Tritium and Carbon-14 Contamination Reshaping the Microbial Community Structure, Metabolic Network, and Element Cycle in the Seawater Environment.

The potential ecological risks caused by entering radioactive wastewater containing tritium and carbon-14 into the sea require careful evaluation. This study simulated seawater's tritium and carbon-14 pollution and analyzed the effects on the seawater and sediment microenvironments. Tritium and carbon-14 pollution primarily altered nitrogen and phosphorus metabolism in the seawater environment. Analysis by 16S rRNA sequencing showed changes in the relative abundance of microorganisms involved in carbon, nitrogen, and phosphorus metabolism and organic matter degradation in response to tritium and carbon-14 exposure. Metabonomics and metagenomic analysis showed that tritium and carbon-14 exposure interfered with gene expression involving nucleotide and amino acid metabolites, in agreement with the results seen for microbial community structure. Tritium and carbon-14 exposure also modulated the abundance of functional genes involved in carbohydrate, phosphorus, sulfur, and nitrogen metabolic pathways in sediments. Tritium and carbon-14 pollution in seawater adversely affected microbial diversity, metabolic processes, and the abundance of nutrient-cycling genes. These results provide valuable information for further evaluating the risks of tritium and carbon-14 in marine environments.

Effects of long-term herbaceous plant restoration on microbial communities and metabolic profiles in coal gangue-contaminated soil.

The soil microbial diversity in the gangue accumulation area is severely stressed by a variety of heavy metals, while the influence of long-term recovery of herbaceous plants on the ecological structure of gangue-contaminated soil is to be explored. Therefore, we analysed the differences in physicochemical properties, elemental changes, microbial community structure, metabolites and expression of related pathways in soils in the 10- and 20-year herbaceous remediation areas of coal gangue. Our results showed that phosphatase, soil urease, and sucrase activities of gangue soils significantly increased in the shallow layer after herbaceous remediation. However, in zone T1 (10-year remediation zone), the contents of harmful elements, such as Thorium (Th; 1.08-fold), Arsenic (As; 0.78-fold), lead (Pb; 0.99-fold), and uranium (U; 0.77-fold), increased significantly, whereas the soil microbial abundance and diversity also showed a significant decreasing trend. Conversely, in zone T2 (20-year restoration zone), the soil pH significantly increased by 1.03- to 1.06-fold and soil acidity significantly improved. Moreover, the abundance and diversity of soil microorganisms increased significantly, the expression of carbohydrates in soil was significantly downregulated, and sucrose content was significantly negatively correlated with the abundance of microorganisms, such as Streptomyces. A significant decrease in heavy metals was observed in the soil, such as U (1.01- to 1.09-fold) and Pb (1.13- to 1.25-fold). Additionally, the thiamin synthesis pathway was inhibited in the soil of the T1 zone; the expression level of sulfur (S)-containing histidine derivatives (Ergothioneine) was significantly up-regulated by 0.56-fold in the shallow soil of the T2 zone; and the S content in the soil significantly reduced. Aromatic compounds were significantly up-regulated in the soil after 20 years of herbaceous plant remediation in coal gangue soil, and microorganisms (Sphingomonas) with significant positive correlations with benzene ring-containing metabolites, such as Sulfaphenazole, were identified.

Degradation of Uranium-Contaminated Decontamination Film by UV Irradiation and Microbial Biodegradation.

This research provides a complete degradation scheme for acrylic copolymer/cellulose acetate butyrate peelable decontamination films. This study analyzed the removal efficiency of uranium by peelable decontamination film. More importantly, the degradability of the films was evaluated by a combined treatment with UV radiation and microbial biodegradation. The results showed that UV radiation would rupture the surface of the decontamination films, which leaded the weight-average molecular weight decreased by 55.3% and number-average molecular weight decreased by 75.83%. Additionally, the microbial flora induced light-degradable decontamination film weight-average molecular weight and number-average molecular weight decreased by 9.3% and 30.73%, respectively. 16S rRNA microbial diversity analysis indicated that Pantoea, Xylella, Cronobacter, and Olivibacter were the major degrading bacteria genera. Among them, 4 key strains that can be stripped of decontamination films have been isolated and identified from the dominant degrading bacteria group. The results show that UV radiation combined with microbial flora can achieve rapid degradation of the decontamination films.

