Non-invasive In Vivo Brain Astrogenesis and Astrogliosis Quantification Using a Far-red E2-Crimson Transgenic Reporter Mouse.

Despite the adaptation of major clinical imaging modalities for small animals, optical bioluminescence imaging technology is the main approach readily reporting gene activity. Yet, in vivo bioluminescence monitoring requires the administration and diffusion of a substrate to the tissues of interest, resulting in experimental variability, high reagent cost, long acquisition time, and stress to the animal. In our study, we avoid such issues upon generating a new transgenic mouse (GFAP-E2crimson) expressing the far-red fluorescent protein E2-crimson under the control of the glial fibrillary acidic protein (GFAP) promoter. Using microscopy, we validated the selective expression of the reporter in the astrocyte cell population and by non-invasive in vivo fluorescence imaging its detection through the scalps and skulls of live animals. In addition, we performed a longitudinal study validating by in vivo imaging that the E2-crimson fluorescence signal is up-regulated, in pups during astrogenesis and in adult mice during astrogliosis upon kainic acid administration. Furthermore, upon crossing GFAP-E2crimson transgenic with 5XFAD Alzheimer's disease mice model, we were able to quantify the chronic inflammation triggered by amyloid deposit and aging over 18 months. As many diseases and conditions can trigger neuroinflammation, we believe that the GFAP-E2crimson reporter mice model delivers tremendous value for the non-invasive quantification of astrogliosis responses in living animals.

Biolum' RGB: A Low-Cost, Versatile, and Sensitive Bioluminescence Imaging Instrument for a Broad Range of Users.

Luminometer and imaging systems are used to detect and quantify low light produced by a broad range of bioluminescent proteins. Despite their everyday use in research, such instruments are costly and lack the flexibility to accommodate the variety of bioluminescence experiment formats that may require top or bottom signal acquisition, high or medium sensitivity, or multiple wavelength detection. To address the growing need for versatile technologies, we developed a highly customizable bioluminescence imager called Biolum' RGB that uses a consumer color digital camera with a high-aperture lens mounted at the bottom or top of a 3D-printed dark chamber and can quantify bioluminescence emission from cells grown in 384-well microplates and Petri dishes. Taking advantage of RGB detectors, Biolum' RGB can distinguish spectral signatures from various bioluminescence probes and quantify bioluminescence resonant energy transfer occurring during protein-protein interaction events. Although Biolum' RGB can be used with any smartphone, in particular for low bioluminescence signals, we recommend the use of recent digital cameras which offer better sensitivity and high signal/noise ratio. Altogether, Biolum' RGB combines the benefits of a plate reader and imager while providing better image resolution and faster acquisition speed, and as such, it offers an exciting alternative for any laboratory looking for a versatile, low-cost bioluminescence imaging instrument.

Optimization of BRET saturation assays for robust and sensitive cytosolic protein-protein interaction studies.

Bioluminescence resonance energy transfer (BRET) saturation is a method of studying protein-protein interaction (PPI) upon quantification of the dependence of the BRET signal on the acceptor/donor (A:D) expression ratio. In this study, using the very bright Nluc/YFP BRET pair acquired respectively with microplate reader and automated confocal microscopy, we significantly improved BRET saturation assay by extending A:D expression detection range and normalizing A:D expression with a new BRET-free probe. We next found that upon using variable instead of fixed amount of donor molecules co-expressed with increasing acceptor concentrations, BRET saturation assay robustness can be further improved when studying cytosolic protein, although the relative amounts of dimers (BRETmax) and the relative dimer affinity (BRET50) remain similar. Altogether, we show that our method can be applied to many PPI networks, involving the NF-κB pathway, high-affinity nanobody, rabies virus-host interactions, mTOR complex and JAK/STAT signaling. Altogether our approach paves the way for robust PPI validation and characterization in living cells.

Proteomic Analysis Uncovers Measles Virus Protein C Interaction With p65-iASPP Protein Complex.

Viruses manipulate the central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two nonstructural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host-interacting proteins are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host-interacting proteins of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines, we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein interaction partners of MV-C were determined by applying protein complementation assay and the bioluminescence resonance energy transfer approach. As a result, we found that MV-C protein specifically interacts with p65-iASPP protein complex that controls both cell death and innate immunity pathways and evaluated the significance of these host factors on virus replication.

Regulation of NF-κB by the p105-ABIN2-TPL2 complex and RelAp43 during rabies virus infection.

At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBß-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNß, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling.

Activation-Induced Cytidine Deaminase Induces DNA Demethylation of Pluripotency Genes in Bovine Differentiated Cells.

Activation-induced cytidine deaminase (AID) is the only enzyme that has been suggested as a putative DNA demethylase in mammals. However, very little is known about AID function as DNA demethylase of bovine differentiated cells toward pluripotent state. To investigate the effect of AID on DNA demethylation, bovine AID complementary DNAs were transfected into bovine differentiated cells, which were mostly methylated in the promoter regions of pluripotency genes. As a result, AID-transfected bovine cells started to transform into colonies at day 19 of transfection. The colonies derived from the transfected cells showed positive alkaline phosphatase (AP) staining and expression of pluripotency genes (OCT-3/4, NANOG, SOX2) and pluripotency-related antigens (SSEA-4, TRA1-60, TRA1-81), which have been widely used to characterize human embryonic stem cells. In particular, the levels of OCT-3/4 and NANOG expression were significantly increased in the AID-transfected cells when compared with the control and empty vector-transfected cells (p < 0.05). Finally, DNA demethylation in the promoter regions of pluripotency genes (OCT-3/4, NANOG) was significantly increased compared with the control (p < 0.05). These results demonstrate that the induction of the AID gene into bovine differentiated cells improves DNA demethylation and expression of pluripotency genes.

Siberian Sturgeon Oocyte Extract Induces Epigenetic Modifications of Porcine Somatic Cells and Improves Developmental Competence of SCNT Embryos.

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with 4 µg/mL of digitonin for 2 min at 4°C in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at 18°C was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.