Notes: Endoplasmic reticulum (ER) and mitochondrial contacts (EMC) allow for cellular communication related to metabolite exchange, apoptosis and intercellular signaling. Dysregulation of EMCs and associated signaling has been linked to neurodegenerative diseases. Here, engineered ascorbate peroxidase (APEX) is utilized to map the EMC proteome in live HEK293 cells. Additionally, a split Renilla luciferase assay was used to monitor ER and mitochondria interactions with the EnduRen™ Live Cell Substrate. (5117)

Notes: A novel maturation-inhibitor (MI), GSK3532795, is characterized for activity against clinical isolates with Gag and PR mutations associated with protease-inhibitor (PI) resistance. A pro-viral plasmid containing Rluc was used to determine viral susceptibility to GSK3532795 with the EnduRen™ Live Cell Substrate. Clinical isolates from patients with PI-resistance did not show decreased sensitivity to GSK3532795, thereby supporting the continued development of GSK3532795 as a treatment for HIV and AIDS. (5115)

Notes: A cocrystal structure of novel therapeutic, JNJ-53718678, for respiratory syncytial virus is presented bound to the respiratory syncytial virus fusion (F) protein (RSVF). Treatment of infected lambs and rodents with JNJ-53718678 lead to efficient inhibition of infection. Additionally, the antiviral activity of JNJ-53718678 against an alternative virus, dengue virus type 2 (DENV-2), was tested using the EnduRen™ Live Cell Substrate. (5116)

Notes: The authors constructed red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) for ratiometric imaging of protein-protein interactions (PPIs) in cell culture and deep tissues of small animals. These BRET systems consist of Renilla reniformis luciferase (RLuc) variants, RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. They used the EnduRen™ Live Cell Substrate in their BRET systems to produce a red-shift emission maxima optimal for deep-tissue imaging. To demonstrate this capability, the authors imaged HT-1080 cells accumulating in the lungs of nude mice. The cells expressed BRET fusion proteins in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. Mice were injected with luciferase substrate at 1.5 hours after cell injection to produce a bioluminescence image. Their data suggest that the BRET systems could be used for drug screening and target validation applications. (4130)

Notes: In this study, the Renilla luciferase gene from the phRL-CMV Vector was incorporated into polioviral RNA in place of a structural protein-coding region. Renilla luciferase activity was then monitored as a convenient indicator of viral replication. Specifically, these authors tested whether the guanine nucleotide exchange factors GBF1 and BIG2 could rescue brefeldin A (BFA)-induced inhibition of polioviral replication in HeLa cells. Cells were transfected with plasmids encoding either GBF1 or BIG2, together with the firefly-luciferase-encoding pGL4.13 Vector, which was used as a control to monitor transfection efficiency. Eighteen hours post-transfection, the cells were transfected with polioviral replicon RNA containing the Renilla luciferase gene. One hour later, normal growth medium containing EnduRen^® Live Cell Substrate and either BFA or solvent alone, was added and Renilla luciferase activity was monitored at hourly intervals for 7 hours. Cells were then lysed and firefly luciferase activity was measured to assess transfection efficiency using the Dual-Glo^® Luciferase Assay System System. In the presence of BFA, GBF1 was able to partially rescue viral replication. In the presence of BIG2 and BFA, no viral replication occurred. (3562)

Notes: Researchers created a recombinant infectious hematopoietic necrosis virus (IHNV_LUC) that contained the Renilla luciferase gene. The IHNV_LUC construct was tested for its ability to infect and kill rainbow trout compared to other IHNV constructs. In other studies, the EnduRen™ Live Cell Substrate was used to monitor points of infection in live fish. It was found that Renilla luciferase could be detected within four days after trout were exposed to 5 x 10⁴ PFU/ml of the IHNV_LUC construct. Areas behind the fins were demonstrated to points of viral entry and infection. (3385)

Notes: Type 1 angiotensin II receptor-Renilla luciferase (AT1R-Rluc), and β-arrestin1 and 2 GFP  fusion constructs (βarr1-GFP and βarr2-GFP) were created for BRET protein interaction assays. Combinations of AT1R-Rluc and β-arrestin-GFP constructs were transfected into COS-7 cells. The COS-7 cell cultures were then activated with 100nM angiotensin II in the presence of 60μM EnduRen™ Live Cell Substrate, and BRET fluorescence readings were taken at 475 and 515 nm over a 1 hour period. The authors also describe analysis of helix I mutants of β-arrestin1 and β-arrestin2 in similar β-arrestin-GFP construct BRET studies.  Data are displayed as a ratio of fluorescence readings with both constructs compared to fluorescence from the AT1R-Rluc construct alone.  (3256)