), velocity at the first applanation(A1V), time at the first applanation(A1T), time at the second applanation(A2T), velocity at the second applanation(A2V), biomechanical corrected IOP(bIOP), and Radius were measured by Corneal Visualization Scheimpflug Technology(Corvis ST). The correlation between SP-A1 and other parameters were analyzed by Spearman rank correlation, and one-way ANOVA was used to analyze the difference of SP-A1 among different age groups or disease severity groups. RESULTS: No statistically differences of gender, visual acuity, IOP, K1, K2, Km, TCT and disease severity classification were found among different age groups(P>0.05). The SP-A1 was positively related to age(rs=0.137, P=0.011). The SP-A1 of keratoconus patients in the ≥31 years group was significantly higher than that in the ≤20 years group and 21-30 years group(P

The microenvironment of oesophageal squamous cell carcinomas (ESCC) is heterogeneous and can strongly impact response to treatment. Here, the authors characterize the ESCC tumour microenvironment with single-cell RNA-seq, finding CST1 + myofibroblasts with potential biological and prognostic significance as well as immunosuppression signatures.

Abstract Background Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post‐translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. Methods Immunoprecipitation and glutathione‐S‐transferase pull‐down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP‐associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF‐mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live‐cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. Results Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti‐AcK471‐Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin‐K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin‐K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin‐K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471‐acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin‐bundling activity. Clinically, high levels of AcK471‐Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease‐free survival of ESCC patients. Conclusions Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin‐K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin‐bundling activity.

Lysyl-oxidase like-2 (LOXL2) regulates extracellular matrix remodeling and promotes tumor invasion and metastasis. Altered metabolism is a core hallmark of cancer, however, it remains unclear whether and how LOXL2 contributes to tumor metabolism. Here, we found that LOXL2 and its catalytically inactive L2Δ13 splice variant boost glucose metabolism of esophageal tumor cells, facilitate tumor cell proliferation and promote tumor development in vivo. Consistently, integrated transcriptomic and metabolomic analysis of a knock-in mouse model expressing L2Δ13 gene revealed that LOXL2/L2Δ13 overexpression perturbs glucose and lipid metabolism. Mechanistically, we identified aldolase A, glyceraldehyde-3-phosphate dehydrogenase and enolase as glycolytic proteins that interact physically with LOXL2 and L2Δ13. In the case of aldolase A, LOXL2/L2Δ13 stimulated its mobilization from the actin cytoskeleton to enhance aldolase activity during malignant transformation. Using stable isotope labeling of amino acids in cell culture (SILAC) followed by proteomic analysis, we identified LOXL2 and L2Δ13 as novel deacetylases that trigger metabolic reprogramming. Both LOXL2 and L2Δ13 directly catalyzed the deacetylation of aldolase A at K13, resulting in enhanced glycolysis which subsequently reprogramed tumor metabolism and promoted tumor progression. High level expression of LOXL2/L2Δ13 combined with decreased acetylation of aldolase-K13 predicted poor clinical outcome in patients with esophageal cancer. In summary, we have characterized a novel molecular mechanism that mediates the pro-tumorigenic activity of LOXL2 independently of its classical amine oxidase activity. These findings may enable the future development of therapeutic agents targeting the metabolic machinery via LOXL2 or L2Δ13. Highlight of the study: LOXL2 and its catalytically inactive isoform L2Δ13 function as new deacetylases to promote metabolic reprogramming and tumor progression in esophageal cancer by directly activating glycolytic enzymes such as aldolase A.

Summary: Obesity is primarily characterized by the dysregulation of lipid metabolism and gut microbiota. Here, we found that the body weight of transgenic mice overexpressing L2Δ13, a selectively spliced isoform of lysyl oxidase-like 2 (LOXL2), was lower than that of wild-type (WT) mice. Numerous microbiotas were significantly changed and most microbial metabolites were abnormal in L2Δ13 mice. Lipid metabolites in feces were negatively correlated with those in plasma, suggesting that L2Δ13 may affect lipid uptake, and potentially, adipose tissue homeostasis. This was supported by the weight loss and decreased area of adipose tissue in L2Δ13 mice. Adipogenic differentiation of primary stromal vascular fraction cells showed that the lipid droplets of L2Δ13 cells were significantly smaller than those of WT cells. Adipocyte differentiation-associated genes were also downregulated in adipose tissue from L2Δ13 mice. Thus, L2Δ13 can induce adipose tissue loss in mice by affecting gut microbiota homeostasis and multi-tissue lipid metabolism.

