Search

Because ADAM17 promotes colonic tumorigenesis, we investigated potential miRNAs regulating ADAM17; and examined effects of diet and tumorigenesis on these miRNAs. We also examined pre-miRNA processing and tumour suppressor roles of several of these miRNAs in experimental colon cancer. Using TargetScan, miR-145, miR-148a, and miR-152 were predicted to regulate ADAM17. miR-143 was also investigated as miR-143 and miR-145 are co-transcribed and associated with decreased tumour growth. HCT116 colon cancer cells (CCC) were co-transfected with predicted ADAM17-regulating miRNAs and luciferase reporters controlled by ADAM17-3’UTR. Separately, pre-miR-143 processing by colonic cells was measured. miRNAs were quantified by RT-PCR. Tumours were induced with AOM/DSS in WT and transgenic mice (Tg) expressing pre-miR-143/miR-145 under villin promoter. HCT116 transfection with miR-145, −148a or −152, but not scrambled miRNA inhibited ADAM17 expression and luciferase activity. The latter was suppressed by mutations in ADAM17-3’UTR. Lysates from colonocytes, but not CCC, processed pre-miR-143 and mixing experiments suggested CCC lacked a competency factor. Colonic miR-143, miR-145, miR-148a, and miR-152 were downregulated in tumours and more moderately by feeding mice a Western diet. Tg mice were resistant to DSS colitis and had significantly lower cancer incidence and tumour multiplicity. Tg expression blocked up-regulation of putative targets of miR-143 and miR-145, including ADAM17, K-Ras, XPO5, and SET. miR-145, miR-148a, and miR-152 directly suppress colonocyte ADAM17 and are down-regulated in colon cancer. This is the first direct demonstration of tumour suppressor roles for miR-143 and miR-145 in an in vivo model of colonic tumorigenesis.

DNA 5-hydroxymethylcytosine (5hmC) modification is known to be associated with gene transcription and frequently used as a mark to investigate dynamic DNA methylation conversion during mammalian development and in human diseases. However, the lack of genome-wide 5hmC profiles in different human tissue types impedes drawing generalized conclusions about how 5hmC is implicated in transcription activity and tissue specificity. To meet this need, we describe the development of a 5hmC tissue map by characterizing the genomic distributions of 5hmC in 19 human tissues derived from ten organ systems. Subsequent sequencing results enabled the identification of genome-wide 5hmC distributions that uniquely separates samples by tissue type. Further comparison of the 5hmC profiles with transcriptomes and histone modifications revealed that 5hmC is preferentially enriched on tissue-specific gene bodies and enhancers. Taken together, the results provide an extensive 5hmC map across diverse human tissue types that suggests a potential role of 5hmC in tissue-specific development; as well as a resource to facilitate future studies of DNA demethylation in pathogenesis and the development of 5hmC as biomarkers., DNA 5-hydroxymethylcytosine (5hmC) modification is associated with gene transcription and used as a mark of mammalian development. Here the authors report a comprehensive 5hmC tissue map and analysis of 5hmC genomic distributions in 19 human tissues derived from 10 organ systems, thus providing insights into the role of 5hmC in tissue-specific development.

Background This study aimed to overexpress or silence connexin 43 (Cx43) and A‐kinase anchoring protein 95 (AKAP95) in human A549 cells to explore their effects on cyclins and on G1/S conversion when the interrelationship of Cx43, AKAP95, and cyclin E1/E2 changes. Methods The study mainly used Western blot analysis and Co‐immuno precipitation to detect the target protein in Cx43/AKAP95 over expressed human A549 cells, and the relationship of proteins Cx43, AKAP95 and Cyclin E during G1‐S phase was explored with qualitative and quantitative analysis. Results The overexpression of Cx43 inhibited the expression of cyclin D1 and E1 by accelerating their degradation and reduced the Cdk2 activity that blocked the DNA transcription activity. However, the overexpression of AKAP95 increased the expression of cyclin D1 and E1 and inhibited their degradation, and enhanced the Cdk2 activity that promoted the DNA transcription activity. Cx43 and AKAP95 competitively bound to cyclin E1/E2, and the competitive binding affected the Cdk2 activity, Rb phosphorylation, DNA transcription activity, and G1/S conversion. Conclusions This study showed that the expression of ERK1/2, PKA, and PKB increased when BEAS‐2B cells were treated with PDGF‐BB, suggesting that ERK1/2, PKA, and PKB might be involved in the binding of AKAP95 with cyclin E, or the separation of AKAP95 from Cx43 from cyclin E1/E2. The specific mechanism underlying this process still needs further exploration.