Toxicity analysis of TNT to alfalfa's mineral nutrition and secondary metabolism.

KEY MESSAGE: Alfalfa has the ability to degrade TNT. TNT exposure caused root disruption of mineral nutrient metabolism. The exposure of TNT imbalanced basal cell energy metabolism. The mechanism of 2,4,6-trinitrotoluene (TNT) toxicity effects was analyzed in alfalfa (Medicago sativa L.) seedlings by examining the mineral nutrition and secondary metabolism of the plant roots. Exposure to 25-100 mg·L-1 TNT in a hydroponic solution for 72 h resulted in a TNT absorption rate of 26.8-63.0%. The contents of S, K, and B in root mineral nutrition metabolism increased significantly by 1.70-5.46 times, 1.38-4.01 times, and 1.40-4.03 times, respectively, after TNT exposure. Non-targeted metabolomics analysis of the roots identified 189 significantly upregulated metabolites and 420 significantly downregulated metabolites. The altered metabolites were primarily lipids and lipid-like molecules, and the most significant enrichment pathways were alanine, aspartate, and glutamate metabolism and glycerophospholipid metabolism. TNT itself was transformed in the root system into several intermediate products, including 4-hydroxylamino-2,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2-hydroxylamino-4,6-dinitrotoluene, 2,4',6,6'-tetranitro-2',4-azoxytoluene, 4,4',6,6'-tetranitro-2,2'-azoxytoluene, and 2,4-dinitrotoluene. Overall, TNT exposure disturbed the mineral metabolism balance, and significantly interfered with basic plant metabolism.

In situ restoration of soil ecological function in a coal gangue reclamation area after 10 years of elm/poplar phytoremediation.

The soil ecological health risks and toxic effects of coal gangue accumulation were examined after 10 years of elm/poplar phytoremediation. The changes in soil enzyme activities, ionome metabolism, and microbial community structure were analyzed at shallow (5-15 cm), intermediate (25-35 cm), and deep (45-55 cm) soil depths. Soil acid phosphatase activity in the restoration area increased significantly by 4.36-7.18 fold (p < 0.05). Soil concentrations of the metal ions Cu, Pb, Ni, Co, Bi, U, and Th were significantly reduced, as were concentrations of the non-metallic element S. The repair effect was shallow > middle > deep. The soil community structure, determined by 16S diversity results, was changed significantly in the restoration area, and the abundance of microorganisms increased at shallow soil depths. Altererythrobacter and Sphingomonas species were at the center of the microbial weight network in the restoration area. Redundancy analysis (RDA) showed that S and Na are important driving forces for the microbial community distributions at shallow soil depths. The KEGG function prediction indicated enhancement of the microbial function of the middle depth soil layers in the restoration area. Overall, phytoremediation enhanced the biotransformation of soil phosphorus in the coal gangue restoration area, reduced the soil content of several harmful metal elements, significantly changed the structure and function of the microbial community, and improved the overall soil ecological environment.

Non-targeted metabolomics reveals the stress response of a cellulase-containing penicillium to uranium.