Proteomics can aid in the identification of molecular subtypes in cancers. Here, the authors perform proteomic profiling of 124 paired oesophageal cancer and adjacent non-tumour tissues and identify two subtypes that are associated with patient survival for therapeutic targeting.

The relevance and underlying molecular mechanisms of epigenetic regulation in squamous cell carcinomas (SCC) await further characterization. Here, the authors show a transcriptional regulatory loop involving SREBF1, TP63 and KLF5 driving tumourigenesis in SCC through fatty acid, ERBB and mTOR pathway regulation.

(1) Background: Esophageal cancer (EC) is an important global health challenge. Due to the lack of necessary biomarkers and therapeutic targets, the survival of EC patients is poor. The EC proteomic data of 124 patients recently published by our group provides a database for research in this field. (2) Methods: Bioinformatics analysis was used to identify DNA replication and repair-related proteins in EC. Proximity ligation assay, colony formation assay, DNA fiber assay, and flow cytometry were used to study the effects of related proteins on EC cells. Kaplan–Meier survival analysis was used to evaluate the relationship between gene expression and the survival time of EC patients. (3) Results: Chromatin assembly factor 1 subunit A (CHAF1A) was highly correlated with proliferating cell nuclear antigen (PCNA) expression in EC. CHAF1A and PCNA colocalized in the nucleus of EC cells. Compared with the knockdown of CHAF1A or PCNA alone, the double knockdown of CHAF1A and PCNA could significantly inhibit EC cell proliferation. Mechanistically, CHAF1A and PCNA synergistically accelerated DNA replication and promoted S-phase progression. EC patients with high expression of both CHAF1A and PCNA had a worse survival rate. (4) Conclusion: we identify CHAF1A and PCNA as key cell cycle-related proteins leading to the malignant progression of EC, and these proteins could serve as important prognostic biomarkers and targets for EC.

AIM:To evaluate the repeatability and comparison of corneal visualization scheimpflug technology(Corvis ST)parameters in keratoconus eyes before and after accelerated transepithelial corneal cross-linking(ATE-CXL).METHODS: Thirty eyes of 30 progressive keratoconus patients were included in the prospective study. Three repeated corneal biomechanical measurements were performed preoperatively and one month postoperatively by Corvis ST. The interclass correlation coefficient(ICC)and 95% confidence interval(CI), Cronbach'α, repeatability coefficient(RC), and coefficient of variation(CV)were used to evaluate the repeatability of Corvis ST parameters. Paired t-test or Wilcoxon rank test was used to evaluate the differences between preoperative and postoperative data.RESULTS: At preoperative, 26 of 39(66.67%)parameters showed good to excellent repeatability, 6(15.38%)showed moderate, and 7(17.95%)showed poor repeatability. Similarly, 34(87.18%)parameters showed good to excellent repeatability, 3(7.69%)showed moderate, and 2(5.13%)showed poor repeatability after ATE-CXL. After ATE-CXL 1mo, the intraocular pressure(IOP), biomechanical corrected IOP(bIOP), first applanation time(A1T), Radius, deformation amplitude at the first applanation(A1DA), deflection length at the maximum deformation(HCDLL)and stiffness parameter at first applanation(SP A1)parameters increased, while the steep keratometry(Ks), flat keratometry(Kf), mean keratometry(Kmean), second applanation time(A2T), DA Ratio Max(2 mm)and integrated radius parameters decreased(all P

