Use of H19 Gene Regulatory Sequences in DNA-Based Therapy for Pancreatic Cancer

Pancreatic cancer is the eighth most common cause of death from cancer in the world, for which palliative treatments are not effective and frequently accompanied by severe side effects. We propose a DNA-based therapy for pancreatic cancer using a nonviral vector, expressing the diphtheria toxin A chain under the control of the H19 gene regulatory sequences. The H19 gene is an oncofetal RNA expressed during embryo development and in several types of cancer. We tested the expression of H19 gene in patients, and found that 65% of human pancreatic tumors analyzed showed moderated to strong expression of the gene. In vitro experiments showed that the vector was effective in reducing Luciferase protein activity on pancreatic carcinoma cell lines. In vivo experiment results revealed tumor growth arrest in different animal models for pancreatic cancer. Differences in tumor size between control and treated groups reached a 75% in the heterotopic model (P = .037) and 50% in the orthotopic model (P = .007). In addition, no visible metastases were found in the treated group of the orthotopic model. These results indicate that the treatment with the vector DTA-H19 might be a viable new therapeutic option for patients with unresectable pancreatic cancer

Adhesion of <i>Pseudomonas fluorescens</i> biofilms to glass, stainless steel and cellulose

Objectives: The adhesion of colloidal probes of stainless steel, glass and cellulose to Pseudomonas fluorescens biofilms was examined using atomic force microscopy (AFM) to allow comparisons between surfaces to which biofilms might adhere. Results: Biofilm was grown on a stainless steel substrate and covered most of the surface after 96 h. AFM approach and retraction curves were obtained when the biofilm was immersed in a tryptone/soy medium. On approach, all the colloidal probes experienced a long non-contact phase more than 100 nm in length, possibly due to the steric repulsion by extracellular polymers from the biofilm and hydrophobic effects. Retraction data showed that the adhesion varied from position to position on the biofilm. The mean value of adhesion of glass to the biofilm (48 ± 7 nN) was the greatest, followed by stainless steel (30 ± 7 nN) and cellulose (7.8 ± 0.4 nN). Conclusion: The method allows understanding of adhesion between the three materials and biofilm, and development of a better strategy to remove the biofilm from these surfaces relevant to different industrial applications

Development of targeted therapy for ovarian cancer mediated by a plasmid expressing diphtheria toxin under the control of H19 regulatory sequences

<p>Abstract</p> <p>Background</p> <p>Ovarian cancer ascites fluid (OCAF), contains malignant cells, is usually present in women with an advanced stage disease and currently has no effective therapy. Hence, we developed a new therapy strategy to target the expression of diphtheria toxin gene under the control of H19 regulatory sequences in ovarian tumor cells. H19 RNA is present at high levels in human cancer tissues (including ovarian cancer), while existing at a nearly undetectable level in the surrounding normal tissue.</p> <p>Methods</p> <p>H19 gene expression was tested in cells from OCAF by the in-situ hybridization technique (ISH) using an H19 RNA probe. The therapeutic potential of the toxin vector DTA-H19 was tested in ovarian carcinoma cell lines and in a heterotopic animal model for ovarian cancer.</p> <p>Results</p> <p>H19 RNA was detected in 90% of patients with OCAF as determined by ISH. Intratumoral injection of DTA-H19 into ectopically developed tumors caused 40% inhibition of tumor growth.</p> <p>Conclusion</p> <p>These observations may be the first step towards a major breakthrough in the treatment of human OCAF, while the effect in solid tumors required further investigation. It should enable us to identify likely non-responders in advance, and to treat patients who are resistant to all known therapies, thereby avoiding treatment failure.</p

Novel Nanohybrids of Silver Particles on Clay Platelets for Inhibiting Silver-Resistant Bacteria