Human industrial activities have caused environmental uranium (U) pollution, resulting in uranium(VI) had radiotoxicity and chemical toxicity. Here, a cellulase-producing Penicillium fungus was screened and characterized by X-ray fluorescence (XRF), and Fourier transform infrared reflection (FT-IR), as well as by GC/MS metabolomics analysis, to study the response to uranium(VI) stress. The biomass of Penicillium decreased after exposure to 100 mg/L U. Uranium combined with carboxyl groups, amino groups, and phosphate groups to form uranium mineralized deposits on the surface of this fungal strain. The α-activity concentration of uranium in the strain was 2.57×106 Bq/kg, and the ß-activity concentration was 2.27×105 Bq/kg. Metabolomics analysis identified 118 different metabolites, as well as metabolic disruption of organic acids and derivatives. Further analysis showed that uranium significantly affected the metabolism of 9 amino acids in Penicillium. These amino acids were related to the TCA cycle and ABC transporter. At the same time, uranium exhibited nucleotide metabolism toxicity to Penicillium. This study provides an in-depth understanding of the uranium tolerance mechanism of Penicillium and provides a theoretical basis for Penicillium to degrade hyper-enriched plants.

Enhancing bile tolerance of Lactobacilli is involved in the hypolipidemic effects of liraglutide.

Liraglutide is an analog of human glucagon-like peptide-1 which play essential roles in regulation of glycolipid metabolism. To investigate role of lactic acid bacteria (LAB) in lipid-lowering effect of liraglutide, 40 mice were divided into normal food diet (NFD), high-fat food (HFD), 10.0 mg/kg/d simvastatin-treated HFD (SIM + HFD), 200 and 400 µg/kg/d liraglutide-treated HFD (LL + HFD and HL + HFD) groups for 5 weeks. We found that liraglutide could upregulate cholesterol 7α-hydroxylase (CYP7A1) and LDL-receptor (LDLR), whereas downregulate 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR). Besides, liraglutide enhance abundance of lactobacillaceae in gut of hyperlipidemic mice and increase bile tolerance ability of LAB by upregulating bile salt hydrolases, and the lysate of liraglutide-sensitive LAB could also directly downregulate HMGCR, the key enzyme in cholesterol synthesis, and inhibit hepatocyte steatosis. These findings might provide new theoretical guidance for clinical application of liraglutide and research and development of antiobesity, hypolipidemic, and cholesterol-lowering drugs or functional foods.

α-Glucosidase Inhibition Action of Major Flavonoids Identified from Hypericum Attenuatum Choisy and Their Synergistic Effects.

Hypericum attenuatum Choisy is a traditional Chinese herbal plant with multiple therapeutic effects. In this study, bioactivity-guided fractionation of Hypericum attenuatum Choisy extracts afforded three major flavonoids (including astragalin, guaijaverin and quercetin), which possessed α-Glucosidase inhibitory activity with IC50 values of 33.90±0.68 µM, 17.23±0.75 µM and 31.90±0.34 µM, respectively. Circular dichroism analysis revealed that all the three compounds could interact with α-glucosidase by inducing conformational changes of the enzyme. Molecular docking results indicated that they could bind to the active site in α-glucosidase, and the binding force was driven mainly by hydrogen bond. Additionally, isobolographic analysis of the interactions between two compounds showed that all the combinations presented a synergistic α-glucosidase inhibitory effect at lower concentrations, and the combination between quercetin and guaijaverin or astragalin exhibited the best synergistic effect. This research might provide a theoretical basis for the application of Hypericum attenuatum Choisy in treating hyperglycemia.

Biodegradation and physiological response mechanism of Bacillus aryabhattai to cyclotetramethylenete-tranitramine (HMX) contamination.

This study aims to reveal the biodegradation and interaction mechanism of cyclotetramethylenete-tranitramine (HMX) by a newly isolated bacteria. In this study, a bacterial strain (Bacillus aryabhattai) with high efficiency for HMX degradation was used as the test organism to analyze the changes in growth status, cell function, and mineral metabolism following exposure to different stress concentrations (0 and 5 mg L-1) of HMX. Non-targeted metabonomics was used to reveal the metabolic response of this strain to HMX stress. The results showed that when the HMX concentration was 5 mg L-1, the removal rate of HMX within 24 h of inoculation with Bacillus aryabhatta was as high as 90.5%, the OD600 turbidity was 1.024, and the BOD5 was 225 mg L-1. Scanning electron microscope (SEM) images showed that the morphology of bacteria was not obvious Variety, Fourier transform infrared spectroscopy (FTIR) showed that the cell surface -OH functional groups drifted, and ICP-MS showed that the cell mineral element metabolism was disturbed. Non-targeted metabonomics showed that HMX induced the differential expression of 254 metabolites (133 upregulated and 221 downregulated). The main differentially expressed metabolites during HMX stress were lipids and lipid-like molecules, and the most significantly affected metabolic pathway was purine metabolism. At the same time, the primary metabolic network of bacteria was disordered. These results confirmed that Bacillus aryabhattai has a high tolerance to HMX and can efficiently degrade HMX. The degradation mechanism involves the extracellular decomposition of HMX and transformation of the degradation products into intracellular purines, amino sugars, and nucleoside sugars that then participate in cell metabolism.

