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Abstract BackgroundAnopheles gambiaedensovirus (AgDNV) is a highly species-specific parvovirus that reaches high titers in adultAnopheles gambiaemosquitoes with few transcriptomic effects and minimal significant fitness effects. Given these characteristics, AgDNV has been proposed as a viral vector for basic research and mosquito control. Previous work created an AgDNV co-expression system with a wild-type AgDNV helper plasmid and a transducing plasmid expressing enhanced green fluorescent protein (EGFP) that can be used to co-transfect cells to generate infectious recombinant transducing AgDNV virions. Generated virions infect theAn. gambiaemidgut, fat body, and ovaries, yet this viral vector system is limited in the size of transgenes that can be expressed due to capsid packaging limitations. MethodsConsidering these size constraints, we created an artificial intron within the EGFP gene of the transducing construct that can express small pieces of genetic material such as microRNAs (miRNAs), microRNA sponges, or other small sequences. Placement of this intron in EGFP created a fluorescent reporter such that incorrect splicing produces a frameshift mutation in EGFP and an early stop codon, whereas correct splicing results in normal EGFP expression and co-transcription of the intronic genetic cargo. A selection of miRNAs with predicted or demonstrated importance in mosquito immunity and reproduction with expression localized to the fat body or ovaries were chosen as intronic cargo. Construct expression and splicing was evaluated, and the impact of miRNA expression on putative miRNA targets was measuredin vitroandin vivo. ResultsThe created intron was correctly spliced in cells and mosquitoes; however, miRNA delivery resulted in inconsistent changes to miRNA and predicted target gene transcript levels—possibly due to organ-specific miRNA expression or inaccurate putative target predictions leading to miRNA–target gene sequence mismatch. ConclusionsAlthough our results on target gene expression were inconsistent, with optimization this viral vector and developed intron have potential as an expression tool withinAn. gambiaemosquitoes or cell lines. Graphical Abstract
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This content will become publicly available on August 26, 2026
NitrOFF: An Engineered Fluorescent Biosensor to Illuminate Nitrate Transport in Living Cells
Abstract The duality of nitrate is nowhere better exemplified than in human physiology—a detrimental pollutant but also a protective nutrient—particularly as connected to nitric oxide. Aside from limited insights into nitrate uptake and storage, foundational nitrate biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real‐time imaging, but such technologies for mammalian cell applications remain rare. Here, we designed and engineered a biosensor fusing the green fluorescent protein EGFP and the nitrate recognition domain NreA fromStaphylococcus carnosus. Seven rounds of directed evolution and 15 mutations resulted in NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn‐off intensiometric response (Kd≈ 9 µM). This was further reinforced by X‐ray crystal structures of apo and nitrate‐bound NitrOFF, which revealed a large‐scale conformational rearrangement changing the relative positioning of the domains by 68.4°. This dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the β7 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, we highlighted the utility of NitrOFF to monitor exogenous nitrate uptake and modulation in the human embryonic kidney (HEK) 293 cell line.
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- Award ID(s):
- 2240095
- PAR ID:
- 10642858
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Angewandte Chemie International Edition
- Volume:
- 64
- Issue:
- 40
- ISSN:
- 1433-7851
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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