Safe and stable generation of induced pluripotent stem cells using doggybone DNA vectors

The application of induced pluripotent stem cells (iPSCs) in advanced therapies is increasing at pace, but concerns remain over their clinical safety profile. We report the first-ever application of doggybone DNA (dbDNA) vectors to generate human iPSCs. dbDNA vectors are closed-capped linear double-stranded DNA gene expression cassettes that contain no bacterial DNA and are amplified by a chemically defined, current good manufacturing practice (cGMP)-compliant methodology. We achieved comparable iPSC reprogramming efficiencies using transiently expressing dbDNA vectors with the same iPSC reprogramming coding sequences as the state-of-the-art OriP/EBNA1 episomal vectors but, crucially, in the absence of p53 shRNA repression. Moreover, persistent expression of EBNA1 from bacterially derived episomes resulted in stimulation of the interferon response, elevated DNA damage, and increased spontaneous differentiation. These cellular activities were diminished or absent in dbDNA-iPSCs, resulting in lines with a greater stability and safety potential for cell therapy

LKB1/KRAS mutant lung cancers constitute a genetic subset of NSCLC with increased sensitivity to MAPK and mTOR signalling inhibition

LKB1/STK11 is a multitasking tumour suppressor kinase. Germline inactivating mutations of the gene are responsible for the Peutz-Jeghers hereditary cancer syndrome. It is also somatically inactivated in approximately 30% of non-small-cell lung cancer (NSCLC). Here, we report that LKB1/KRAS mutant NSCLC cell lines are sensitive to the MEK inhibitor CI-1040 shown by a dose-dependent reduction in proliferation rate, whereas LKB1 and KRAS mutations alone do not confer similar sensitivity. We show that this subset of NSCLC is also sensitised to the mTOR inhibitor rapamycin. Importantly, the data suggest that LKB1/KRAS mutant NSCLCs are a genetically and functionally distinct subset and further suggest that this subset of lung cancers might afford an opportunity for exploitation of anti-MAPK/mTOR-targeted therapies

Underground operation of the ICARUS T600 LAr-TPC: first results

Open questions are still present in fundamental Physics and Cosmology, like the nature of Dark Matter, the matter-antimatter asymmetry and the validity of the particle interaction Standard Model. Addressing these questions requires a new generation of massive particle detectors exploring the subatomic and astrophysical worlds. ICARUS T600 is the first large mass (760 ton) example of a novel detector generation able to combine the imaging capabilities of the old famous "bubble chamber" with an excellent energy measurement in huge electronic detectors. ICARUS T600 now operates at the Gran Sasso underground laboratory, studying cosmic rays, neutrino oscillation and proton decay. Physical potentialities of this novel telescope are presented through few examples of neutrino interactions reconstructed with unprecedented details. Detector design and early operation are also reported.Comment: 14 pages, 8 figures, 2 tables. Submitted to Jins

Anti-EGFR Antibody Efficiently and Specifically Inhibits Human TSC2−/− Smooth Muscle Cell Proliferation. Possible Treatment Options for TSC and LAM

BACKGROUND: Tuberous sclerosis complex (TSC), a tumor syndrome caused by mutations in TSC1 or TSC2 genes, is characterized by the development of hamartomas. We previously isolated, from an angiomyolipoma of a TSC2 patient, a homogenous population of smooth muscle-like cells (TSC2(-/-) ASM cells) that have a mutation in the TSC2 gene as well as TSC2 loss of heterozygosity (LOH) and consequently, do not produce the TSC2 gene product, tuberin. TSC2(-/-) ASM cell proliferation is EGF-dependent. METHODS AND FINDINGS: Effects of EGF on proliferation of TSC2(-/-) ASM cells and TSC2(-/-) ASM cells transfected with TSC2 gene were determined. In contrast to TSC2(-/-) ASM cells, growth of TSC2-transfected cells was not dependent on EGF. Moreover, phosphorylation of Akt, PTEN, Erk and S6 was significantly decreased. EGF is a proliferative factor of TSC2(-/-) ASM cells. Exposure of TSC2(-/-) ASM cells to anti-EGFR antibodies significantly inhibited their proliferation, reverted reactivity to HMB45 antibody, a marker of TSC2(-/-) cell phenotype, and inhibited constitutive phosphorylation of S6 and ERK. Exposure of TSC2(-/-) ASM cells to rapamycin reduced the proliferation rate, but only when added at plating time. Although rapamycin efficiently inhibited S6 phosphorylation, it was less efficient than anti-EGFR antibody in reverting HMB45 reactivity and blocking ERK phosphorylation. In TSC2(-/-) ASM cells specific PI3K inhibitors (e.g. LY294002, wortmannin) and Akt1 siRNA had little effect on S6 and ERK phosphorylation. Following TSC2-gene transfection, Akt inhibitor sensitivity was observed. CONCLUSION: Our results show that an EGF independent pathway is more important than that involving IGF-I for growth and survival of TSC(-/-) ASM cells, and such EGF-dependency is the result of the lack of tuberin