Human Papillomavirus E6/E7 and Long Noncoding RNA TMPOP2 Mutually Upregulated Gene Expression in Cervical Cancer Cells.

TMPOP2 was previously suggested to be an oncogenic long noncoding RNA which is excessively expressed in cervical cancer cells and inhibits E-cadherin gene expression by recruiting transcription repressor EZH2 to the gene promoter. So far, the function and regulation of TMPOP2 in cervical cancer remain largely unknown. Herein, we found that TMPOP2 expression was correlated with human papillomavirus 16/18 (HPV16/18) E6 and E7 in cervical cancer cell lines CaSki and HeLa. Tumor suppressor p53, which is targeted for degradation by HPV16/18, was demonstrated to associate with two p53 response elements in the TMPOP2 promoter to repress the transcription of the TMPOP2 gene. Reciprocally, ectopic expression of TMPOP2 was demonstrated to sequester tumor repressor microRNAs (miRNAs) miR-375 and miR-139 which target HPV16/18 E6/E7 mRNA and resulted in an upregulation of HPV16/18 E6/E7 genes. Thereby, HPV16/18 E6/E7 and the long noncoding RNA (lncRNA) TMPOP2 form a positive feedback loop to mutually derepress gene expression in cervical cancer cells. Moreover, results of RNA sequencing and cell cycle analysis showed that knockdown of TMPOP2 impaired the expression of cell cycle genes, induced cell cycle arrest, and inhibited HeLa cell proliferation. Together, our results indicate that TMPOP2 and HPV16/18 E6/E7 mutually strengthen their expression in cervical cancer cells to enhance tumorigenic activities.IMPORTANCE Human papillomaviruses 16 and 18 (HPV16/18) are the main causative agents of cervical cancer. Viral proteins HPV16/18 E6 and E7 are constitutively expressed in cancer cells to maintain oncogenic phenotypes. Accumulating evidences suggest that HPVs are correlated with the deregulation of long noncoding RNAs (lncRNAs) in cervical cancer, although the mechanism was unexplored in most cases. TMPOP2 is a newly identified lncRNA excessively expressed in cervical cancer. However, the mechanism for the upregulation of TMPOP2 in cervical cancer cells remains largely unknown and its relationship with HPVs is still elusive. The significance of our research is in revealing the mutual upregulation of HPV16/18 E6/E7 and TMPOP2 with the molecular mechanisms explored. This study will expand our understandings of the oncogenic activities of human papillomaviruses and lncRNAs.

Integrated metabonomic-proteomic analysis reveals the effect of glucose stress on metabolic adaptation of Lactococcus lactis ssp. lactis CICC23200.

