A numerical study of residual flow induced by eddy viscosity-shear covariance in a tidally energetic estuary

Abstract(#br)The inner regime of an estuary has unique tidal mixing processes but received relatively less attention. A numerical model was developed to investigate the tidal variability of vertical mixing and the residual flow induced by eddy viscosity–shear covariance (ESCO) in the inner regime of a tidally energetic estuary in Southeastern China. Because of migration of the saltwater/freshwater interface, the water column in the inner regime undergoes a saltwater-dominant high-water period and a freshwater-dominant low-water period during a tidal cycle. The different mixing processes of high- and low-water periods led to typical (reverse) internal tidal asymmetry, i.e. stronger (weaker) mixing during flood tides than ebb tides when the tidal range was large (small). Tidal straining was the main driver of internal tidal asymmetry during the high-water period, while the asymmetries of duration and current velocity between flood and ebb were the main drivers during the low-water period. For typical internal tidal asymmetry, the ESCO stress was negative and the ESCO flow had a two-layer structure with landward flow near the bottom and seaward flow near the surface. For reverse internal tidal asymmetry, the ESCO stress was positive and the vertical pattern of the ESCO flow was reversed. The magnitude of the ESCO flow was several times greater than that of the density-driven flow. The reverse internal tidal asymmetry occurred in the freshwater-dominant low-water period indicates that the ESCO stress could be an important driver of tidal rectification flow in homogeneous coastal waters

MicroRNAs Up-Regulated by CagA of Helicobacter pylori Induce Intestinal Metaplasia of Gastric Epithelial Cells

CagA of Helicobacter pylori is a bacterium-derived oncogenic protein closely associated with the development of gastric cancers. MicroRNAs (miRNAs) are a class of widespread non-coding RNAs, many of which are involved in cell growth, cell differentiation and tumorigenesis. The relationship between CagA protein and miRNAs is unclear. Using mammalian miRNA profile microarrays, we found that miRNA-584 and miRNA-1290 expression was up-regulated in CagA-transformed cells, miRNA-1290 was up-regulated in an Erk1/2-dependent manner, and miRNA-584 was activated by NF-κB. miRNA-584 sustained Erk1/2 activities through inhibition of PPP2a activities, and miRNA-1290 activated NF-κB by knockdown of NKRF. Foxa1 was revealed to be an important target of miRNA-584 and miRNA-1290. Knockdown of Foxa1 promoted the epithelial-mesenchymal transition significantly. Overexpression of miRNA-584 and miRNA-1290 induced intestinal metaplasia of gastric epithelial cells in knock-in mice. These results indicate that miRNA-584 and miRNA-1290 interfere with cell differentiation and remodel the tissues. Thus, the miRNA pathway is a new pathogenic mechanism of CagA

DataSheet_1_Single-cell transcriptome analysis reveals heterogeneity and convergence of the tumor microenvironment in colorectal cancer.zip

IntroductionColorectal cancer (CRC) ranks second for mortality and third for morbidity among the most commonly diagnosed cancers worldwide. We aimed to investigate the heterogeneity and convergence of tumor microenvironment (TME) in CRC.MethodsWe analyzed the single-cell RNA sequencing data obtained from the Gene Expression Omnibus (GEO) database and identified 8 major cell types and 25 subgroups derived from tumor, para-tumor and peripheral blood.ResultsIn this study, we found that there were significant differences in metabolic patterns, immunophenotypes and transcription factor (TF) regulatory patterns among different subgroups of each major cell type. However, subgroups manifested similar lipid metabolic patterns, immunosuppressive functions and TFs module at the end of the differentiation trajectory in CD8+ T cells, myeloid cells and Fibroblasts. Meanwhile, TFs regulated lipid metabolism and immunosuppressive ligand-receptor pairs were detected by tracing the differentiation trajectory. Based on the cell subgroup fractions calculated by CIBERSORTx and bulk RNA-sequencing data from The Cancer Genome Atlas (TCGA), we constructed an immune risk model and clinical risk model of CRC which presented excellent prognostic value.ConclusionThis study identified that the differentiation was accompanied by remodeling of lipid metabolism and suppression of immune function, which suggest that lipid remodeling may be an important trigger of immunosuppression. More importantly, our work provides a new perspective for understanding the heterogeneity and convergence of the TME and will aid the development of prognosis and immunotherapies of CRC patients.</p

