550 research outputs found

    THE ROLE OF NOTCH PATHWAY IN MULTIPLE MYELOMA-ASSOCIATED DRUG RESISTANCE

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    BACKGROUND: Multiple myeloma (MM) is an incurable hematological malignancy characterized by drug resistance, intrinsic or induced by bone marrow stromal cells (BMSCs). In MM cells, Notch pathway may be aberrantly activated due to the hyperexpression of Notch1, Notch2 or Jagged1 and 2 ligands. This effect may be attributed to genetic mutation only in part (i.e. translocations involving the MAF transcription factors may increase the transcriptional activity on their target gene Notch2). MM cells settle in the bone marrow (BM) and the BM microenvironment may be another player contributing to Notch signaling activation by triggering Notch receptors through BMSC-derived ligands or other stimuli including hypoxia. Indeed, recent evidences indicate that both hypoxic stimuli and Notch signaling activation are involved in cancer stem cell maintenance and self-renewal, thereby contributing to drug resistance due to the resilience of this cancer subpopulation. MM stem cells (MM-SCs) have been characterized as a CD138- subpopulation. AIM: The aim of this study was to investigate the outcome of Notch signaling hyper-activation in intrinsic and BMSC-mediated drug resistance in MM cells and MM-SCs. METHODS: I assessed the effect of Jagged ligands by an inhibitory approach on MM cells. This was carried out by silencing Jagged1 and 2 through specific siRNAs or lentivirally expressed shRNAs. The study got advantage of OPM2 and U266 cell lines and MM primary cells from 10 patients. MM cells were cultured alone, to assess the effect of Jagged silencing on intrinsic drug resistance, or co-cultured with BMSCs, to investigate the effect of Jagged inhibition on BMSC-mediated drug resistance. The BMSC models used were: i) the human HS5GFP+ cell line that, when cultured with MM cell lines, enabled a flow cytometric analysis of variations in drug resistance and anti-apoptotic proteins expressed by MM cells, along with changes in BMSC-production of pro-tumor cytokines (i.e. IL-6 and SDF-1\uf061); ii) the murine fibroblasts NIH3T3. These cell lines mimic BMSCs and, when cultured with MM cell lines, enabled to confirm changes in key proteins by gene expression analysis through RT-PCR using species-specific primers to distinguish the contribution of MM cells (human) or NIH3T3 cells (murine); iii) experiments on primary CD138- MM cells were carried out using primary BMSCs stained with PKH26 as feeder cells. Intrinsic and BMSC-induced drug resistance was analyzed by challenging MM cells cultured alone or in co-culture systems with standard-of care drugs, i.e. Bortezomib. Apoptosis was assessed by detection of the percentage of AnnexinV+ cells through flow cytometry. Hypoxic BM microenvironment was mimicked by using cobalt chloride (CoCl2), while Notch pathway activation was inhibited using DAPT (a \u3b3-secretase inhibitor). MM-SCs were analyzed in H929 cell line by flow cytometric analysis of the CD138- subpopulation. The effect of hypoxia on protein expression changes of Notch pathway members (i.e. Notch2 and Jagged1) was assessed by Western blot assay, while changes of Notch transactivation activity were assessed by dual luciferase Notch reporter assay in OPM2 cells and HEK293 cells. The high transfectability level of HEK293 cell line also enabled its transfection with multiple plasmids to assess the specific effect of CoCl2 treatment on the transcriptional activity of Notch1 and Notch2. RESULTS: The results of this work demonstrate that Jagged1 and 2 increased expression levels affect MM cell biology maintaining high levels of intrinsic drug resistance through the expression of anti-apoptotic genes, i.e. BCL2, Survivin and ABCC1, BCLXL, SDF-1\u3b1, CXCR4, with the consequent increase of MM cell sensitivity to standard-of-care drugs. Concerning the interaction of MM cells and BMSCs, MM cells stimulate the protective behavior of BMSCs, by inducing Notch activation through tumor-derived Jagged1 and 2, with a consequent increase of drug resistance due to: i) release of pro-tumor soluble factors by BMSCs, i.e. SDF-1\u3b1 and VEGF; ii) the induction of an elevated anti-apoptotic background in MM cells due to an increased expression of anti-apoptotic genes such as BCL2, Survivin and ABCC1. In vitro results were confirmed by co-cultures of primary MM cells. Finally, I verified that an hypoxic stimulus, mimicked by CoCl2, may be a cause of Notch activation in MM cells by increasing the transcriptional activity of Notch1 and Notch2, supposedly through interaction with HIF-1\u3b1 that prevents ICN proteosomal degradation. Notch signaling activated by CoCl2 positively regulates the MM-SC population. The resilience that characterizes MM-SCs suggests that hypoxia-mediated activation of Notch signaling may be a further mechanism by which the BM microenvironment may induce the acquisition of drug resistance in MM. CONCLUSION: The evidences that Jagged1 and 2 silencing affects the intrinsic and acquired drug resistance in MM cells support the rationale for a Notch-tailored approach to overcome MM patients relapse

    Electrochemically Generated Luminescence of Luminol and Luciferin in Ionic Liquids

