Comparison of DC Bead-irinotecan and DC Bead-topotecan drug eluting beads for use in locoregional drug delivery to treat pancreatic cancer

DC Bead is a drug delivery embolisation system that can be loaded with doxorubicin or irinotecan for the treatment of a variety of liver cancers. In this study we demonstrate that the topoisomerase I inhibitor topotecan hydrochloride can be successfully loaded into the DC Bead sulfonate-modified polyvinyl alcohol hydrogel matrix, resulting in a sustained-release drug eluting bead (DEBTOP) useful for therapeutic purposes. The in vitro drug loading capacity, elution characteristics and the effects on mechanical properties of the beads are described with reference to our previous work with irinotecan hydrochloride (DEBIRI). Results showed that drug loading was faster when the solution was agitated compared to static loading and a maximum loading of ca. 40–45 mg topotecan in 1 ml hydrated beads was achievable. Loading the drug into the beads altered the size, compressibility moduli and colour of the bead. Elution was shown to be reliant on the presence of ions to perform the necessary exchange with the electrostatically bound topotecan molecules. Topotecan was shown by MTS assay to have an IC50 for human pancreatic adenocarcinoma cells (PSN-1) of 0.22 and 0.27 lM compared to 28.1 and 19.2 lM for irinotecan at 48 and 72 h, respectively. The cytotoxic efficacy of DEBTOP on PSN-1 was compared to DEBIRI. DEPTOP loaded at 6 & 30 mg ml-1, like its free drug form, was shown to be more potent than DEBIRI of comparable doses at 24, 48 & 72 h using a slightly modified MTS assay. Using a PSN-1 mouse xenograft model, DEBIRI doses of 3.3–6.6 mg were shown to be well tolerated (even with repeat administration) and effective in reducing the tumour size. DEBTOP however, was lethal after 6 days at doses of 0.83–1.2 mg but demonstrated reasonable efficacy and tolerability (again with repeat injection possible) at 0.2–0.4 mg doses. Care must therefore be taken when selecting the dose of topotecan to be loaded into DC Bead given its greater potency and potential toxicity

Placement of an aortomonoiliac stent graft without femorofemoral revascularization in endovascular aneurysm repair: a case report

<p>Abstract</p> <p>Introduction</p> <p>Endovascular aortic repair, if technically feasible, is the treatment of choice for patients with a contained ruptured aortic aneurysm who are unfit for open surgery.</p> <p>Case presentation</p> <p>We report the case of an 80-year-old Caucasian man who presented with an unusually configured, symptomatic infrarenal aortic aneurysm. His aneurysm showed an erosion of the fourth lumbar vertebra and a severely arteriosclerotic pelvic axis. A high thigh amputation of his right leg had been performed 15 months previously. On his right side, occlusion of his external iliac artery, common femoral artery, and deep femoral artery had occurred. His aneurysm was treated by a left-sided aortomonoiliac stent graft without femorofemoral revascularization, resulting in occlusions of both internal iliac arteries. No ischemic symptoms appeared, although perfusion of his right side was maintained only over epigastric collaterals.</p> <p>Conclusions</p> <p>The placement of aortomonoiliac stent grafts for endovascular treatment of infrarenal aortic aneurysms without contralateral revascularization is a feasible treatment option in isolated cases. In this report, access problems and revascularization options in endovascular aneurysm repair are discussed.</p

Fluorescent Protein-Based Methods for On-Plate Screening of Gene Insertion

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening

Problem formulation in the environmental risk assessment for genetically modified plants

Problem formulation is the first step in environmental risk assessment (ERA) where policy goals, scope, assessment endpoints, and methodology are distilled to an explicitly stated problem and approach for analysis. The consistency and utility of ERAs for genetically modified (GM) plants can be improved through rigorous problem formulation (PF), producing an analysis plan that describes relevant exposure scenarios and the potential consequences of these scenarios. A properly executed PF assures the relevance of ERA outcomes for decision-making. Adopting a harmonized approach to problem formulation should bring about greater uniformity in the ERA process for GM plants among regulatory regimes globally. This paper is the product of an international expert group convened by the International Life Sciences Institute (ILSI) Research Foundation

Prostate transglutaminase (TGase-4) antagonizes the anti-tumour action of MDA-7/IL-24 in prostate cancer

