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Objective: To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG, to validate its expression in 293T cells, and to purify the secretory protein with biological activity. Methods: The DSG2 extracellular domain fragment gene (DSG2ex), was amplified by PCR, and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1. The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein. The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA. Results: The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed. The highest protein expression level was obtained with 293T cells after 96 h of transfection. The relative molecular mass of the purified product was between 100 and 130 kDa estimated by SDS-PAGE, which was consistent with the expectation. The yield of the purified protein reached 0.8 mg/mL with a purity over 90%. The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting. Moreover, the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein (HAdV-55).Conclusion: A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established, and biologically active DSG2 extracellular domain protein was purified, which laid the foundation for the later study of its protein function and anti-adenovirus drugs. [ABSTRACT FROM AUTHOR]

Background: CD73 promotes progression in several malignancies and is considered as a novel immune checkpoint. However, the function of CD73 in intrahepatic cholangiocarcinoma (ICC) remains uncertain. In this study, we aim to investigate the role of CD73 in ICC. Methods: Multi-omics data of 262 ICC patients from the FU-iCCA cohort were analyzed. Two single-cell datasets were downloaded to examine the expression of CD73 at baseline and in response to immunotherapy. Functional experiments were performed to explore the biological functions of CD73 in ICC. The expression of CD73 and HHLA2 and infiltrations of CD8 + , Foxp3 + , CD68 + , and CD163 + immune cells were evaluated by immunohistochemistry in 259 resected ICC samples from Zhongshan Hospital. The prognostic value of CD73 was assessed by Cox regression analysis. Results: CD73 correlated with poor prognosis in two ICC cohorts. Single-cell atlas of ICC indicated high expression of CD73 on malignant cells. TP53 and KRAS gene mutations were more frequent in patients with high CD73 expression. CD73 promoted ICC proliferation, migration, invasion, and epithelial-mesenchymal transition. High CD73 expression was associated with a higher ratio of Foxp3 + /CD8 + tumor-infiltrating lymphocytes (TILs) and CD163 + /CD68 + tumor-associated macrophages (TAMs). A positive correlation between CD73 and CD44 was observed, and patients with high CD73 expression showed elevated expression of HHLA2. CD73 expression in malignant cells was significantly upregulated in response to immunotherapy. Conclusions: High expression of CD73 is associated with poor prognosis and a suppressive tumor immune microenvironment in ICC. CD73 could potentially be a novel biomarker for prognosis and immunotherapy in ICC. [ABSTRACT FROM AUTHOR]

Background: GATA family of transcription factors are critical for organ development and associated with progression of various cancer types. However, their expression patterns and prognostic values for hepatocellular carcinoma (HCC) are still largely unknown. Methods: Expression of GATA transcription factors in HCC cell lines and tissues (n = 240) were evaluated by RT-qPCR, western blot and immunohistochemistry. Cellular proliferation, migration and invasion of HepG2 was evaluated by CCK-8 kit, scratch wound assay and transwell matrigel invasion assay, respectively. Results: GATA2 expression was decreased in HCC cell lines (p = 0.056 for mRNA, p = 0.040 for protein) and tissues (p = 1.27E-25) compared with normal hepatocytes. Decreased expression of intratumoral GATA2 protein significantly correlated with elevated alpha feto-protein (p = 2.7E-05), tumor size >5 cm (p = 0.049), absence of tumor capsule (p = 0.002), poor differentiation (p = 0.005), presence of tumor thrombi (p = 0.005) and advanced TNM stage (p = 0.001) and was associated with increased recurrence rate and decreased overall survival rate by univariate (p = 1.6E-04 for TTR, p = 1.7E-04 for OS) and multivariate analyses (HR = 0.63, 95% CI = 0.43–0.90, p = 0.012 for TTR; HR = 0.67, 95% CI = 0.47–0.95, p = 0.026 for OS). RNAi-mediated knockdown of GATA2 expression significantly enhanced proliferation, migration and invasion of HepG2 cell in vitro. Conclusions: Decreased expression of hematopoietic factor GATA2 was associated with poor prognosis of HCC following resection. [ABSTRACT FROM AUTHOR]