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    Electrochemiluminescence (ECL) is the generation of light triggered by an electrochemical reaction. ECL has been extensively studied in solvent-based electrolytes, but there is a lack of data on using electrode reactions to populate an excited-state light emitter in room temperature ionic liquids (RTILs). This work explores the current response, light intensity (photon counting), and spectral signatures of the cathodic ECL of luminol and firefly's luciferin in imidazolium-based RTILs. We have demonstrated that the cathodic (superoxide-triggered) ECL of both luminol and adenylate-ester of firefly's luciferin is viable in RTILs, explored the effect of water contaminations, and importantly, shown that the ECL signal persists for up to about 700 s after the removal of the external cathodic pulse, which is probably due to the stabilization of superoxide by double-layer cation-rich structures. Long-lived RTIL double-layer structures and their endogenous fields are detected as stable and discrete open-circuit potential plateaus

    Coupled Electronic and Nuclear Motions during Azobenzene Photoisomerization Monitored by Ultrafast Electron Diffraction

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    Ultrafast electron diffraction is a powerful technique that can resolve molecular structures with femtosecond and angstrom resolutions. We demonstrate theoretically how it can be used to monitor conical intersection dynamics in molecules. Specific contributions to the signal are identified which vanish in the absence of vibronic coherence and offer a direct window into conical intersection paths. A special focus is on hybrid scattering from nuclei and electrons, a process that is unique to electron (rather than X-ray) diffraction and monitors the strongly coupled nuclear and electronic motions in the vicinity of conical intersections. An application is made to the cis to trans isomerization of azobenzene, computed with exact quantum dynamics wavepacket propagation in a reactive two-dimensional nuclear space

    Assessment of Mesophotic Coral Ecosystem Connectivity for Proposed Expansion of a Marine Sanctuary in the Northwest Gulf of Mexico: Larval Dynamics

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    In coral reef ecosystems, mesophotic coral habitat (\u3e30 m to the end of the photic zone) are extensions of shallow reefs and contribute to the persistence of coral reef populations. In the North West Gulf of Mexico (NW GOM), the Flower Garden Banks National Marine Sanctuary (FGBNMS) is an isolated reef ecosystem comprising contiguous shallow and mesophotic reefs habitats on two central banks along the margin of the continental shelf. A future expansion of the sanctuary is proposed to include additional mesophotic banks and aims at building a network of protected areas in the NW GOM to ensure the persistence of the coral reef populations inhabiting the sanctuary. To evaluate the feasibility of this expansion and investigate the overall dynamics of coral species in the region, we studied the patterns of larval connectivity of Montastraea cavernosa, a common depth generalist coral species, using a larval dispersal modeling approach. Our results highlighted larval exports from the NW GOM banks to the northeastern and southwestern GOM, larval connectivity between all banks investigated in this study, and the potential for exporting larvae from mesophotic to shallower reefs. Our study associated with Studivan and Voss (2018; associate manuscript) demonstrates the relevance of combining modeling and genetic methods to consider both demographic and genetic timescales for the evaluation of the connectivity dynamics of marine populations. In the case of the NW GOM, both studies support the future management plan for expanding FGBNMS

    Spectroscopic fingerprints of DNA/RNA pyrimidine nucleobases in third-order nonlinear electronic spectra

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    Accurate ab initio modeling of spectroscopic signals in nonlinear electronic spectra, such as bidimensional (2D) spectra, requires the computation of the electronic transitions induced by the incoming pump/probe pulses, resulting in a challenging calculation of many electronic excited states. A protocol is thus required to evaluate the variations of spectral properties, like transition energies and dipole moments, with the computational level, and to estimate the sensitivity of the spectra to these variations. Such a protocol is presented here within the framework of complete and restricted active space self-consistent field (CASSCF/RASSCF) theory and its second-order perturbation theory extensions (CASPT2/RASPT2). The electronic excited-state manifolds of pyrimidine nucleobases (thymine, uracil, and cytosine) are carefully characterized in vacuo employing high-level RAS(0,0|10,8|2,12)//SS-RASPT2 calculations. The results provide a reference data set that can be used for optimizing computational efforts and costs, as required for studying computationally more demanding multichromophoric systems (e.g., di- and oligonucleotides). The spectroscopic signatures of the 2D electronic spectrum of a perfectly stacked uracil–cytosine dimer model are characterized, and experimental setups are proposed that can resolve non-covalent interchromophoric interactions in canonical pyrimidine nucleobase-stacked dimers

    Time-Resolved Optical Pump-Resonant X-ray Probe Spectroscopy of 4-Thiouracil: A Simulation Study

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    We theoretically monitor the photoinduced ∗ → n∗ internal conversion process in 4-thiouracil (4TU), triggered by an optical pump. The element-sensitive spectroscopic signatures are recorded by a resonant X-ray probe tuned to the sulfur, oxygen, or nitrogen K-edge. We employ high-level electronic structure methods optimized for core-excited electronic structure calculation combined with quantum nuclear wavepacket dynamics computed on two relevant nuclear modes, fully accounting for their quantum nature of nuclear motions. We critically discuss the capabilities and limitations of the resonant technique. For sulfur and nitrogen, we document a pre-edge spectral window free from ground-state background and rich with ∗ and n∗ absorption features. The lowest sulfur K-edge shows strong absorption for both ∗ and n*. In the lowest nitrogen K-edge window, we resolve a state-specific fingerprint of the ∗ and an approximate timing of the conical intersection via its depletion. A spectral signature of the n∗ transition, not accessible by UV-vis spectroscopy, is identified. The oxygen K-edge is not sensitive to molecular deformations and gives steady transient absorption features without spectral dynamics. The */n∗ coherence information is masked by more intense contributions from populations. Altogether, element-specific time-resolved resonant X-ray spectroscopy provides a detailed picture of the electronic excited-state dynamics and therefore a sensitive window into the photophysics of thiobases
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