A three-drug nanoscale drug delivery system designed for preferential lymphatic uptake for the treatment of metastatic melanoma

Metastatic melanoma has a high mortality rate due to lymphatic progression of the disease. Current treatment is surgery followed by radiation and intravenous chemotherapy. However, drawbacks for current chemotherapeutics lie in the fact that they develop resistance and do not achieve therapeutic concentrations in the lymphatic system. We hypothesize that a three-drug nanoscale drug delivery system, tailored for lymphatic uptake, administered subcutaneously, will have decreased drug resistance and therefore offer better therapeutic outcomes. We prepared and characterized nanoparticles (NPs) with docetaxel, everolimus, and LY294002 in polyethyleneglycol-block-poly(ε-caprolactone) (PEG-PCL) polymer with different charge distributions by modifying the ratio of anionic and neutral end groups on the PEG block. These NPs are similarly sized (~48nm), with neutral, partially charged, or fully charged surface. The NPs are able to load ~2mg/mL of each drug and are stable for 24h. The NPs are assessed for safety and efficacy in two transgenic metastatic melanoma mouse models. All the NPs were safe in both models based on general appearance, weight changes, death, and blood biochemical analyses. The partially charged NPs are most effective in decreasing the number of melanocytes at both the proximal (sentinel) lymph node (LN) and the distal LN from the injection site. The neutral NPs are efficacious at the proximal LN, while the fully charged NPs have no effect on either LNs. Thus, our data indicates that the NP surface charge and lymphatic efficacy are closely tied to each other and the partially charged NPs have the highest potential in treating metastatic melanoma

The Signaling Petri Net-Based Simulator: A Non-Parametric Strategy for Characterizing the Dynamics of Cell-Specific Signaling Networks

Reconstructing cellular signaling networks and understanding how they work are major endeavors in cell biology. The scale and complexity of these networks, however, render their analysis using experimental biology approaches alone very challenging. As a result, computational methods have been developed and combined with experimental biology approaches, producing powerful tools for the analysis of these networks. These computational methods mostly fall on either end of a spectrum of model parameterization. On one end is a class of structural network analysis methods; these typically use the network connectivity alone to generate hypotheses about global properties. On the other end is a class of dynamic network analysis methods; these use, in addition to the connectivity, kinetic parameters of the biochemical reactions to predict the network's dynamic behavior. These predictions provide detailed insights into the properties that determine aspects of the network's structure and behavior. However, the difficulty of obtaining numerical values of kinetic parameters is widely recognized to limit the applicability of this latter class of methods

Integrating BRAF/MEK inhibitors into combination therapy for melanoma

The discovery of BRAF mutations in melanoma has not yet translated into clinical success, suggesting that BRAF/MEK inhibitors will need to be combined with other agents. In the current review, we discuss other pathways likely to be important for melanoma progression and suggest possible drug combinations for future clinical testing

Synergistic antitumour effects of rapamycin and oncolytic reovirus.

There are currently numerous oncolytic viruses undergoing clinical trial evaluation in cancer patients and one agent, Talimogene laherparepvec, has been approved for the treatment of malignant melanoma. This progress highlights the huge clinical potential of this treatment modality, and the focus is now combining these agents with conventional anticancer treatments or agents that enhance viral replication, and thereby oncolysis, in the tumour microenvironment. We evaluated the combination of reovirus with rapamycin in B16F10 cell, a murine model of malignant melanoma, based on potential mechanisms by which mTOR inhibitors might enhance viral oncolysis. Rapamycin was not immunomodulatory in that it had no effect on the generation of an antireovirus-neutralising antibody response in C57/black 6 mice. The cell cycle effects of reovirus (increase G0/G1 fraction) were unaffected by concomitant or sequential exposure of rapamycin. However, rapamycin attenuated viral replication if given prior or concomitantly with reovirus and similarly reduced reovirus-induced apoptotic cell death Annexin V/PI and caspase 3/7 activation studies. We found clear evidence of synergistic antitumour effects of the combination both in vitro and in vivo, which was sequence dependent only in the in vitro setting. In conclusion, we have demonstrated synergistic antitumour efficacy of reovirus and rapamycin combination