Long noncoding RNAs (lncRNAs) have important regulatory roles in cancer biology. Although some lncRNAs have well‐characterized functions, the vast majority of this class of molecules remains functionally uncharacterized. To systematically pinpoint functional lncRNAs, a computational approach was proposed for identification of lncRNA‐mediated competing endogenous RNAs (ceRNAs) through combining global and local regulatory direction consistency of expression. Using esophageal squamous cell carcinoma (ESCC) as model, we further identified many known and novel functional lncRNAs acting as ceRNAs (ce‐lncRNAs). We found that most of them significantly regulated the expression of cancer‐related hallmark genes. These ce‐lncRNAs were significantly regulated by enhancers, especially super‐enhancers (SEs). Landscape analyses for lncRNAs further identified SE‐associated functional ce‐lncRNAs in ESCC, such as HOTAIR, XIST, SNHG5, and LINC00094. THZ1, a specific CDK7 inhibitor, can result in global transcriptional downregulation of SE‐associated ce‐lncRNAs. We further demonstrate that a SE‐associated ce‐lncRNA, LINC00094 can be activated by transcription factors TCF3 and KLF5 through binding to SE regions and promoted ESCC cancer cell growth. THZ1 downregulated expression of LINC00094 through inhibiting TCF3 and KLF5. Our data demonstrated the important roles of SE‐associated ce‐lncRNAs in ESCC oncogenesis and might serve as targets for ESCC diagnosis and therapy.

Keratoconus is a corneal disease characterized by corneal ectasia, progressive corneal thinning, conical protrusion and irregular astigmatism. Several studies have indicated that keratoconus is a complex disease with genetic heterogeneity. Genetic studies on keratoconus involving nuclear genome, mitochondrial genome and epigenetics were increasing. This article reviews the recent progress in genetic research on keratoconus.

Lysyl oxidase-like 2 (LOXL2) is a metalloenzyme that catalyzes the oxidative deamination ε-amino group of lysine. It is found that LOXL2 is a promotor for the metastasis and invasion of cancer cells. Disulfide bonds are important components in LOXL2, and they play a stabilizing role for protein structure or a functional role for regulating protein bioactivity. The redox potential of disulfide bond is one important property to determine the functional role of disulfide bond. In this study, we have calculated the reduction potential of all the disulfide bonds in LOXL2 by non-equilibrium alchemical simulations. Our results show that seven of seventeen disulfide bonds have high redox potentials between −182 and −298 mV and could have a functional role, viz., Cys573–Cys625, Cys579–Cys695, Cys657–Cys673, and Cys663–Cys685 in the catalytic domain, Cys351–Cys414, Cys464–Cys530, and Cys477–Cys543 in the scavenger receptor cysteine-rich (SRCR) domains. The disulfide bond of Cys351–Cys414 is predicted to play an allosteric function role, which could affect the metastasis and invasion of cancer cells. Other functional bonds have a catalytic role related to enzyme activity. The rest of disulfide bonds are predicted to play a structural role. Our study provides an important insight for the classification of disulfide bonds in LOXL2 and can be utilized for the drug design that targets the cysteine residues in LOXL2.

Marine nature products are unique compounds that are produced by the marine environment including plants, animals, and microorganisms. The wide diversity of marine natural products have great potential and are versatile in terms of drug discovery. In this paper, we use state-of-the-art computational methods to discover inhibitors from marine natural products to block the function of Fascin, an overexpressed protein in various cancers. First, virtual screening (pharmacophore model and molecular docking) was carried out based on a marine natural products database (12015 molecules) and provided eighteen molecules that could potentially inhibit the function of Fascin. Next, molecular mechanics generalized Born surface area (MM/GBSA) calculations were conducted and indicated that four molecules have higher binding affinities than the inhibitor NP-G2-029, which was validated experimentally. ADMET analyses of pharmacokinetics demonstrated that one of the four molecules does not match the criterion. Finally, ligand Gaussian accelerated molecular dynamics (LiGaMD) simulations were carried out to validate the three inhibitors binding to Fascin stably. In addition, dynamic interactions between protein and ligands were analyzed systematically. Our study will accelerate the development of the cancer drugs targeting Fascin.

Development of chemoresistance remains a major challenge in treating esophageal squamous cell carcinoma (ESCC) patients despite treatment advances. However, the role of RAC1 in chemoresistance of ESCC and the underlying mechanisms remain largely unknown. In this study, we found that higher levels of RAC1 expression were associated with poorer prognosis in ESCC patients. Enhanced RAC1 expression increased cell proliferation, migration, and chemoresistance in vitro. Combination therapy using RAC1 inhibitor EHop‐016 and cisplatin significantly promoted cell viability inhibition, G2/M phase cycle arrest, and apoptosis when compared to each monotherapy. Mechanistically, glycolysis was significantly downregulated in the RAC1 inhibitor monotherapy group and the combination group via inhibiting AKT/FOXO3a signaling when compared to the control group. Moreover, the silencing of RAC1 inhibited AKT/FOXO3a signaling and cell glycolysis while the upregulation of RAC1 produced an opposite effect. In murine xenograft models, the tumor volume and the expression of glycolytic enzymes were significantly reduced in combination therapy when compared to each monotherapy group. Overall, our study demonstrates that targeting RAC1 with an inhibitor overcomes cisplatin resistance in ESCC by suppressing glycolytic enzymes, which provides a promising strategy for treatment of ESCC in clinical practice.