Additional file 2: of Up-regulated NRIP2 in colorectal cancer initiating cells modulates the Wnt pathway by targeting RORβ

Analysis of the relationship between NRIP2, RORB and clinical parameters. (DOCX 1238 kb

miRNA-1290 was up-regulated in an Erk1/2-dependent manner and miRNA-584 was activated indirectly by miRNA-1290. A. Determination of transactivities of Elk-1.

<p>AGS cells were transiently transfected with pGL3-Elk-1 plasmid for 24 h and subsequently infected with <i>H</i>.<i>pylori</i> including NCTC11637 and 7 <i>cagA</i>+ isolates for additional 7 h (MOI≈50) respectively. Cells were lysed and the lysates were collected for determination of luminescent intensities by dual-luciferase ÿreporter assay. The results showed that transactivities of Elk-1 was up-regulated after infection of <i>cagA</i>+ <i>H</i>.<i>pylori</i> (<i>P</i> <0.01, ANOVA). <b>B. Determination of luciferase activations containing promoter region of miRNA-584 and miRNA-1290.</b> Plasmids containing <i>Elk</i>-1 or <i>cagA</i> were co-transfected with recombinant luciferase reporters containing promoter regions of miRNA-584 and miRNA-1290 (≈2000 bp) respectively into 293T cells for 24 h (<i>Elk-</i>1/<i>cagA</i>: luciferase reporters: Renilla luciferase = 3∶1∶0.01). Cells were lysed and the lysates were collected for determination of luminescent intensities by dual-luciferase_reporter assay. The results showed that miRNA-1290 was up-regulated by both Elk-1 and CagA (<i>P</i> <0.01, ANOVA), whereas miRNA-584 was not affected. <b>C. Determination of miRNA-584 in stable miRNA-1290–expressing cells.</b> To determine miRNA-584 in stable miRNA-1290–expressing cells, total RNA from stable miRNA-1290–expressing cells were isolated and real-time PCR was performed for miRNA-584. The results showed that miRNA-584 was up-regulated in stable miRNA-1290–expressing cells. <b>D. Expression of miRNA-1290 enhanced NF-κB activities.</b> miRNA-1290 was co-transfected with NF-κB reporter into SGC7901 cells for 24 h (miRNA-1290: luciferase reporters: Renilla luciferase = 3∶1∶0.01). Cells were lysed and the lysates were collected for determination of luminescent intensities by dual- luciferase_reporter assay. The results showed that miRNA-1290 enhanced NF-κB activities (<i>P</i> <0.01, ANOVA). <b>E. Detection of NKRF protein in transfected cells.</b> miRNA-1290-expressing gastric carcinoma SGC7901 cells were lysed and subsequently were subjected to western blot analysis to probe with anti-NKRF antibodies. SGC7901 cells expressing the empty vector were also established as a control. <b>F. Knockdown of NKRF increased NF-κB activities... </b><i>NKRF</i> shRNA plasmid (Santa Cruz) was co-transfected with NF-κB reporters into SGC7901 cells for 48 h (<i>NKRF</i> shRNA plasmid: NF-κB reporters: Renilla luciferase = 3∶1∶ 0.1). Cells were lysed and the lysates were collected for determination of luminescent intensities. The results showed that knockdown of <i>NKRF</i> increased NF-κB activities (<i>P</i> <0.01, ANOVA). <b>G. Activation of NF-κB upregulated miRNA-584... </b><i>NKRF</i> shRNA plasmid was co-transfected with recombinant luciferase reporters containing promoter regions of miRNA-584 into SGC7901 cells for 48 h (<i>NKRF</i> shRNA plasmid: luciferase reporters: Renilla luciferase = 3∶1∶ 0.1). Cells were lysed and the lysates were collected for determination of luminescent intensities. The results showed that NF-κB up-regulated miRNA-584 (<i>P</i> <0.01, ANOVA). <b>H. Overexpression of c-Rel upregulated miRNA-584... </b><i>c-Rel</i> plasmid was co-transfected with recombinant luciferase reporters containing promoter regions of miRNA-584 into SGC7901 cells for 48 h (<i>c-Rel</i> plasmid: luciferase reporters: Renilla luciferase = 3∶1∶ 0.1). Cells were lysed and the lysates were collected for determination of luminescent intensities. The results showed that Overexpression of c-Rel upregulated miRNA-584 (<i>P</i> <0.01, ANOVA). Data are represented as mean +/− s.e.m.</p

