Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy

International audienceWe demonstrate that simultaneous second-harmonic generation (SHG) and two-photon-excited fluorescence (TPEF) can be used to rapidly image biological membranes labeled with a styryl dye. The SHG power is made compatible with the TPEF power by use of near-resonance excitation, in accord with a model based on the theory of phased-array antennas, which shows that the SHG radiation is highly structured. Because of its sensitivity to local asymmetry, SHG microscopy promises to be a powerful tool for the study of membrane dynamics

G9a/GLP targeting in MM promotes autophagy-associated apoptosis and boosts proteasome inhibitor-mediated cell death

Multiple myeloma (MM) is an (epi)genetic highly heterogeneous plasma cell malignancy that remains mostly incurable. Deregulated expression and/or genetic defects in epigenetic-modifying enzymes contribute to high-risk disease and MM progression. Overexpression of the histone methyltransferase G9a was reported in several cancers, including MM, correlating with disease progression, metastasis, and poor prognosis. However, the exact role of G9a and its interaction partner G9a-like protein (GLP) in MM biology and the underlying mechanisms of action remain poorly understood. Here, we report that high G9a RNA levels are associated with a worse disease outcome in newly diagnosed and relapsed MM patients. G9a/GLP targeting using the specific G9a/GLP inhibitors BIX01294 and UNC0638 induces a G 1 -phase arrest and apoptosis in MM cell lines and reduces primary MM cell viability. Mechanistic studies revealed that G9a/GLP targeting promotes autophagy-associated apoptosis by inactivating the mTOR/4EBP1 pathway and reducing c-MYC levels. Moreover, genes deregulated by G9a/GLP targeting are associated with repressive histone marks. G9a/GLP targeting sensitizes MM cells to the proteasome inhibitors (PIs) bortezomib and carfilzomib, by (further) reducing mTOR signaling and c-MYC levels and activating p-38 and SAPK/JNK signaling. Therapeutic treatment of STGM1 mice with BIX01294 delayed in vivo MM tumor growth, and cotreatment with bortezomib resulted in a further reduction in tumor burden and a significantly prolonged survival. In conclusion, we provide evidence that the histone methyltransferases G9a/GLP support MM cell growth and survival by blocking basal autophagy and sustaining high c-MYC levels. G9a/GLP targeting represents a promising strategy to improve PI-based treatment in patients with high G9a/GLP levels

Maternal embryonic leucine zipper kinase is a novel target for diffuse large B cell lymphoma and mantle cell lymphoma

Diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are among the most aggressive B cell non-Hodgkin lymphomas. Maternal embryonic leucine zipper kinase (MELK) plays a role in cancer cell cycle progression and is associated with poor prognosis in several cancer cell types. In this study, the role of MELK in DLBCL and MCL and the therapeutic potential of MELK targeting is evaluated. MELK is highly expressed in DLBCL and MCL patient samples, correlating with a worse clinical outcome in DLBCL. Targeting MELK, using the small molecule OTSSP167, impaired cell growth and survival and induced caspase-mediated apoptosis in the lymphoma cells. Western blot analysis revealed that MELK targeting decreased the phosphorylation of FOXM1 and the protein levels of EZH2 and several mitotic regulators, such as Cdc25B, cyclin B1, Plk-1, and Aurora kinases. In addition, OTSSP167 also sensitized the lymphoma cells to the clinically relevant Bcl-2 inhibitor venetoclax by strongly reducing Mcl1 levels. Finally, OTSSP167 treatment of A20-inoculated mice resulted in a significant prolonged survival. In conclusion, targeting MELK with OTSSP167 induced strong anti-lymphoma activity both in vitro and in vivo. These findings suggest that MELK could be a potential new target in these aggressive B cell malignancies

The International DORIS Service (IDS) - Recent Developments in Preparation for ITRF2013

The International DORIS Service (IDS) was created in 2003 under the umbrella of the International Association of Geodesy (IAG) to foster scientific research related to the French DORIS tracking system and to deliver scientific products, mostly related to the International Earth rotation and Reference systems Service (IERS). We first present some general background related to the DORIS system (current and planned satellites, current tracking network and expected evolution) and to the general IDS organization (from Data Centers, Analysis Centers and Combination Center). Then, we discuss some of the steps recently taken to prepare the IDS submission to ITRF2013 (combined weekly time series based on individual solutions from several Analysis Centers). In particular, recent results obtained from the Analysis Centers and the Combination Center show that improvements can still be made when updating physical models of some DORIS satellites, such as Envisat, Cryosat-2 or Jason-2. The DORIS contribution to ITRF2013 should also benefit from the larger number of ground observations collected by the last generation of DGXX receivers (first instrument being onboard Jason-2 satellite). In particular for polar motion, sub-millarcsecond accuracy seems now to be achievable. Weekly station positioning internal consistency also seems to be improved with a larger DORIS constellation

The anaphase-promoting complex/cyclosome : a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma

BACKGROUND: The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL. METHODS: The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot. RESULTS: We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax. CONCLUSION: We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL

Clinical Correlations of Polycomb Repressive Complex 2 in Different Tumor Types

PRC2 (Polycomb repressive complex 2) is an evolutionarily conserved protein complex required to maintain transcriptional repression. The core PRC2 complex includes EZH2, SUZ12, and EED proteins and methylates histone H3K27. PRC2 is known to contribute to carcinogenesis and several small molecule inhibitors targeting PRC2 have been developed. The present study aimed to identify the cancer types in which PRC2 targeting drugs could be beneficial. We queried genomic and transcriptomic (cBioPortal, KMplot) database portals of clinical tumor samples to evaluate clinical correlations of PRC2 subunit genes. EZH2, SUZ12, and EED gene amplification was most frequently found in prostate cancer, whereas lymphoid malignancies (DLBCL) frequently showed EZH2 mutations. In both cases, PRC2 alterations were associated with poor prognosis. Moreover, higher expression of PRC2 subunits was correlated with poor survival in renal and liver cancers as well as gliomas. Finally, we generated a Python application to analyze the correlation of EZH2/SUZ12/EED gene knockouts by CRISPR with the alterations detected in the cancer cell lines using DepMap data. As a result, we were able to identify mutations that correlated significantly with tumor cell sensitivity to PRC2 knockout, including SWI/SNF, COMPASS/COMPASS-like subunits and BCL2, warranting the investigation of these genes as potential markers of sensitivity to PRC2-targeting drugs

Membrane imaging by second-harmonic generation microscopy

International audienceWe present a detailed analysis of the generation of second-harmonic radiation from biological membranes labeled with a styryl dye. In particular, we consider the high-numerical-aperture limit appropriate to high-resolution microscopy in which an excitation beam is tightly focused from the side onto a membrane surface. In this limit the active surface area that contributes to second-harmonic generation (SHG) depends only on the tightness of the beam focus and the SHG radiation is confined by phase matching into two well-defined off-axis lobes. We derive expressions for the SHG radiation power, angular distribution, and polarization dependence in the cases of ideal or nonideal molecular alignment in the membrane and uniaxiality of the molecular hyperpolarizability. We define an SHG cross section similar to that used in two-photon-excited fluorescence (TPEF) to permit direct comparison of the two imaging modalities. Finally, we corroborate our results with experiments based on the excitation of a styryl dye in giant unilamellar vesicles with a mode-locked Ti:sapphire laser