BACKGROUND As mechanisms of neoplasia in patients with ulcerative colitis (UC) remain poorly understood, we sought to identify pathways of carcinogenesis in this high-risk population. METHODS MicroRNA (miRNA) and mRNA expression was examined in nondysplastic rectosigmoid mucosa from UC patients with (n = 19) or without remote colon neoplasia (n = 23). We developed a method to identify miRNA-regulated pathways based on differentially expressed miRNAs and their putative mRNAs targets in the same samples. One key pathway identified in the analysis, miR-4728-3p regulation of focal adhesion signaling was further evaluated in vitro and through examination of expression in UC-cancers. RESULTS There were 101 significantly up-regulated and 98 down-regulated miRNAs (adjusted P < 0.05) in the rectal mucosa of UC patients harboring proximal neoplasia. Bioinformatic analysis identified miR-4728-3p as a regulator of 3 proteins involved in focal adhesion signaling, CAV1, THBS2, and COL1A2. Real-time PCR validated down-regulation of miR-4728-3p in nondysplastic tissue remote from UC-neoplasia and in UC-associated colon cancers. miR-4728-3p transfection into colon cancer cells down-regulated expression levels and decreased luciferase activities in cells expressing a wild type 3' untranslated region compared with a mutant 3' untranslated region for all 3 genes. Exogenous transfected miR-4728-3p also delayed wound healing and decreased formation of focal adhesion complexes. CONCLUSIONS Patients with long-standing UC who harbor neoplasia can be identified based on miRNA and mRNA profiles in nondysplastic tissue. Using a method to analyze miRNA and mRNA expression from the same tissues, we identified that miR-4728-3p is likely an important tumor suppressor in UC-associated colon carcinogenesis.

Horseradish peroxidase (HRP)-mediated crosslinking of poly(amido amine) (PAA) copolymers was successfully applied in the preparation of in situ forming degradable hydrogels under physiological conditions. PAA copolymers containing different amounts of tyramine residues (termed as pAEEOL/TA) could be synthesized through Michael-type addition between methylenebisacryamide and amine mixture of 2-(2-aminoethoxy) ethanol and tyramine (TA). Depending on the amount of TA residue, the HRP, and H2O2 concentration, the gelation times could be varied from about 50 to 350 s. The swelling and degradation experiments indicated under physiological conditions the pAEEOL/TA-based hydrogels are completely degradable within 6–8 days. Rheological analysis revealed that storage modulus of the hydrogels increased from 2500 to 4100 Pa when increasing HRP concentrations. Importantly, pAEEOL/TA copolymers have low cytotoxicity. Moreover, NIH 3T3 (mouse embryonic fibroblast) cells exposed in the degradation products of pAEEOL/TA-based hydrogels retained high cell viability, implying that the hydrogels are cyto-biocompatible. In vitro release of methylene blue and IgG protein from pAEEOL/TA-based hydrogels could be effectively sustained by encapsulation of the drug in the hydrogels. The results indicate that HRP-crosslinked, degradable pAEEOL/TA-based hydrogels are promising for biomedical applications. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

This paper demonstrates a novel strategy for site-selective cell adhesion and in situ cultivation of living cells, integrated with real-time monitoring of cellular small biomolecules based on dual functional protein microarrays. The protein microarrays have been produced on the superhydrophobic|philic Au-TiO2 micropatterns, through further modification of L-cysteine (Cys) and followed by successive immobilization of a model protein, cytochrome c (cyt c). Experimental results have revealed that the created cyt c microarrays play dual functions: one is employed as a robust substrate for site-selective cell adhesion and in situ cultivation of living cells, because the protein microarrays exhibit high selectivity and bioaffinity toward cells, as well as long biostability under cell culture condition up to 7 days. Meanwhile, the cyt c microarrays can also serve as sensing elements for hydrogen peroxide (H2O2) due to the inherent enzymatic activity of the heme center in cyt c. Direct electron transfer of cyt c has been enhanced at the Cys-modified Au-TiO2 (Au-TiO2/Cys) microarrays, and the electrochemical behavior can be tuned by varying the width and spacing of the microband arrays. Furthermore, cyt c is stably immobilized on the Au-TiO2/Cys microarrays and maintains its enzymatic activity after confined on the microarrays. Thus, the optimized cyt c microarrays show striking analytical performance for H2O2 determination, e.g., high sensitivity and selectivity, broad linear range from 10(-9) M to 10(-2) M, low detection limit down to 2 nM, and short response time within 5 s. As a result, the excellent analytical properties of the cyt c microarrays, as well as the characteristic of the protein microarrays themselves, including high selectivity, long biostability, and good bioaffinity, opens up a method for selective in situ cultivation of cells integrated with real-time detection of signaling biomolecules such as H2O2 released from living cells, which shows potential for physiological and pathological investigations.