The Murine Double Minute 2 (MDM2) protein is a key regulator of cell proliferation and apoptosis that acts primarily by inhibiting the p53 tumor suppressor. Similarly, the PI3-Kinase (PI3K)/AKT pathway is critical for growth factor-mediated cell survival. Additionally, it has been reported that AKT can directly phosphorylate and activate MDM2. In this study, we show that IGF-1 up-regulates MDM2 protein levels in a PI3K/AKT-dependent manner. Inhibition of mTOR by rapamycin or expression of a dominant negative eukaryotic initiation factor 4E binding protein 1 (4EBP1) mutant protein, as well as ablation of eukaryotic initiation factor 4E (eIF4E), efficiently abolishes IGF-1-mediated up-regulation of MDM2. In addition, we show that rapamycin effectively inhibits MDM2 expression and sensitizes cancer cells to chemotherapy. Taken together, this study reveals a novel mechanism by which IGF-1 activates MDM2 via the mTOR pathway, and that pharmacologic inhibition of mTOR combined with chemotherapy may be more effective in treatment of a subset of cancers harboring increased MDM2 activation. [ABSTRACT FROM AUTHOR]

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A hydroponic experiment was conducted to study the effects of applying 100% NO3--N,100% NH4+-N, and 75% NO3--N+25% NH4+-N on the nitrogen metabolism and the nitrate reductase (NR) and glutamine synthetase (GS) gene expression of cherry tomato during its fruit development. Applying 75% NO3--N+25% NH4+-N slightly increased the single fruit mass, and increased the fruit NH4+-N, total amino acid, and total N contents and N accumulation significantly, compared with applying 100% NO3--N. In treatments 100% NO3--N and 75% NO3--N+25% NH4+-N, the fruit NR activity and its gene expression had no significant difference, but were higher than those in treatment 100% NH4+-N. The fruit GS activity was significantly higher in treatment 75% NO3--N+25% NH4+-N than in treatment 100% NO3--N. In the three treatments, isozyme GS1 (Cytosolic type GS) and GS2 (Chloroplast type GS) expression was inconsistent with GS activity, suggesting that the effects of applied N on GS activity could be mainly reflected at posttranscriptional level. [ABSTRACT FROM AUTHOR]

The Gadd45γ (growth arrest and DNA damage-inducible) gene is activated transcriptionally by at least two kinds of agents: DNA damaging agent such as methyl methanesulfonate (MMS) and UV radiation, or cytokines such as interleukin (IL)-6, IL-2 and granulocyte colony-stimulating factor (G-CSF). To investigate the sequences and transcription factors involved in induction of Gadd45γ after treatment with IL-6, the human gene was cloned and sequenced. We found C/EBP (CCAAT/enhancer-binding protein) family proteins, major transcription factors in the IL-6 signal transduction pathway, could regulate the transcriptional activity of the Gadd45γ promoter. In addition, a noncanonical C/EBP-binding site within the Gadd45γ promoter where C/EBPβ and C/EBPδ could bind, was identified by electrophoretic mobility shift assay (EMSA) and reporter gene analysis. Furthermore, we found a coordinated expression profile between Gadd45γ mRNA and C/EBPs (β and δ) protein during the differentiation of M1 cells: the amount of Gadd45γ transcripts became maximal when both C/EBPβ and C/EBPδ levels were high, on day 1 of differentiation of M1 cells after treatment with IL-6. These findings suggest that mitotic growth arrest coupled to M1 cell differentiation is mediated by C/EBPs stimulation of growth arrest-associated genes such as Gadd45γ. [ABSTRACT FROM AUTHOR]