Screening and identification of miRNA-584 and miRNA-1290 in CagA-transfected cells. A. Detection of <i>cagA</i> DNA fragments in transfected cells.

<p>The standard calcium phosphate precipitation method was used to establish stable CagA-expressing gastric carcinoma AGS cells. Transfection was validated using PCR and DNA sequencing after cell clones had grown. AGS cells stably expressing the empty <i>pEGFP-c</i>1 vector were also established as a control. <b>B. Detection of CagA protein in transfected cells.</b> Stable CagA-expressing gastric carcinoma AGS cells were lysed with RIPA buffer. The lysates were subjected to SDS-PAGE and subsequently to western blot analysis to probe with anti-GFP antibodies. AGS cells stably expressing the empty <i>pEGFP-c</i>1 vector were also established as a control. <b>C. Screening and identification of miRNA-84 and miRNA-290 in stable CagA-expressing AGS cells.</b> Total RNA was extracted from stable CagA-expressing AGS cells and control cells. Mammalian miRNA expression profile microarray scanning and TaqMan real-time PCR were performed. The results showed that both miRNA-584 and miRNA-1290 were up-regulated in stable CagA-expressing AGS cells. <b>D. Identification of miRNA-584 and miRNA-1290 in gastric carcinoma SGC7901 cells transiently expressing CagA.</b> Total RNA from gastric carcinoma SGC7901 cells transfected with <i>cagA</i>/<i>pEGFP-c</i>1 plasmid was extracted and real-time PCR was performed. Both miRNA-584 and miRNA-1290 were up-regulated in transiently CagA-expressing SGC7901 cells. <b>E. Identification of miRNA-584 and miRNA-1290 in gastric carcinoma AGS cells infected with </b><b><i>H</i></b><b>.</b><b><i>pylori</i></b><b>.</b> Total RNA was extracted from AGS cells infected with <i>H</i>.<i>pylori</i> including NCTC11637 and 9 <i>cagA</i>– isolates for 7 h (MOI≈50) respectively, and TaqMan real-time PCR was performed. Both miRNA-584 and miRNA-1290 were up-regulated after infection of <i>cagA</i>+ <i>H</i>.<i>pylori</i> (all <i>P</i> <0.01, ANOVA). Data are represented as mean +/− s.e.m.</p

<i>Foxa</i>1 is a target of miRNA-584 and miRNA-1290 and knockdown of <i>Foxa</i>1 promotes EMT. A. Schematic of potential target screening strategy. B. Transcription factor targets of miRNA-584 and miRNA-1290.

<p>Transcription factors targeted by miRNA-584 and miRNA-1290 expressed in AGS cells were predicted by TargetScan (www. targetscan.org). <b>C. Determination of pMIR-report luciferase activity.</b> The recombinant plasmid pMIR-<i>Foxa</i>1 report was co-transfected with pcDNA6.2-GW/EmGFP-miR containing miRNA-584 or miRNA-1290 into AGS gastric cancer cells for 48 h. Cells were lysed and luciferase activity was determined by dual-luminescence reporter assay. Co-transfection of miRNA-584 or miRNA-1290 with pMIR-<i>Foxa</i>1 inhibited more than 90% of firefly luciferase activity (all <i>P</i> <0.001, ANOVA), indicating that miRNA-584 and miRNA-1290 target <i>Foxa</i>1 mRNA. <b>D. Detection of Foxa1 in cells transiently expressing miRNA-584 and miRNA-1290.</b> pcDNA6.2-GW/EmGFP-miR containing miRNA-584 or miRNA -1290 was transfected into SW620 colon cancer cells for 48 h. Cells (1×10⁶) were lysed and the supernatant was subjected to western blot analysis. The results showed that the expression of Foxa1 protein was down-regulated about 50% after transfection of miRNA-584 or miRNA-1290. These results indicate that Foxa1 is a target of miRNA-584 and miRNA-1290. <b>E. Analysis of the percentage of CD44⁺CD133⁺ cells.</b> SW620 colon cancer cells were infected with recombinant <i>Foxa</i>1 shRNA lentivirus. Infected SW620 cells were digested and resuspended at 1×10⁶ per 100 µL of PBS containing 2% FBS, and labeling antibodies (APC-CD44 and PE-CD133) were added at 4°C for 30 min in the dark. After two washes, cells were analyzed using a FACSAria. An empty vector control was also established. The percentage of CD44⁺CD133⁺ cells significantly decreased after <i>Foxa</i>1 knockdown (<i>P</i> <0.05, ANOVA). The supernatant from SW620 colon cancer cells with <i>Foxa</i>1 knockdown was subjected to western blot for detection of Bmi1. <b>F. </b><b><i>Foxa</i></b><b>1 knockdown promotes EMT.</b> The supernatant from SW620 colon cancer cells with <i>Foxa</i>1 knockdown was subjected to SDS-PAGE, and proteins were transferred onto a nitrocellulose membrane for western blotting for Foxa1, E-cadherin, and vimentin. <b>G. Detection of E-cadherin in cells with stable expression of miRNA-584 or/and miRNA-1290.</b> pcDNA6.2-GW/EmGFP-miR containing miRNA-584 or/and miRNA -1290 was transfected into SW620 colon cancer cells. Cells were separated at 24 h after transfection and screened by 5 µg/mL puromycin for 7 days. Survival clones were picked up and amplified respectively for 4 weeks. Cells were lysed and the supernatant was detected for E-cadherin. GAPDH was as controls.</p

Overexpression of miRNA-584 and miRNA-1290 induced intestinal metaplasia in knock-in mice. A. Determination of miRNA-584 and miRNA-1290 in knock-in mice.

<p>Total RNA was extracted from minced tissues of mice by Trizol reagent. Real-time PCR was performed for determination of miRNA-584 and miRNA-1290. The results showed miRNA-584 and miRNA-1290 increased in knock-in mice. Data are represented as mean +/− s.e.m. <b>B. Morphological observation of knock-in mice.</b> Obvious morphological alterations were not observed at the 12th week in knock-in mice. However, gastric mucosa layers were significantly thinned and flattened, and rugal folds had disappeared at the 72nd week <b>C. H&E staining of gastric mucosa.</b> Obvious morphological alterations were not observed at the 12th week in knock-in mice. However, gastric epitheliums were replaced by intestinal metaplasia under light microscopy (×100). <b>D. Determination of </b><b><i>Muc</i></b><b> 2 and </b><b><i>Muc</i></b><b> 6.</b> Total RNA was extracted from minced gastric tissues of mice by Trizol reagent. Real-time PCR was performed for determination of <i>Muc</i>2 and <i>Muc</i>6. The results showed <i>Muc</i>2 was significantly increased while <i>Muc</i>6 was significantly decreased in transgenic mice (all <i>P</i> <0.01, ANOVA). Data are represented as mean +/−s.e.m. <b>E. PAS staining.</b> Gastric mucosa from transgenic mice at 72nd W were checked by PAS staining (×100). The results showed that PAS-positive cells were increased in in transgenic mice.</p