Metabolomics is an emerging field that quantifies numerous metabolites systematically. The key purpose of metabolomics is to identify the metabolites corresponding to each biological phenotype, and then provide an analysis of the mechanisms involved. Although metabolomics is important to understand the involved biological phenomena, the approach’s ability to obtain an exhaustive description of the processes is limited. Thus, an analysis-integrated metabolomics, transcriptomics, proteomics, and other omics approach is recommended. Such integration of different omics data requires specialized statistical and bioinformatics software. This review focuses on the steps involved in metabolomics research and summarizes several main tools for metabolomics analyses. We also outline the most abnormal metabolic pathways in several cancers and diseases, and discuss the importance of multi-omics integration algorithms. Overall, our goal is to summarize the current metabolomics analysis workflow and its main analysis software to provide useful insights for researchers to establish a preferable pipeline of metabolomics or multi-omics analysis.

Abstract Metastasis accounts for 90% of cancer death worldwide, and effective therapeutic strategies are lacking. The aim of this work is to identify the key drivers in tumor metastasis and screen therapeutics for treatment of esophageal squamous cell carcinoma (ESCC). Gene Ontology analysis of The Cancer Genome Atlas (TCGA) gene expression datasets of ESCC patients with or without lympy metastasis identifies that TGFβ2 is highly enriched in the pathways essential for tumor metastasis and upregulates in the metastatic ESCC tumors. High TGFβ2 expression in ESCC correlates with metastasis and patient survival, and functionally contributes to tumor metastasis via activating extracellular signal‐regulated kinases (ERK) signaling. By screening of a library consisting of 429 bioactive compounds, imperatorin is verified as a novel TGFβ2 inhibitor, with robustly suppressive effect on tumor metastasis in multiple mice models. Mechanistically, direct binding of imperatorin and CREB1 inhibits phosphorylation, nuclear translocation of CREB1, and its interaction with TGFβ2 promoter, represses TGFβ2 expression and fibroblasts‐secreted CCL2, and then inactivates ERK signaling to block cancer invasion and abrogates the paracrine effects of fibroblasts on tumor angiogenesis and metastasis. Overall, the findings suggest the use of TGFβ2 as a diagnostic and prognostic biomarker and therapeutic target in ESCC, and supports the potential of imperatorin as a novel therapeutic strategy for cancer metastasis.

Background: Early detection would improve upper gastrointestinal cancer prognosis. We aimed to identify serum protein biomarker for the detection of early-stage upper gastrointestinal cancer. Methods: We performed a three-tiered study including 2028 participants from three medical centres. First, we applied two different antibody arrays to screen candidate serum proteins that increased in 20 patients with oesophageal squamous cell carcinoma (ESCC) compared with 20 normal controls. We then evaluated the selected protein by enzyme-linked immunosorbent assay in 1064 participants including 731 upper gastrointestinal cancer patients (287 ESCCs, 237 oesophagogastric junction adenocarcinomas (EJAs), and 207 stomach cancers) and 333 normal controls. The diagnostic value of the selected protein was finally validated in two independent cohorts of ESCC patients and controls (n=472 and 452, respectively). The receiver operating characteristic was used to calculate diagnostic accuracy. Findings: Serum insulin-like growth factor binding protein-1 (IGFBP-1) identified in both antibody arrays showed significantly elevated levels in upper gastrointestinal cancers, compared with normal controls. Serum IGFBP-1 provided high diagnostic accuracy of early-stage ESCC, EJA, stomach and cancer (areas under the curve: 0·898, 0·936 and 0·864, respectively). This protein maintained diagnostic performance for early-stage ESCC in independent cohorts 1 and 2 (0·849 and 0·911, respectively). Additionally, serum levels of IGFBP-1 dropped significantly after surgical resection of primary tumours, compared with the corresponding pre-operative ESCC samples (p < 0·05). Interpretation: Serum IGFBP-1 represents a promising diagnostic biomarker to detect early-stage upper gastrointestinal cancer. Keywords: Upper gastrointestinal cancer, IGFBP-1, Early diagnosis, Sensitivity, Specificity