Background Transglutamiase-4 (TGase-4), also known as prostate transglutaminase, belongs to the TGase family and is uniquely expressed in the prostate gland. The functions of this interesting protein are not clearly defined. In the present study, we have investigated an unexpected link between TGase-4 and the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24), a cytokine known to regulate the growth and apoptosis of certain cancer and immune cells. Methods Frozen sections of normal and malignant human prostate tissues and human prostate cancer (PCa) cell lines PC-3 and CA-HPV-10, cell lines expressing low and high levels of TGase-4, and recombinant MDA-7/IL-24 (rhMDA-7/IL-24) were used. Expression construct for human TGase-4 was generated using a mammalian expression vector with full length human TGase-4 isolated from normal human prostate tissues. PC-3 cells were transfected with expression construct or control plasmid. Stably transfected cells for control transfection and TGase-4 over expression were created. Similarly, expression of TGase-4 in CA-HPV-10 cells were knocked down by way of ribozyme transgenes. Single and double immunofluorescence microscopy was used for localization and co-localization of TGase-4 and MDA-7/IL-24 in PCa tissues and cells with antibodies to TGase-4; MDA-7/IL-24; IL-20alpha; IL-20beta and IL-22R. Cell-matrix adhesion, attachment and migration were by electric cell substrate impedance sensing and growth by in vitro cell growth assay. A panel of small molecule inhibitors, including Akt, was used to determine signal pathways involving TGase-4 and MDA-7/IL-24. Results We initially noted that MDA-7 resulted in inhibition of cell adhesion, growth and migration of human PCa PC-3 cells which did not express TGase-4. However, after the cells over-expressed TGase-4 by way of transfection, the TGase-4 expressing cells lost their adhesion, growth and migratory inhibitory response to MDA-7. On the other hand, CA-HPV-10 cells, a cell type naturally expressing high levels of TGase-4, had a contrasting response to MDA-7 when compared with PC-3 cells. Inhibitor to Akt reversed the inhibitory effect of MDA-7, only in PC-3 control cells, but not the TGase-4 expressing PC-3 cells. In human prostate tissues, TGase-4 was found to have a good degree of co-localization with one of the MDA-7 receptor complexes, IL-20Ra. Conclusion The presence of TGase-4 has a biological impact on a prostate cancer cell's response to MDA-7. TGase-4, via mechanism(s) yet to be identified, blocked the action of MDA-7 in prostate cancer cells. This has an important implication when considering the use of MDA-7 as a potential anticancer cytokine in prostate cancer therapies

Analysis of the anti-tumor effect of cetuximab using protein kinetics and mouse xenograft models

<p>Abstract</p> <p>Background</p> <p>The binding of EGFR and its ligands leads to autophosphorylation of receptor tyrosine kinase as well as subsequent activation of signal transduction pathways that are involved in regulating cellular proliferation, differentiation, and survival. An EGFR inhibitor, cetuximab binds to EGFR and consequently blocks a variety of cellular processes. <it>KRAS</it>/<it>BRAF </it>mutations are known to be associated with a low response rate to cetuximab. In the present study, to clarify the anti-tumor mechanisms of cetuximab, we evaluated the <it>KRAS</it>/<it>BRAF </it>status, phosphorylation level of the EGFR pathway, and the tumor suppression effect in vivo, using a human colon cancer cell line HT29, which exhibited the highest EGFR expression in response to the cetuximab therapy among the 6 colorectal cancer cell lines tested.</p> <p>Findings</p> <p>The conventional growth suppression assay did not work efficiently with cetuximab. EGF, TGF-α, and IGF activated the EGFR/MAPK cell signaling pathway by initiating the phosphorylation of EGFR. Cetuximab partially inhibited the EGFR/MAPK pathway induced by EGF, TGF-α, and IGF. However, cetuximab exposure induced the EGFR, MEK, and ERK1/2 phosphorylation by itself. Mouse xenograft tumor growth was significantly inhibited by cetuximab and both cetuximab-treated and -untreated xenograft specimens exhibited phosphorylations of the EGFR pathway proteins.</p> <p>Conclusions</p> <p>We have confirmed that cetuximab inhibited the EGFR/MAPK pathway and reduced tumor growth in the xenografts while the remaining tumor showed EGFR pathway activation. These results suggest that: ( i ) The effect of cetuximab in growth signaling is not sufficient to induce complete growth suppression in vitro; ( ii ) time-course monitoring may be necessary to evaluate the effect of cetuximab because EGFR signaling is transmitted in a minute order; and ( iii ) cetuximab treatment may have cells acquired resistant selectively survived in the heterogeneous cancer population.</p

Cell-to-Cell Transformation in Escherichia coli: A Novel Type of Natural Transformation Involving Cell-Derived DNA and a Putative Promoting Pheromone

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as ‘cell-to-cell transformation’. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10–5–10–6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria