Differential chemosensitivity to antifolate drugs between RAS and BRAF melanoma cells.

BACKGROUND: The importance of the genetic background of cancer cells for the individual susceptibility to cancer treatments is increasingly apparent. In melanoma, the existence of a BRAF mutation is a main predictor for successful BRAF-targeted therapy. However, despite initial successes with these therapies, patients relapse within a year and have to move on to other therapies. Moreover, patients harbouring a wild type BRAF gene (including 25% with NRAS mutations) still require alternative treatment such as chemotherapy. Multiple genetic parameters have been associated with response to chemotherapy, but despite their high frequency in melanoma nothing is known about the impact of BRAF or NRAS mutations on the response to chemotherapeutic agents. METHODS: Using cell proliferation and DNA methylation assays, FACS analysis and quantitative-RT-PCR we have characterised the response of a panel of NRAS and BRAF mutant melanoma cell lines to various chemotherapy drugs, amongst them dacarbazine (DTIC) and temozolomide (TMZ) and DNA synthesis inhibitors. RESULTS: Although both, DTIC and TMZ act as alkylating agents through the same intermediate, NRAS and BRAF mutant cells responded differentially only to DTIC. Further analysis revealed that the growth-inhibitory effects mediated by DTIC were rather due to interference with nucleotide salvaging, and that NRAS mutant melanoma cells exhibit higher activity of the nucleotide synthesis enzymes IMPDH and TK1. Importantly, the enhanced ability of RAS mutant cells to use nucleotide salvaging resulted in resistance to DHFR inhibitors. CONCLUSION: In summary, our data suggest that the genetic background in melanoma cells influences the response to inhibitors blocking de novo DNA synthesis, and that defining the RAS mutation status could be used to stratify patients for the use of antifolate drugs

Tyrosine photophysics during the early stages of β-amyloid aggregation leading to Alzheimer's

We have monitored the formation of toxic β-amyloid oligomers leading to Alzheimer's disease by detecting changes in the fluorescence decay of intrinsic tyrosine. A new approach based on the non-Debye model of fluorescence kinetics resolves the complexity of the underlying photophysics. The gradual disappearance of nonmonotonic fluorescence decay rates, at the early stages of aggregation as larger, tighter-packed oligomers are formed, is interpreted in terms of tyrosine-peptide dielectric relaxation influencing the decay. The results demonstrate the potential for a new type of fluorescence lifetime sensing based on dual excited-state/dielectric relaxation, with application across a broad range of biological molecules. The results also reconcile previously conflicting models of protein intrinsic fluorescence decay based on rotamers or dielectric relaxation by illustrating conditions under which both are manifest

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

<p>Abstract</p> <p>Background</p> <p>Melanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the <it>Xiphophorus </it>melanoma model system, a mutated version of the EGF receptor Xmrk (<it>Xiphophorus </it>melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation.</p> <p>Methods</p> <p>Here, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene <it>FOSL1 </it>was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated.</p> <p>Results</p> <p>Genes with the strongest upregulation after receptor activation were FOS-like antigen 1 (<it>Fosl1</it>), early growth response 1 (<it>Egr1</it>), osteopontin (<it>Opn</it>), insulin-like growth factor binding protein 3 (<it>Igfbp3</it>), dual-specificity phosphatase 4 (<it>Dusp4</it>), and tumor-associated antigen L6 (<it>Taal6</it>). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that <it>FOSL1</it>, <it>OPN</it>, <it>IGFBP3</it>, <it>DUSP4</it>, and <it>TAAL6 </it>also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of <it>FOSL1 </it>in human melanoma cell lines reduced their proliferation and migration.</p> <p>Conclusion</p> <p>Altogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.</p

Negative ion chemistry in Titan's upper atmosphere

International audienceThe Electron Spectrometer (ELS), one of the sensors making up the Cassini Plasma Spectrometer (CAPS) revealed the existence of numerous negative ions in Titan's upper atmosphere. The observations at closest approach (not, vert, similar1000 km) show evidence for negatively charged ions up to not, vert, similar10,000 amu/q, as well as two distinct peaks at 22±4 and 44±8 amu/q, and maybe a third one at 82±14 amu/q. We present the first ionospheric model of Titan including negative ion chemistry. We find that dissociative electron attachment to neutral molecules (mostly HCN) initiates the formation of negative ions. The negative charge is then transferred to more acidic molecules such as HC3N, HC5N or C4H2. Loss occurs through associative detachment with radicals (H and CH3). We attribute the three low mass peaks observed by ELS to CN−, C3N−/C4H− and C5N−. These species are the first intermediates in the formation of the even larger negative ions observed by ELS, which are most likely the precursors to the aerosols observed at lower altitudes

