Zinc Finger Nuclease mediated knockout of ADP dependent Glucokinase in Cancer cell lines: Effects on cell survival and Mitochondrial Oxidative Metabolism

<div><p>Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of <i>ADPGK</i>, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of <i>ADPGK,</i> and were ADPGK-null by immunoblotting. <i>ADPGK</i> knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when <i>ADPGK</i> was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of <i>ADPGK</i> in HCT116 cells caused few changes in global gene expression, knockout of <i>ADPGK</i> in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the <i>ADPGK</i> knockouts, in some cases markedly so. Collectively, the results demonstrate that <i>ADPGK</i> can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of <i>ADPGK</i> is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.</p></div

A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.)

The complex allotetraploid genome is one of major challenges in cotton for repressing gene expression. Developing site-specific DNA mutation is the long-term dream for cotton breeding scientists. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as a robust biotechnology for targeted-DNA mutation. In this study, two sgRNAs, GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, were designed in the identical genomic regions of GhMYB25-like A and GhMYB25-like D, which were encoded by cotton A subgenome and the D subgenome, respectively, was assembled to direct Cas9-mediated allotetraploid cotton genome editing. High proportion (14.2–21.4%) CRISPR/Cas9-induced specific truncation events, either from GhMYB25-like A DNA site or from GhMYB25-like D DNA site, were detected in 50% examined transgenic cotton through PCR amplification assay and sequencing analyses. Sequencing results also demonstrated that 100% and 98.8% mutation frequency were occurred on GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2 target site respectively. The off-target effect was evaluated by sequencing two putative off-target sites, which have 3 and 1 mismatched nucleotides with GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, respectively; all the examined samples were not detected any off-targetcaused mutation events. Thus, these results demonstrated that CRISPR/Cas9 is qualified for generating DNA level mutations on allotetraploid cotton genome with high-efficiency and high-specificity.ECU Open Access Publishing Support Fun

Dendrophthoe pentandra (L.) Miq extract effectively inhibits inflammation, proliferation and induces p53 expression on colitis-associated colon cancer

Background: Indonesian mistletoe grows on various trees. Mango Mistletoes (Dendrophthoe pentandra) is one type of mistletoe that grown on mango tree (.benalu mangga in bahasa Indonesia). Our study used mistletoe as a parasitic plant that has been used for traditional medicine. It has been known that Dendrophtoe pentandra extract (DPE) anti-inflammatory and anticancer. Furthermore, it is necessary to follow-up study in vivo to evaluate the response to treatment of new cancer therapeutic agents. This research aimed to determine the levels of IL-22, myeloperoxide (MPO), proliferation and wild-type p53 expression after the administration of DPE to murine models of CAC. Methods: Mouse colitis associated colon cancer (CAC) was induced firstly by azoxymethane (AOM) and followed by administration of drinking water containing 5 % dextran sodium sulfate (DSS) in a cycle protocol, each cycle consisted of seven days of 5 % DSS in the drinking water and followed by seven days of regular water. This study consists of five treatment groups: I was treated water only (control), II was administrated by (DSS only, without DPE), (III-V) were administrated by DPE (125 mg/kg BW, 250 mg/kg BW and 500 mg/kg BW) respectively. The administrated of DPE were started from the 8th weeks, were continued until 21 weeks. At the end of 21 weeks of the experiment, mice were sacrificed, colon tissue was removed, and then subjected to ELISA, flow cytometry, real-time PCR and histology examination. Results: Administration of DPE 250 mg/kgBW significantly reduce the levels of IL-22 and MPO compared with DSS only group (p < 0.001; p < 0.001). Colonic epithelial cells proliferation of group IV (DPE 250 mg/kgBW) were significantly lower than III and V groups. There was no significant change in the S phase in mice were treated DPE 125 mg/kg BW and 500 mg/kg BW, while administration of DPE 250 mg/kg BW was able to increase the percentage of cells in S phase. The expression of mRNA p53 was up regulated in mice received DPE 125 mg/kg BW. Conclusion: These findings indicate that the DPE could inhibit colonic epithelial cells proliferation through p53 pathway independently. This study also showed that DPE could be potential sources of new therapy

Design of a 5 GHz Reflectarray with Reduced Size Unit Cell and Extremely Low Phase Sensitivity

This article presents a novel, compact reflectar-ray antenna operating at 5 GHz. The array consists of two types of phasing element - square ring and complementary square ring. The complementary square ring is used for the phase values that are not covered by the single ring element. The grid size of the array is 0.28 lambda x 0.28 lambda at operating frequency 5 GHz that is much smaller than the conventional periodicity 0.5 lambda x 0.5 lambda. The aim of unit cell de sign is to have a slower slope of the reflection phase graph without sacrificing the phase range of 360 degrees where in general there is a trade-off between these two goals. The maximum slope in reflection phase graph obtained here is 34 degrees/mm. The proposed array is fabricated on a low loss PTFE substrate of thickness 3.175 mm (0.053 lambda at 5 GHz) and illuminated by a horn antenna. Radiation pattern results show a very precise far-field beam with 3-dB beam-width of 7 degrees and 7.3 degrees for two principal planes respectively. The gain of the antenna is 26 dBi at 5 GHz