A combined proteomic and metabonomic approach was used to investigate the metabolism of Lactococcus lactis ssp. lactis subjected to glucose stress treatment. A proteomic method was used to determine 1,427 altered proteins, including 278 proteins with increased expression and 255 proteins with decreased expression. A metabonomic approach was adopted to identify 98 altered metabolites, including 62 metabolites with increased expression and 26 metabolites with decreased expression. The integrated analysis indicated that the RNA and DNA mismatch repair process and energy metabolism were enhanced in response to high-glucose stress in L. lactis. Lactococcus lactis responded to glucose stress by up-regulating oxidoreductase activity, which acted on glycosyl bonds, hydrolase activity, and organic acid transmembrane transporter activity. This led to an improvement in the metabolic flux from glucose to pyruvate, lactate, acetate, and maltose. Down-regulation of amino acid transmembrane transporter, aminoacyl-transfer RNA ligase, hydroxymethyl-, formyl-, and related transferase activities resulted in a decrease in the nitrogen metabolism-associated metabolic pathway, which might be related to inhibition of the production of biogenic amines. Overall, we highlight the response of metabolism to glucose stress and provide potential possibilities for the reduced formation of biogenic amines in improved level of sugar in the dairy fermentation industry. Moreover, according to the demand for industrial production, sugar concentration in fermented foods should be higher, or lower, than a set value that is dependent on bacterial strain and biogenic amine yield.

Assessment of cyto- and genotoxic effects of Cesium-133 in Vicia faba using single-cell gel electrophoresis and random amplified polymorphic DNA assays.

The aim of this study was to evaluate the ecotoxic effect of high concentration cesium (Cs) exposure on plant root growth and its toxicological mechanism. The radicle of broad bean (Vicia faba) was selected as experimental material. The cytotoxic and genotoxic effects of plants exposed to different Cs levels (0.19-1.5 mM) for 48 h were evaluated using scanning electron microscopy (SEM), X-ray fluorescence (XRF) analysis, single-cell gel electrophoresis (SCGE) and random amplified polymorphic DNA (RAPD) assays. The results showed that radicle elongation decreased clearly after 48 h of exposure treatment with different concentrations of Cs solution. The root cell structure was obviously damaged in the Cs treatment groups (0.19-1.5 mM). At a Cs concentration of 1.5 mM, the percentages of viable non-apoptotic cells, viable apoptotic cells, non-viable apoptotic cells, and non-viable cells were 40.09%, 20.67%, 28.73%, and 10.52%, respectively. SCGE showed DNA damage in radicle cells 48 h after Cs exposure. Compared with the control group, the percentage of tail DNA in Cs exposed group (0.38-1.5 mM) increased by 0.56-1.12 times (P < 0.05). RAPD results showed that the genomic stability of V. faba radicles decreased by 4.44%-15.56%. This study confirmed that high concentration Cs exposure had cytotoxicity and genotoxicity effects on plants.

Structure-based engineering of heparinase I with improved specific activity for degrading heparin.

BACKGROUND: Heparinase I from Pedobacter heparinus (Ph-HepI), which specifically cleaves heparin and heparan sulfate, is one of the most extensively studied glycosaminoglycan lyases. Enzymatic degradation of heparin by heparin lyases not only largely facilitates heparin structural analysis but also showed great potential to produce low-molecular-weight heparin (LMWH) in an environmentally friendly way. However, industrial applications of Ph-HepI have been limited by their poor yield and enzyme activity. In this work, we improve the specific enzyme activity of Ph-HepI based on homology modeling, multiple sequence alignment, molecular docking and site-directed mutagenesis. RESULTS: Three mutations (S169D, A259D, S169D/A259D) exhibited a 50.18, 40.43, and 122.05% increase in the specific enzyme activity and a 91.67, 108.33, and 75% increase in the yield, respectively. The catalytic efficiencies (kcat/Km) of the mutanted enzymes S169D, A259D, and S169D/A259D were higher than those of the wild-type enzyme by 275, 164, and 406%, respectively. Mass spectrometry and activity detection showed the enzyme degradation products were in line with the standards of the European Pharmacopoeia. Protein structure analysis showed that hydrogen bonds and ionic bonds were important factors for improving specific enzyme activity and yield. CONCLUSIONS: We found that the mutant S169D/A259D had more industrial application value than the wild-type enzyme due to molecular modifications. Our results provide a new strategy to increase the catalytic efficiency of other heparinases.

Comparative transcriptomics analysis of potassium uptake pathways mediated cesium accumulation differences and related molecular mechanisms in Brassica juncea and Vicia faba.