We develop a novel nanohybrid showing a strong antibacterial activity on all of the tested pathogens, including methicillin-resistant Staphylococcus auerus and silver-resistant E. coli. The nanohybrid consists of silver nanoparticles (AgNPs) supported on 1 nm-thick silicate platelets (NSPs). The AgNP/NSP nanohybrid enables to encapsulate bacteria and triggers death signals from the cell membrane. The geographic shape of the NSPs concentrates AgNPs but impedes their penetration into attached cells, mitigating the detrimental effect of silver ion deposition in applied tissues. Moreover, the tightly tethered AgNPs on NSP surface achieve a stronger biocidal effect than silver nitrate, but bypassing Ag+ mechanism, on silver-resistant bacteria. This nanohybrid presents an effective and safe antimicrobial agent in a new perspective

Automated Force Volume Image Processing for Biological Samples

Atomic force microscopy (AFM) has now become a powerful technique for investigating on a molecular level, surface forces, nanomechanical properties of deformable particles, biomolecular interactions, kinetics, and dynamic processes. This paper specifically focuses on the analysis of AFM force curves collected on biological systems, in particular, bacteria. The goal is to provide fully automated tools to achieve theoretical interpretation of force curves on the basis of adequate, available physical models. In this respect, we propose two algorithms, one for the processing of approach force curves and another for the quantitative analysis of retraction force curves. In the former, electrostatic interactions prior to contact between AFM probe and bacterium are accounted for and mechanical interactions operating after contact are described in terms of Hertz-Hooke formalism. Retraction force curves are analyzed on the basis of the Freely Jointed Chain model. For both algorithms, the quantitative reconstruction of force curves is based on the robust detection of critical points (jumps, changes of slope or changes of curvature) which mark the transitions between the various relevant interactions taking place between the AFM tip and the studied sample during approach and retraction. Once the key regions of separation distance and indentation are detected, the physical parameters describing the relevant interactions operating in these regions are extracted making use of regression procedure for fitting experiments to theory. The flexibility, accuracy and strength of the algorithms are illustrated with the processing of two force-volume images, which collect a large set of approach and retraction curves measured on a single biological surface. For each force-volume image, several maps are generated, representing the spatial distribution of the searched physical parameters as estimated for each pixel of the force-volume image

Atomic force microscopy studies of bioprocess engineering surfaces - imaging, interactions and mechanical properties mediating bacterial adhesion

The detrimental effect of bacterial biofilms on process engineering surfaces is well documented. Thus, interest in the early stages of bacterial biofilm formation; in particular bacterial adhesion and the production of anti-fouling coatings has grown exponentially as a field. During this time, Atomic force microscopy (AFM) has become an essential tool for the evaluation of bacterial adhesion. Due to its versatility AFM offers not only insight into the topographical landscape and mechanical properties of the engineering surfaces, but elucidates, through direct quantification the topographical and biomechnical properties of the foulants The aim of this paper is to collate the current research on bacterial adhesion, both theoretical and practical, and outline how AFM as a technique is uniquely equipped to provide further insight into the nanoscale world at the bioprocess engineering surface

Enhancing adipose stem cell chondrogenesis: A study on the roles of dexamethasone, transforming growth factor β3 and ascorbate supplements and their combination

Varied exogenous chondrogenic factors (CFs) are implicated in promoting differentiation of stem cells along a chondrocyte lineage in the field of regenerative tissue engineering for articular cartilage repair. The effects of dexamethasone, transforming growth factor &beta;3 (TGF-&beta;3), ascorbate, and their combinations, on mRNA expression in micromass-cultured human adipose derived stem cells (hADSCs) were investigated as a function of time. Indices include chondrogenic, hypertrophic, angiogenic, fibrogenic and osteogenic markers along with mechanical properties, assessed by atomic force microscopy. Early in the culture, i.e., at day three, no significant differences in mRNA expression of SOX9, aggrecan, lubricin, Col XI, Col X, vascular endothelial growth factor, Col I, and alkaline phosphatase were observed among samples treated with different CFs. However, significant differences in mRNA expression levels of pre-mentioned markers among samples treated with each CF exist when samples were supplied with the CFs for more than three days. A new indexing scheme summing expression of chondrogenic and subtracting non-chondrogenic angiogenic, fibrogenic and osteogenic marker levels shows dexamethasone is the overall leading CF among the factors and their combinations. Based on this scheme, we have projected not only the possible signaling pathways which might be affected by addition of CFs but also hypothetical indexes that may occur upon temporal variation of growth factor regimens