Here, we report on a novel superoxide anion (O2(*-)) biosensor based on direct electron transfer of copper, zinc-superoxide dismutase (Cu, Zn-SOD) at zinc oxide nanodisks surface for in vivo tracking of O2(*-) in bean sprouts. Direct electron transfer of SOD is achieved at ZnO nanodisks film prepared by a one-step electrodeposited method, with a high heterogeneous electron rate constant of 17 +/- 2 s(-1). Spectroscopic data demonstrate that SOD strongly immobilized onto the nanostructured ZnO surfaces processes its inherent activity toward O2(*-) dismutation. A combination of the facilitated direct electron transfer and the bifunctional enzymatic catalytic activities of the SOD substantially provides a dual electrochemical approach to determination of O2(*-) with high selectivity, wide linear range, long stability, and good reproducibility. In particular, SOD adsorbed on the ZnO nanodisks film is capable of sensing O2(*-) cathodically at a very positive potential, 0 mV (vs Ag|AgCl), where the common interfering species such as hydrogen peroxide, uric acid, ascorbic acid, and 3,4-dihydroxyphenylacetic acid were effectively avoided. The excellent analytical performance of the present O2(*-) biosensor, combined with the remarkable characteristics of nanostructured ZnO films, such as biocompatibility, ease of preparation, and facile to miniaturize, paves an electrochemical way for reliable and durable in vivo determination of O2(*-) in bean sprouts.

This communication describes a novel and facile approach to direct electron transfer of copper, zinc–superoxide dismutase (Cu, Zn–SOD) at a physical vapor deposited zinc oxide (ZnO) nanoparticles surface. SOD exhibits quasi-reversible electrochemical behavior in phosphate buffer solution (PBS, pH 7.25), with apparent formal potential of 195.2 ± 4.6 mV vs. Ag|AgCl and high heterogeneous electron rate constant of 10.4 ± 1.8 s−1. Electrochemical results indicate that SOD stably immobilizes on the nanostructured ZnO film and processes its intrinsic enzymatic activity after adsorbed on ZnO nanoparticles. An electrostatic interaction between SOD with low isoelectric point (pI = 4.9) and ZnO nanoparticles with surface pKa of 9.35 is suggested to be the main force for the adsorption of SOD molecules on the nanostructured ZnO surface, and as a consequence, ZnO nanoparticles surface is favor for direct electron transfer of SOD. The enhanced electron transfer of SOD, together with intrinsic bidirectional catalytic activity of SOD toward superoxide anion (O2-) dismutation paves a way for the development of a third-generation O2- biosensor. Keywords: Electron transfer, Superoxide dismutase, ZnO nanoparticles, Superoxide anion, Physical vapor deposition

The WO 3 nanoparticles film is first employed as a support matrix for confining cytochrome c (cyt. c ), an excellent model for studying electron transfer between the redox enzymes and the electrode. The surface p K a of nanostructured WO 3 film is estimated to be ∼2.74 using electrochemical method. The present WO 3 surface with negative charge at the neutral solution is very benefit for the adsorption of cyt. c with positive charge and facilitates electron transfer of cyt. c . As a result, direct and fast electron transfer of cyt. c is realized at the nanostructured WO 3 surface with the redox formal potential ( E 0 ′) of −133.5 ± 1.7 mV ( n = 4) versus Ag/AgCl and heterogeneous electron transfer rate constant of 5.57 ± 0.54 s −1 . Experimental data indicate that cyt. c is stably confined onto the WO 3 nanoparticles film, possibly due to the electrostatic interaction between WO 3 nanostructures and cyt. c , and processes its enzymatic activity toward H 2 O 2 . Based on these results, the third-generation biosensor for H 2 O 2 is developed with high selectivity, free from not only common anodic interferences like ascorbic acid, uric acid, 3,4-dihydroxyphenylacetic acid, and so on, but also cathodic interference—O 2 . The remarkable analytical advantages, as well as the characteristic of WO 3 nanoparticles film such as biocompatibility, low-cost, and facile to miniature give a strong basis for continuous, on-line detection of H 2 O 2 under pathophysiological conditions.

The morphology-dependent electrochemistry and electrocatalytical activity of cytochrome c (cyt. c) were investigated at pyramidal, rodlike, and spherical gold nanostructures directly electrodeposited onto sputtered gold surfaces. Direct, reversible electron transfer of cyt. c, for the first time, was realized at nanorod-like and nanopyramidal gold surfaces without any mediators or promoters, while no redox reaction was observed at the nanospherical gold electrode. The electrochemical properties of cyt. c vary with the shape of gold nanostructures with respect to the reversibility of electrode reactions, kinetic parameters, the formal potentials (E0'), and charge-transport resistance (Rct), suggesting shape-dependent mechanisms for the electrode reactions of cyt. c. The experimental results manifest that cyt. c was stably immobilized on the nanostructured gold electrodes with different conformational changes of the heme microenvironment. Consequently, not only the electroactivity, but also the inherent biological activity of the immobilized cyt. c strongly depended on the shape of the electrode surfaces. The facilitated electron transfer combined with the intrinsic catalytical activity of cyt. c substantially constructed a third-generation H2O2 biosensor with high selectivity, quick response time, large linear range, and good sensitivity. The electrocatalytical activity of the immobilized cyt. c toward H2O2 was also found to be morphology dependent, and the linear range of H2O2 detection could be tuned by means of employing the nanostructured gold surfaces with different shapes.