To analyze the differences between high- and low-accumulation plants in cesium (Cs) uptake and its related mechanism, Brassica juncea (a hyperaccumulation plant for Cs) and Vicia faba (a low-accumulation plant for Cs) were selected as comparative experimental materials. The contributions to Cs uptake of a K-transporter-mediated high-affinity transport system and a K-channel-mediated low-affinity transport system in the two plants were compared and analyzed. The difference between the two plants in the mechanism of Cs uptake was further analyzed using transcription sequence technology. The results show that the transfer characteristics of Cs in the two plants had a similar distribution relationship with K. The contribution rate of the K-channel pathway to Cs uptake was 32.00% in the V. faba seedling roots, which was significantly higher than for B. juncea (9.81%) (P < 0.01); the contribution rate of the K-transporter pathway to Cs uptake of the B. juncea seedlings was 32.08%, which was significantly higher than that of the V. faba seedlings (17.13%)(P < 0.05). Other uptake pathways also mediated the uptake of Cs by roots in B. juncea and V. faba (contribution rate: 54.92-60.09% and 42.18-59.73%, respectively). The transcriptome sequencing results confirmed that Cs-induced treatment significantly inhibited the expression of the K-transporter protein and K-channel protein-related genes in the V. faba roots, but it had no significant effect on the expression of related genes in the B. juncea roots. Thus, one reason for the significant difference between the two plant in the accumulation of Cs is that Cs inhibited the expression of related transporter protein genes in the V. faba roots.

High-efficiency antioxidant system, chelating system and stress-responsive genes enhance tolerance to cesium ionotoxicity in Indian mustard (Brassica juncea L.).

Indian mustard (Brassica juncea L.) was more tolerance to Cs than some sensitive plants, such as Arabidopsis thaliana and Vicia faba, and may have a special detoxification mechanism. In this study, the effects on reactive oxygen species (ROS) content, the antioxidant enzyme system and chelation system in Indian mustard were studied by observing different plant physiological responses. In addition, we focused on the analysis of gene regulatory networks related to ROS formation, ROS scavenging system, and other stress-response genes to Cs exposure using a transcriptome-sequencing database. The results showed that ROS and malonaldehyde content in seedlings increased significantly in Cs-treatment groups. The enzyme activities of superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase were increased, and the synthesis of antioxidants glutathione, phytochelatin and metallothionein also increased under Cs treatment. Further analysis showed that ROS formation pathways were primarily the photosynthetic electron transport chain process and photorespiration process in the peroxisome. Antioxidant enzyme systems and the respiratory burst oxidase homolog protein-mediated signal transduction pathway played a key role in ROS scavenging. In summary, one of the mechanisms of tolerance and detoxification of Indian mustard to Cs was that it enhanced the scavenging ability of antioxidant enzymes to ROS, chelated free Cs ions in cells and regulated the expression of related disease-resistant genes.

Stable expression of infliximab in CRISPR/Cas9-mediated BAK1-deficient CHO cells.

OBJECTIVES: To establish stable infliximab-expressing Chinese hamster ovary (CHO) cells with high tolerance to serum-free culture. RESULTS: Bcl-2 antagonist/killer 1 (BAK1), which is a key mediator of the apoptosis pathway, was disrupted, and infliximab, which is a broadly used monoclonal antibody for the treatment of rheumatoid arthritis and other autoimmune diseases, was incorporated into the BAK1 locus of the CHO chromosome using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas genome-editing technique. The activating effects of serum starvation on BAK1 and cytochrome C (CytC) were suppressed in the genome-edited cells, and the ability of the cells to resist the serum starvation-induced loss of mitochondrial membrane potential and apoptosis was increased, as indicated by the results of polymerase chain reaction (PCR), flow cytometry, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) analysis. In addition, during subsequent passages, infliximab could be stably produced in the genome-edited CHO cells, and the recombinant antibody could effectively antagonize the cytotoxic effect of tumor necrosis factor α (TNFα). CONCLUSIONS: A CHO cell line capable of stably expressing infliximab and adapting to serum-free culture was constructed. This work lays the foundation for the development of infliximab biosimilars.

