Simkins, J. W. | Search bioRxiv

bioRxiv2023

Engineering luminopsins with improved coupling efficiencies

SignificanceLuminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. AimWe investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. ApproachWe quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons. ResultsLuciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. ConclusionsWhole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Forster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

molecular biology↗

bioRxiv2018

Prenatal aromatase inhibition alters postnatal immune function in domestic chickens (Gallus gallus)

In birds, exposure to testosterone during embryonic development can suppress immune function; however, it is unclear whether this is caused by direct stimulation of androgen receptors. Estradiol is synthesized from testosterone by the enzyme aromatase, and this conversion is a necessary step in many signaling pathways that are ostensibly testosterone-dependent. Many lines of evidence in mammals indicate that estradiol can affect immune function. We tested the hypothesis that immunosuppressive effects of avian in ovo testosterone exposure are mediated by conversion to estradiol by aromatase, using Fadrozole to inhibit aromatization of endogenous testosterone during a crucial period of embryonic immune system development in domestic chickens (Gallus gallus). We then measured total IgY antibody count, response to PHA challenge, mass of thymus and bursa of Fabricius, and plasma testosterone post-hatch on days 3 and 18. We predicted that if immunomodulation by testosterone is dependent on aromatization, then Fadrozole treatment would lead to elevated immune activity by inhibiting estrogen production. Conversely, if testosterone inhibits immune function directly by binding to androgen receptors, then Fadrozole treatment would likely not alter immune function. Fadrozole treated birds had decreased day 3 plasma IgY antibody titers but there was a strong trend towards increased day 18 thymic mass. Furthermore, Fadrozole treatment generated a positive relationship between testosterone and thymic mass in males, and tended to increase day 18 IgY levels for a given bursal mass in females. There was no effect on PHA response, bursal mass, or plasma testosterone at either age. Overall, Fadrozole treated birds tended to have elevated indicators of immune function, implicating aromatization as a relevant pathway through which developmental exposure to testosterone can affect immunity.

developmental biology↗