Lung cancer stem cells (CSCs) play a pivotal role in tumor development, drug resistance, metastasis and recurrence of lung cancer. Thus, it is of great importance to study the mechanism by which CSCs are regulated. In this study, we demonstrate that the deubiquitinase USP4 is critically important in promoting lung cancer stemness. Silencing of USP4 leads to reduction of Oct4 and Sox2 expression, decreased CD133+ cell population and inhibition of tumorsphere formation. Conversely, ectopic expression of USP4 significantly enhances lung cancer cell stemness, which is effectively rescued by simultaneous silencing of Twist1. Mechanistically, we identified USP4 as a novel deubiquitinase of Twist1. USP4 binds to, deubiquitinates and stabilizes Twist1 protein. Furthermore, we show that USP4 expression is elevated in human lung cancer specimens and is positively correlated with Twist1 expression. High expression of USP4/Twist1 is associated with poor clinical outcomes of lung cancer patients. Together, this study highlights an important role for USP4 in lung cancer stemness and suggests USP4 as a potential target for lung cancer diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Abstract: The tumor suppressor p53 is pivotal in cell growth arrest and apoptosis upon cellular stresses including DNA damage. Mounting evidence indicates that p63 proteins, which are homologs of p53, are also involved in apoptosis under certain circumstances. In this study, we found that treatment with DNA damage agents leads to down-regulation of ΔNp63α and induces apoptosis in FaDu and HaCat cells carrying mutant p53. Further study shows that DNA damage reduces steady-state mRNA level of ΔNp63α, but has little effect on its protein stability. In addition, knockdown of endogenous ΔNp63α directly induces apoptosis and sensitizes cells to DNA damage, while exogenous expression of ΔNp63α partially confers cellular resistance to DNA damage. Together, these data suggest that DNA damage down-regulates ΔNp63α, which may directly contribute to DNA damage-induced apoptosis. [Copyright &y& Elsevier]

Technologies to assess the molecular targets of biomolecules inliving cells are lacking. We have developed a technology calledmagnetism-based interaction capture (MAGIC) that identifies moleculartargets on the basis of induced movement of superparamagneticnanoparticles inside living cells. Efficient intracellular uptake ofsuperparamagnetic nanoparticles (coated with a small molecule ofinterest) was mediated by a transducible fusogenic peptide. Thesenanoprobes captured the small molecule's labeled target protein and weretranslocated in a direction specified by the magnetic field. Use ofMAGIC in genome-wide expression screening identified multiple proteintargets of a drug. MAGIC was also used to monitor signal-dependentmodification and multiple interactions of proteins. [ABSTRACT FROM AUTHOR]

Maize is an important food crop in the world, but the yield and quality of maize have been significantly reduced due to the impact of insect pests. In order to address this issue, the cry1Ah gene was subjected to error-prone PCR for mutagenesis, and subsequently, the mutant cry1Ah-1 gene was introduced into maize inbred line GSH9901 callus using the Agrobacterium -mediated method. The T2 generation transformed plants were obtained by subculture, and 9 transgenic positive plants were obtained by molecular detection which was carried out by PCR, qRT-PCR, Bt gold-labeled immunoassay test strips, Western blot and ELISA. It was found that the Cry1Ah-1 gene could be transcribed normally in maize leaves, of which OE1 and OE3 had higher relative expression levels and could successfully express proteins of 71.94 KD size. They were expressed in different tissues at the 6-leaf stage, heading stage and grain-filling stage, and could ensure the protection of maize from corn borer throughout the growth period. The biological activities of OE1 and OE3 were tested indoors and in the field, and the results showed that in indoors, the corn borer that fed on OE1 and OE3 corn leaves had a mortality rate of 100 % after 3 days; in the field, OE1 and OE3 had strong insecticidal activity against corn borer , reaching a high resistance level. In conclusion, the transgenic cry1Ah-1 maize has a strong insecticidal effect on corn borer , and has a good prospect of commercialization. • cry1Ah-1 mutant gene was introduced into maize callus by Agrobacterium-mediated method. • This gene is expressed in different tissues at different times, which can ensure the protection of maize from corn borer. [ABSTRACT FROM AUTHOR]