Knockdown of DNMT1 and DNMT3a Promotes the Angiogenesis of Human Mesenchymal Stem Cells Leading to Arterial Specific Differentiation.

Human mesenchymal stem cells (hMSCs) possess the potential to differentiate into endothelial cells (EC). DNA methylation plays an important role in cell differentiation during development. However, the role of the DNA methyltransferases Dnmt1 and Dnmt3a in specific arterial differentiation of hMSCs is not clear. Here, we show that the CpG islands in the promoter regions of the EC specification and arterial marker genes were highly methylated in hMSCs based on bisulfite genomic sequencing. Treatment with the DNMT inhibitor 5-aza-dc induced the reactivation of EC specification and arterial marker genes by promoting demethylation of these genes as well as stimulating tube-like structure formation. The hMSCs with stable knockdown of Dnmt1/Dnmt3a were highly angiogenic and expressed several arterial specific transcription factors and marker genes. A Matrigel plug assay confirmed that Dnmt1/Dnmt3a stable knockdown hMSCs enhanced blood vessel formation compared with WT MSCs. We also identified that the transcription factor E2F1 could upregulate the transcription of arterial marker genes by binding to the promoters of arterial genes, suggesting its critical role for arterial specification. Moreover, miRNA gain/loss-of-function analyses revealed that miR152 and miR30a were involved in endothelial differentiation of hMSCs by targeting Dnmt1 and Dnmt3a, respectively. Taken together, these data suggest that Dnmt1 and Dnmt3a are critical regulators for epigenetic silencing of EC marker genes and that E2F1 plays an important role in promoting arterial cell determination. Stem Cells 2016;34:1273-1283.

Fusion with pep-1, a cell-penetrating peptide, enhances the transmembrane ability of human epidermal growth factor.

Administration of macromolecule compositions in medicine and cosmetics always exhibited low bioavailability due to the limitation of transmembrane transport. Here, human epidermal growth factor (hEGF) was fused with glutathione S-transferase (GST) and Pep-1, the first commercial cell-penetrating peptide, in Escherichia coli. The fusion protein was firstly purified with the affinity chromatography, and then the GST tag was released by TEV protease. Final purification was achieved by the ion exchange chromatography. The biological activities and the transmembrane ability of the obtained products were determined using scratch wound-healing assay, MTT analysis, and immunofluorescence assay. The results showed that both rhEGF and Pep-1-fused hEGF were soluble expressed in E. coli. The fusion of Pep-1 could markedly increase the transmembrane ability of EGF, whereas it did not interfere with the growth-stimulating and migration-promoting functions of hEGF on fibroblasts. This research provided a novel strategy for the transmembrane transport of protein-derived cosmetics or drugs.

Transcriptional factors p300 and MRTF-A synergistically enhance the expression of migration-related genes in MCF-7 breast cancer cells.

The transcriptional coactivator p300 is highly expressed in breast cancer tissues. MRTF-A is a transcription factor governed by the Rho-GTPase-actin signaling pathway. The purpose of this study was to explore the role of p300 in breast cancer metastasis. Here we showed that the motility of breast cancer cells was enhanced by the overexpression of p300, meanwhile, the transcription of migration-related genes was upregulated. Depletion of p300 downregulated the migration-related genes and slowed down the migration of breast cancer cells. p300 worked synergistically with MRTF-A to activate the transcription of MYH9, MYL9 and CYR61. As identified by co-IP, p300 interacted with the C-terminal TAD domain of MRTF-A. And together with MRTF-A, p300 was associated with the target gene promoters. Furthermore, MRTF-A was found to be acetylated in MCF-7 breast cancer cells. These results demonstrated the involvement of p300 in the MRTF-A mediated gene regulation and breast cancer cell migration.