Cuccarolo P.,Istituto Nazionale per la Ricerca sul Cancro Centro Biotecnologie Avanzate |
Cuccarolo P.,University of Genoa |
Viaggi S.,University of Genoa |
Viaggi S.,Advanced Diagnostic Technologies LLC |
Degan P.,Istituto Nazionale per la Ricerca sul Cancro Centro Biotecnologie Avanzate
Fanconi's anemia (FA) patients face severe pathological consequences. Bone marrow failure, the major cause of death in FA, accounting for as much as 80-90% of FA mortality, appears to be significantly linked to excessive apoptosis of hematopoietic cells induced by oxidative stress. However, 20-25% of FA patients develop malignancies of myeloid origin. A survival strategy for bone marrow and hematopoietic cells under selective pressure evidently exists. This study reports that lymphoblastoid cell lines derived from two FA patients displayed significant resistance to oxidative stress induced by treatments with H 2O2 and various glutathione (GSH) inhibitors that induce production of reactive oxygen species, GSH depletion and mitochondrial membrane depolarization. Among the various GSH inhibitors employed, FA cells appear particularly resistant to menadione (5 μm) and ethacrynic acid (ETA, 50 μm), two drugs that specifically target mitochondria. Even after pre-treatment with buthionine sulfoximine, a GSH synthesis inhibitor that induces enhanced induction of reactive oxygen species, FA cells maintain significant resistance to these drugs. These data suggest that the resistance to oxidative stress and the altered mitochondrial and metabolic functionality found in the FA mutant cells used in this study may indicate the survival strategy that is adopted in FA cells undergoing transformation. The study of redox and mitochondria regulation in FA may be of assistance in diagnosis of the disease and in the care of patients. © 2012 FEBS. Source
Ahlford A.,Uppsala University |
Kjeldsen B.,Technical University of Denmark |
Reimers J.,Technical University of Denmark |
Lundmark A.,Uppsala University |
And 4 more authors.
We present an optimized procedure for freeze-drying and storing reagents for multiplex PCR followed by genotyping using a tag-array minisequencing assay with four color fluorescence detection which is suitable for microfluidic assay formats. A test panel was established for five cancer mutations in three codons (175, 248 and 273) of the tumor protein gene (TP53) and for 13 common single nucleotide polymorphisms (SNPs) in the TP53 gene. The activity of DNA polymerase was preserved for six months of storage after freeze-drying, and the half-life of activities of exonuclease I and shrimp alkaline phosphatase were estimated to 55 and 200 days, respectively. We conducted a systematic genotyping comparison using freeze-dried and liquid reagents. The accuracy of successful genotyping was 99.1% using freeze-dried reagents compared to liquid reagents. As a proof of concept, the genotyping protocol was carried out with freeze-dried reagents stored in reaction chambers fabricated by micromilling in a cyclic olefin copolymer substrate. The results reported in this study are a key step towards the development of an integrated microfluidic device for point-of-care DNA-based diagnostics. © 2010 The Royal Society of Chemistry. Source
Lencioni R.,Advanced Diagnostic Technologies LLC
Tumor cells express vascular endothelial growth factor (VEGF) that can activate VEGF receptors (VEGFRs) on or within tumor cells to promote growth in an angiogenesis-independent fashion; however, this autocrine VEGF pathway has not been reported in hepatocellular carcinoma (HCC). Sorafenib, an angiogenic inhibitor, is the only drug approved for use in advanced HCC patients. Yet the treatment efficacy is diverse and the mechanism behind it remains undetermined. Our aims were to study the molecular mechanisms underlying autocrine VEGF signaling in HCC cells and evaluate the critical role of autocrine VEGF signaling on sorafenib treatment efficacy. By immunohistochemistry, we found robust nuclear and cytoplasmic staining for active, phosphorylated VEGF receptor 1 (pVEGFR1) and phosphorylated VEGF receptor 2 (pVEGFR2), and by western blotting we found that membrane VEGFR1 and VEGFR2 increased in HCC tissues. We showed that autocrine VEGF promoted phosphorylation of VEGFR1 and VEGFR2 and internalization of pVEGFR2 in HCC cells, which was both pro-proliferative through a protein lipase C-extracellular kinase pathway and self-sustaining through increasing VEGF, VEGFR1, and VEGFR2 mRNA expressions. In high VEGFR1/2-expressing HepG2 cells, sorafenib treatment inhibited cell proliferation, reduced VEGFR2 mRNA expression in vitro, and delayed xenograft tumor growth in vivo. These results were not found in low VEGFR1/2-expressing Hep3B cells. In an advanced HCC population on sorafenib treatment for postoperative recurrence, we found that the absence of VEGFR1 or VEGFR2 expression in resected tumor tissues before sorafenib treatment was associated with poorer overall survival. Conclusion: Autocrine VEGF signaling directly promotes HCC cell proliferation and affects the sorafenib treatment outcome in vitro and in vivo, which may enable better stratification for clinical treatment decisions. © 2014 by the American Association for the Study of Liver Diseases. Source
Agency: Department of Defense | Branch: Defense Health Program | Program: SBIR | Phase: Phase II | Award Amount: 717.46K | Year: 2014
Surgical training requires an understanding of the human body"s complex 3-dimensional anatomical structures and the spatial relationships of nerves, blood vessels and other vital structures. Learning these spatial skills requires a lengthy training period and much practice that usually takes place in the operating room while under the supervision of a senior surgeon at great financial cost and potential risk to patients. Alternatively, there exist expensive models that may be used before a student can master the necessary skills. Advanced Diagnostic Technologies (ADxT) has developed a novel, low cost, 3D printer for synthetic tissue deposition. This 3D printing tool is useful for a wide range of applications including surgery simulation, endoscopic and intravascular procedures, as well as clinical task training, basic medical skills development, medical device design verification or visualization.
Agency: National Aeronautics and Space Administration | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 99.98K | Year: 2011
NASA has a serious unmet need for miniature biologic sensors capable of on orbit sample analysis and in situ, real time analysis of astronaut health. We propose an alternative way to monitor the health or illness of the crew members that would provide continuous monitoring of blood analytes. This information can be transmitted back to ground control in real time. The method could bypass the need to preserve biological specimens. This novel multivariate system would measure the optical rotation of laser polarized light reflected from human skin as well as scattering and absorption characteristics directly related to the concentration of certain blood analytes that reliably indicate the state of health of the individual.