Master regulator transcription factors TP63 and SOX2 have been reported to overlap in genomic occupancy in squamous cell carcinomas (SCCs). Here, the authors demonstrate that TP63 and SOX2 promote co-operatively long non-coding RNA CCAT1 expression through activating its super-enhancer, and CCAT1 forms a complex with TP63 and SOX2, which regulates EGFR super-enhancers and enhances both the MEK/ERK1/2 and PI3K/AKT signaling pathways in SCC.

Abstract Background Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal carcinoma in China. This study was to develop a staging model to predict outcomes of patients with ESCC. Methods Using Cox regression analysis, principal component analysis (PCA), partitioning clustering, Kaplan–Meier analysis, receiver operating characteristic (ROC) curve analysis, and classification and regression tree (CART) analysis, we mined the Gene Expression Omnibus database to determine the expression profiles of genes in 179 patients with ESCC from GSE63624 and GSE63622 dataset. Results Univariate cox regression analysis of the GSE63624 dataset revealed that 2404 protein-coding genes (PCGs) and 635 long non-coding RNAs (lncRNAs) were associated with the survival of patients with ESCC. PCA categorized these PCGs and lncRNAs into three principal components (PCs), which were used to cluster the patients into three groups. ROC analysis demonstrated that the predictive ability of PCG-lncRNA PCs when applied to new patients was better than that of the tumor-node-metastasis staging (area under ROC curve [AUC]: 0.69 vs. 0.65, P

Abstract Background Increasing evidence shows that dysregulated long non-coding RNAs (lncRNAs) can serve as potential biomarkers for cancer prognosis. However, lncRNA signatures, as potential prognostic biomarkers for esophageal squamous cell carcinoma (ESCC), have been seldom reported. Methods Based on our previous transcriptome RNA sequencing analysis from 15 paired ESCC tissues and adjacent normal tissues, we selected 10 lncRNAs with high score rank and characterized the expression of those lncRNAs, by qRT-PCR, in 138 ESCC and paired adjacent normal samples. These 138 patients were divided randomly into training (n = 77) and test (n = 59) groups. A prognostic signature of lncRNAs was identified in the training group and validated in the test group and in an independent cohort (n = 119). Multivariable Cox regression analysis evaluated the independence of the signature in overall survival (OS) and disease-free survival (DFS) prediction. GO and KEGG pathway analysis, combined with cell transwell and proliferation assays, are applied to explore the function of the three lncRNAs. Results A novel three-lncRNA signature, comprised of RP11-366H4.1.1 (ENSG00000248370), LINC00460 (ENSG00000233532) and AC093850.2 (ENSG00000230838), was identified. The signature classified patients into high-risk and low-risk groups with different overall survival (OS) and disease-free survival (DFS). For the training group, median OS: 23.1 months vs. 39.1 months, P

Vertical deflection of a frame beam is an important indicator in the limit-state analysis of frame structures, particularly for steel–concrete composite beams, which are usually designed with large spans and heavy loads. In this study, the equivalent flexural stiffness of composite frame beams is analysed to evaluate their vertical deflection. A theoretical beam model with a spring constraint boundary and varied stiffness segments is established to consider the influence of both the rotation restraint stiffness at the beam ends and the cracked section in the negative moment region, such that the inelastic bending deformation of the composite beams can be elaborately described. By an extensive parametric analysis, a fitting formula for evaluating the equivalent flexural stiffness of the composite beams, including the effects of the rotational constraint and the concrete cracking, is obtained. The validity of the proposed formula is demonstrated by comparing its calculation accuracy with those of existing design formulas for analysing the equivalent flexural stiffness of the composite beam members. Moreover, its utility is further verified by conducting non-linear finite element simulations of structural systems to examine the serviceability limit state and the entire process evolution of beam deflections under vertical loading. Finally, to facilitate the practical application of the proposed formula in engineering design, a simplified method to calculate the deflection of composite beams, which utilises the internal force distribution of elastic analysis, is presented based on the concept of equivalent flexural stiffness.

