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Abstract Background Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC) to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Gag interactions in living cells by reconstitution of yellow fluorescent protein (YFP) from its N- or C-terminal fragments. Results The results obtained with catalytic domain 1 and 2 (CD1 and CD2) mutants indicate that A3G-A3G and A3G-Gag multimerization is dependent on an intact CD1 domain, which is required for RNA binding. A mutant HIV-1 Gag that exhibits reduced RNA binding also failed to reconstitute BiFC with wild-type A3G, indicating a requirement for both HIV-1 Gag and A3G to bind to RNA for their multimerization. Addition of a non-specific RNA binding peptide (P22) to the N-terminus of a CD1 mutant of A3G restored BiFC and virion incorporation, but failed to inhibit viral replication, indicating that the mutations in CD1 resulted in additional defects that interfere with A3G's antiviral activity. Conclusion These studies establish a robust BiFC assay for analysis of intracellular interactions of A3G with other macromolecules. The results indicate that in vivo A3G is a monomer that forms multimers upon binding to RNA. In addition, we observed weak interactions between wild-type A3G molecules and RNA binding-defective mutants of A3G, which could explain previously described protein-protein interactions between purified A3G molecules.

Purpose: Genomic profiling of biopsied tissue is the basis for precision cancer therapy. However, biopsied materials may not contain sufficient amounts of tumor deoxyribonucleonic acid needed for the analysis. We propose a method to determine the adequacy of specimens for performing genomic profiling by quantifying their metabolic activity. Methods: We estimated the average density of tumor cells in biopsy specimens needed to successfully perform genomic analysis following the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK‐IMPACT) protocol from the minimum amount of deoxyribonucleonic acid needed and the volume of tissue typically used for analysis. The average 18F‐FDG uptake per cell was assessed by incubating HT‐29 adenocarcinoma tumor cells in 18F‐FDG containing solution and then measuring their activity with a scintillation well counter. Consequently, we evaluated the response of two devices around the minimum expected activities which would indicate genomic profiling adequacy of biopsy specimens obtained under 18F‐FDG PET/CT guidance. Surrogate samples obtained using 18G core needle biopsies of gels containing either 18F‐FDG‐loaded cells in the expected concentrations or the corresponding activity were measured using autoradiography and a scintillation well counter. Autoradiography was performed using a CCD‐based device with real‐time image display as well as with digital autoradiography imaging plates following a 30‐min off‐line protocol for specimen activity determination against previously established calibration. Results: Cell incubation experiments and estimates obtained from quantitative autoradiography of biopsy specimens (QABS) indicate that specimens acquired under 18F‐FDG PET/CT guidance that contained the minimum amount of cells needed for genomic profiling would have an average activity concentration in the range of about 3 to about 9 kBq/mL. When exposed to specimens with similar activity concentration, both a CCD‐based autoradiography device and a scintillation well counter produced signals with sufficient signal‐to‐background ratio for specimen genomic adequacy identification in less than 10 min, which is short enough to allow procedure guidance. Conclusion: Scintillation well counter measurements and CCD‐based autoradiography have adequate sensitivity to detect the tumor burden needed for genomic profiling during 18F‐FDG PET/CT‐guided 18G core needle biopsies of liver adenocarcinoma metastases. [ABSTRACT FROM AUTHOR]

Here, we report, for the first time, pre-biopsy PET/MR imaging and subsequent ex vivo dual fluorescence (two-fluorescent channel, dual fluorophore) imaging of PSMA-positive PCa. Our initial experience with hybrid modality imaging agents demonstrates potential in precisely detecting PSMA-expressing tumors pre-biopsy and margins after biopsy which could provide for both PET and fluorescence imaging guidance for PCa. [Extracted from the article]