Hepatic stellate cells (HSCs) play vital roles in tumorigenesis and progression of hepatocellular carcinoma (HCC). However, there remains a lack of high-throughput studies on gene expression alterations in HCC cells in response to direct interactions with HSCs. In this study, we established a direct co-culture model of HSCs and HCC cells. We found that the expression of a set of miRNAs, most notably miR-1246, was triggered by HSCs. RORα was confirmed as the target gene of miR-1246. Either overexpression of miR-1246 or knockdown of RORα enhanced the proliferation, invasiveness, and metastatic capability of HCC both in vitro and in vivo, through Wnt/β-catenin pathway activation and promotion of epithelial-mesenchymal transition (EMT). Moreover, upregulation of miR-1246 and repression of RORα were prominent features of aggressive clinical HCC. The miR-1246-RORα-Wnt/β-catenin axis is a novel pathway through which HSCs accelerate HCC progression. [ABSTRACT FROM AUTHOR]

Abnormal expressions of sodium channel SCN1A and SCN3A genes alter neural excitability that are believed to contribute to the pathogenesis of epilepsy, a long-term risk of recurrent seizures. Ketogenic diet (KD), a high-fat and low-carbohydrate treatment for difficult-to-control (refractory) epilepsy in children, has been suggested to reverse gene expression patterns. Here, we reveal a novel role of GAPDH on the posttranscriptional regulation of mouse Scn1a and Scn3a expressions under seizure and KD conditions. We show that GAPDH binds to a conserved region in the 3′ UTRs of human and mouse SCN1A and SCN3A genes, which decreases and increases genes' expressions by affecting mRNA stability through SCN1A 3′ UTR and SCN3A 3′ UTR, respectively. In seizure mice, the upregulation and phosphorylation of GAPDH enhance its binding to the 3′ UTR, which lead to downregulation of Scn1a and upregulation of Scn3a . Furthermore, administration of KD generates β-hydroxybutyric acid which rescues the abnormal expressions of Scn1a and Scn3a by weakening the GAPDH's binding to the element. Taken together, these data suggest that GAPDH-mediated expression regulation of sodium channel genes may be associated with epilepsy and the anticonvulsant action of KD. [ABSTRACT FROM AUTHOR]

Laminaria japonica , a brown seaweed, has been used in Traditional Chinese Medicine (TCM) to treat a variety of diseases including lung cancer. To demonstrate the effects of Fucoxanthin (FX), a major active component extracted from Laminaria japonica on metastasis and Gefitinib (Gef) sensitivity in human lung cancer cells both in vitro and in vivo. Invasion and migration of lung cancer cells were detected using the wound healing assay and transwell assay. Epithelial-to-mesenchymal transition (EMT) factors and PI3K/AKT/NF-κB pathways were analyzed by western blotting. RNA interference (RNAi) technology was used to silence TIMP-2 gene expression in A549 cells. The anti-metastatic effect of FX was evaluated in vivo in an experimental lung metastatic tumor model. On the other hand, cell counting kit-8 assay was used to study the cell viability of human lung cancer PC9 cells and Gef resistant PC9 cells (PC9/G) after Gef, FX or FX combined with Gef treatment. PC9 xenograft model was established to explore the anti-tumor effect of FX or combined with Gef. Immunohistochemistry staining assay and immunofluorescence staining assay were used to reveal the effects of FX on lung cancer cell proliferation and apoptosis. FX was able to significantly inhibit lung cancer cells migration and invasion in vitro. FX suppressed the expressions of Snail, Twist, Fibronectin, N-cadherin, MMP-2, PI3K, p-AKT and NF-κB, and increased the expression of TIMP-2. Furthermore, knockdown of TIMP-2 attenuated FX-mediated invasion inhibition. Additionally, we demonstrated that FX inhibited lung cancer cells metastasis in vivo. The anti-metastatic effects of FX on lung cancer cells might be attributed to inhibition of EMT and PI3K/AKT/NF-κB pathway. We further demonstrated that the anti-tumor activity of FX was not only limited to the drug sensitive cell lines, but also prominent on lung cancer cells with Gef resistant phenotype. Furthermore, in vivo xenograft assay confirmed that FX inhibited tumor growth and enhanced the sensitivity of lung cancer cells to Gef and this effect may be due to inhibition of tumor cell proliferation and activation of apoptosis. Collectively, our findings suggested that FX suppresses metastasis of lung cancer cells and overcomes EGFR TKIs resistance. Thus, FX is worthy of further investigation as a drug candidate for the treatment of lung cancer. Image 1 [ABSTRACT FROM AUTHOR]