Esophageal squamous cell carcinoma is a leading cause of cancer death. Mapping the transcriptional landscapes such as isoforms, fusion transcripts, as well as long noncoding RNAs have played a central role to understand the regulating mechanism during malignant processes. However, canonical methods such as short-read RNA-seq are difficult to define the entire polyadenylated RNA molecules. Here, we combined single-molecule real-time sequencing with RNA-seq to generate high-quality long reads and to survey the transcriptional program in esophageal squamous cells. Compared with the recent annotations of human transcriptome (Ensembl 38 release 91), single-molecule real-time data identified many unannotated transcripts, novel isoforms of known genes and an expanding repository of long intergenic noncoding RNAs (lincRNAs). By integrating with annotation of lincRNA catalog, 1,521 esophageal-cancer-specific lincRNAs were defined from single-molecule real-time reads. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that these lincRNAs and their target genes are involved in a variety of cancer signaling pathways. Isoform usage analysis revealed the shifted alternative splicing patterns, which can be recaptured from clinical samples or supported by previous studies. Utilizing vigorous searching criteria, we also detected multiple transcript fusions, which are not documented in current gene fusion database or readily identified from RNA-seq reads. Two novel fusion transcripts were verified based on real-time PCR and Sanger sequencing. Overall, our long-read single-molecule sequencing largely expands current understanding of full-length transcriptome in esophageal cells and provides novel insights on the transcriptional diversity during oncogenic transformation.

To comprehensively evaluate the association between rs7517847 and rs2201841 polymorphisms in the Interleukin-23 (IL-23) receptor gene and ankylosing spondylitis (AS), a meta-analysis was performed. The Pubmed, Embase, MEDLINE, Cochrane, China National Knowledge Infrastructure (CNKI), VIP, Wanfang and China Biology Medicine disc (CBMdisc) databases were searched to identify eligible studies on rs7517847 and rs2201841 polymorphisms in the IL-23 receptor gene and AS that were published through September 2014. Data of interest were extracted from each study, and the meta-analysis was performed using STATA 12.0. Four studies were eligible for the meta-analysis and included a total patient population of 2,465. With regards to rs7517847, the current study showed that the genotype GG and allele G might play a protective role during AS (OR = 0.76, 95% CI [0.59–0.99]; OR = 0.88, 95% CI [0.78–0.99] for homozygote and allelic models, respectively). However, according to the meta-analysis, there was no statistical association between the genotype or allele of rs2201841 and an individual’s susceptibility to AS in all genetic models. In conclusion, it was the IL-23 rs7517847 polymorphism rather than the rs2201841 polymorphism that had a statistical association with AS. Nevertheless, more evidence is needed to confirm this result. Consequently, it is necessary to carry out more high-quality studies to confirm the associations between these two single nucleotide polymorphisms and AS.

The membrane-cytoskeleton link organizer ezrin may be the most "dramatic" tumor marker, being strongly over-expressed in nearly one-third of human malignancies. However, the molecular mechanisms of aberrant ezrin expression still need to be clarified. Ezrin, encoded by the VIL2 gene, has two transcript variants that differ in the transcriptional start site (TSS): V1 and V2. Both V1 and V2 encode the same protein. Here, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) induced over-expression of human VIL2 in esophageal squamous cell carcinoma (ESCC) cells. Furthermore, VIL2 V1 but not V2 was up-regulated after TPA stimulation in a time-dependent manner. AP-1 and Sp1 binding sites within the promoter region of VIL2 V1 acted not only as basal transcriptional elements but also as a composite TPA-responsive element (TRE) for the transcription of VIL2 V1. TPA stimulation enhanced c-Jun and Sp1 binding to the TRE via activation of the ERK1/2 pathway and increased protein levels of c-Jun, c-Fos, and Sp1, resulting in over-expression of VIL2 V1, whereas the MEK1/2 inhibitor U0126 blocked these events. Finally, we showed that TPA promoted the migration of ESCC cells whereas MEK1/2 inhibitor or ezrin silencing could partially inverse this alteration. Taken together, these results suggest that TPA is able to induce VIL2 V1 over-expression in ESCC cells by activating MEK/ERK1/2 signaling and increasing binding of Sp1 and c-Jun to the TRE of the VIL2 V1 promoter, and that VIL2 is an important TPA-induced effector.