Photoelectrons in the Enceladus plume

The plume of Enceladus is a remarkable plasma environment containing several charged particle species. These include cold magnetospheric electrons, negative and positive water clusters, charged nanograins, and “magnetospheric photoelectrons” produced from ionization of neutrals throughout the magnetosphere near Enceladus. Here we discuss observations of a population newly identified by the Cassini Plasma Spectrometer (CAPS) electron spectrometer instrument—photoelectrons produced in the plume ionosphere itself. These were found during the E19 encounter, in the energetic particle shadow where penetrating particles are absent. Throughout E19, CAPS was oriented away from the ram direction where the clusters and nanograins are observed during other encounters. Plume photoelectrons are also clearly observed during the E9 encounter and are also seen at all other Enceladus encounters where electron spectra are available. This new population, warmer than the ambient plasma population, is distinct from, but adds to, the magnetospheric photoelectrons. Here we discuss the observations and examine the implications, including the ionization source these electrons provide

Osteoblasts contribute to a protective niche that supports melanoma cell proliferation and survival

Melanoma is the deadliest form of skin cancer; a primary driver of this high level of morbidity is the propensity of melanoma cells to metastasize. When malignant tumours develop distant metastatic lesions the new local tissue niche is known to impact on the biology of the cancer cells. However, little is known about how different metastatic tissue sites impact on frontline targeted therapies. Intriguingly, melanoma bone lesions have significantly lower response to BRAF or MEK inhibitor therapies. Here, we have investigated how the cellular niche of the bone can support melanoma cells by stimulating growth and survival via paracrine signalling between osteoblasts and cancer cells. Melanoma cells can enhance the differentiation of osteoblasts leading to increased production of secreted ligands, including RANKL. Differentiated osteoblasts in turn can support melanoma cell proliferation and survival via the secretion of RANKL that elevates the levels of the transcription factor MITF, even in the presence of BRAF inhibitor. By blocking RANKL signalling, either via neutralizing antibodies, genetic alterations or the RANKL receptor inhibitor SPD304, the survival advantage provided by osteoblasts could be overcome

An empirical approach to modeling ion production rates in Titan's ionosphere II: Ion production rates on the nightside

Ionization of neutrals by precipitating electrons and ions is the main source of Titan's nightside ionosphere. This paper has two goals: (1) characterization of the role of electron impact ionization on the nightside ionosphere for different magnetospheric conditions and (2) presentation of empirical ion production rates determined using densities measured by the Cassini Ion and Neutral Mass Spectrometer on the nightside. The ionosphere between 1000 and 1400 km is emphasized. We adopt electron fluxes measured by the Cassini Plasma Spectrometer-Electron Spectrometer and the Magnetospheric Imaging Instrument as classified by Rymer et al. (2009). The current paper follows an earlier paper (Paper I), in which we investigated sources of Titan's dayside ionosphere and demonstrated that the photoionization process is well understood. The current paper (Paper II) demonstrates that modeled and empirical ionization rates on the nightside are in agreement with an electron precipitation source above 1100 km. Ion production rate profiles appropriate for different Saturnian magnetospheric conditions, as outlined by Rymer et al., are constructed for various magnetic field topologies. Empirical production rate profiles are generated for deep nightside flybys of Titan. The results also suggest that at lower altitudes (below 1100 km) another source, such as ion precipitation, is probably needed

Quantum key distribution and 1 Gbit/s data encryption over a single fibre

We perform quantum key distribution (QKD) in the presence of 4 classical channels in a C-band dense wavelength division multiplexing (DWDM) configuration using a commercial QKD system. The classical channels are used for key distillation and 1 Gbps encrypted communication, rendering the entire system independent from any other communication channel than a single dedicated fibre. We successfully distil secret keys over fibre spans of up to 50 km. The separation between quantum channel and nearest classical channel is only 200 GHz, while the classical channels are all separated by 100 GHz. In addition to that we discuss possible improvements and alternative configurations, for instance whether it is advantageous to choose the quantum channel at 1310 nm or to opt for a pure C-band configuration.Comment: 9 pages, 7 figure

Probing beta amyloid aggregation using fluorescence anisotropy : experiments and simulation

The aggregation of beta amyloid (Ab) protein is associated with the development of Alzheimer's disease. In this work we monitor Ab aggregation using fluorescence anisotropy, a technique that provides information on the rotational diffusion of the fluorescing tyrosine (Tyr) side chains. We also perform Monte Carlo (MC) and fully atomistic Molecular Dynamics (MD) simulations to interpret the experiments. The experimental results show that there are two different rotational timescales contributing to the anisotropy. Our MC simulation captures this behaviour in a coarse-scale manner, and, more importantly, shows that the Tyr side chains must have their movements restricted in order to reproduce the anisotropy. The MD simulations provide a molecular scale view, and indeed show that aggregation restricts the Try side chains to yield anisotropy in line with the experimental results. This combination of experiment and simulation therefore provides a unique insight into the aggregation process, and we suggest how this approach might be used to gain further information on aggregating protein systems