LOXL2 (lysyl oxidase-like 2), an enzyme that catalyzes oxidative deamination of lysine residue, is upregulated in esophageal squamous cell carcinoma (ESCC). A LOXL2 splice variant LOXL2-e13 and its wild type were overexpressed in ESCC cells followed by microarray analyses. In this study, we explored the potential role and molecular mechanism of LOXL2-e13 based on known protein-protein interactions (PPIs), following microarray analysis of KYSE150 ESCC cells overexpressing a LOXL2 splice variant, denoted by LOXL2-e13, or its wild-type counterpart. The differentially expressed genes (DEGs) of LOXL2-WT and LOXL2-e13 were applied to generate individual PPI subnetworks in which hundreds of DEGs interacted with thousands of other proteins. These two DEG groups were annotated by Functional Annotation Chart analysis in the DAVID bioinformatics database and compared. These results found many specific annotations indicating the potential specific role or mechanism for LOXL2-e13. The DEGs of LOXL2-e13, comparing to its wild type, were prioritized by the Random Walk with Restart algorithm. Several tumor-related genes such as ERO1L, ITGA3, and MAPK8 were found closest to LOXL2-e13. These results provide helpful information for subsequent experimental identification of the specific biological roles and molecular mechanisms of LOXL2-e13. Our study also provides a work flow to identify potential roles of splice variants with large scale data.

BACKGROUND:Esophageal squamous cell carcinoma (ESCC) has the highest mortality rates in China. The 5-year survival rate of ESCC remains dismal despite improvements in treatments such as surgical resection and adjuvant chemoradiation, and current clinical staging approaches are limited in their ability to effectively stratify patients for treatment options. The aim of the present study, therefore, was to develop an immunohistochemistry-based prognostic model to improve clinical risk assessment for patients with ESCC. METHODS:We developed a molecular prognostic model based on the combined expression of axis of epidermal growth factor receptor (EGFR), phosphorylated Specificity protein 1 (p-Sp1), and Fascin proteins. The presence of this prognostic model and associated clinical outcomes were analyzed for 130 formalin-fixed, paraffin-embedded esophageal curative resection specimens (generation dataset) and validated using an independent cohort of 185 specimens (validation dataset). RESULTS:The expression of these three genes at the protein level was used to build a molecular prognostic model that was highly predictive of ESCC survival in both generation and validation datasets (P = 0.001). Regression analysis showed that this molecular prognostic model was strongly and independently predictive of overall survival (hazard ratio = 2.358 [95% CI, 1.391-3.996], P = 0.001 in generation dataset; hazard ratio = 1.990 [95% CI, 1.256-3.154], P = 0.003 in validation dataset). Furthermore, the predictive ability of these 3 biomarkers in combination was more robust than that of each individual biomarker. CONCLUSIONS:This technically simple immunohistochemistry-based molecular model accurately predicts ESCC patient survival and thus could serve as a complement to current clinical risk stratification approaches.

BACKGROUND: Esophageal Squamous Cell Carcinoma (ESCC) is a major subtype of esophageal cancer causing significant morbility and mortality in Asia. Mechanism of initiation and progression of this disease is unclear. Tumor initiating cells (TICs) are the subpopulation of cells which have the ability to self-renew, as well as, to drive initiation and progression of cancer. Increasing evidence has shown that TICs exist in a variety of tumors. However, the identification and characterization of TICs in esophageal carcinoma has remained elusive. METHODOLOGY/PRINCIPAL FINDINGS: to identify TICs in ESCC, ESCC cell lines including two primary cells were used for screening suitable surface marker. Then colony formation assay, drug resistant assay and tumorigenicity assay in immune deficient mice were used to characterize TICs in ESCC. We found that just the CD44 expression correlated with tumorigenicity in ESCC cell lines. And then induced differentiation of ESCC cells by all-trans retinoic acid treatment led to decreased expression of CD44. The FACS isolated cell subpopulations with high CD44 expression showed increased colony formation and drug resistance in vitro, as well as significantly enhanced tumorigenicity in NOD/SICD mice, as compared to the low expressing CD44 ESCC cells. CONCLUSIONS/SIGNIFICANCE: our study has discovered a novel TIC surface marker, CD44, which can be utilized to enrich efficiently the TICs in ESCC. These findings will be useful for further studies of these cells and exploring therapeutic approaches.

Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1 -methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily. [ABSTRACT FROM AUTHOR]