Tsukamoto, H. | Search bioRxiv

bioRxiv2023

A self-inactivating invertebrate opsin with resistance to retinal depletion optically drives biased signaling toward Gβγ-dependent ion channel modulation

Animal opsins, light-sensitive G protein-coupled receptors (GPCRs), have been utilized for optogenetic tools to control G protein-dependent signaling pathways. Upon G protein activation, the Ga and G{beta}{gamma} subunits drive different intracellular signaling pathways, leading to complex cellular responses. For some purposes, Ga-, G{beta}{gamma}-dependent signaling needs to be separately modulated, but these responses are simultaneously evoked due to the 1:1 stoichiometry of Ga and G{beta}{gamma}. Nevertheless, we show temporal activation of G protein using a self-inactivating invertebrate opsin, Platynereis c-opsin1, drives biased signaling for G{beta}{gamma}-dependent GIRK channel activation in a light-dependent manner by utilizing the kinetic difference between G{beta}{gamma}-dependent and Ga-dependent responses. The opsin-induced transient Gi/o activation preferably causes activation of the kinetically-fast G{beta}{gamma}-dependent GIRK channels rather than slower Gi/o-dependent adenylyl cyclase inhibition. Although similar G{beta}{gamma}-biased signaling properties were observed in a selfinactivating vertebrate visual pigment, Platynereis c-opsin1 needs fewer retinal molecules to evoke cellular responses. Furthermore, the G{beta}{gamma}-biased signaling properties of Platynereis c-opsinl are enhanced by genetically fused with RGS8 protein which accelerates G protein inactivation. The self-inactivating invertebrate opsin and its RGS8-fusion protein can function as optical control tools biased for G{beta}{gamma}-dependent ion channel modulation.

biophysics↗

bioRxiv2022

Microaerophilic activated sludge system for ammonia recovery from high-strength nitrogenous wastewater: Performance and microbial communities

A transition to ammonia recovery from wastewater has started; however, a technology for sustainable nitrogen retention in the form of ammonia is still in development. This study validated a microaerophilic activated sludge (MAS) system to efficiently retain ammonia from high-strength nitrogenous wastewater. The MAS is based on conventional activated sludge (CAS) with aerobic and settling compartments. Low dissolved oxygen (DO) concentrations (<0.1 mg/L) and short solid retention times (SRTs) (<5 d) eliminated nitrifying bacteria. The two parallel MASs were successfully operated for 300 d and had ammonia retention of 101.7 {+/-} 24.9% and organic carbon removal of 85.5 {+/-} 8.9%. The MASs mitigated N2O emissions with an emission factor of <0.23%, much lower than the default value of CAS (1.6%). A short-term step-change test demonstrated that N2O indicated the initiation of nitrification and the completion of denitrification in the MAS. The parallel MASs had comparable microbial diversity, promoting organic carbon oxidation while inhibiting ammonia-oxidizing microorganisms (AOMs), as revealed by 16S rRNA gene amplicon sequencing, qPCR of functional genes, and fluorescent in situ hybridization of {beta}-Proteobacteria AOB. The microbial analyses also uncovered that filamentous bacteria were positively correlated with effluent turbidity. Together, controlling DO and SRT achieved successful ammonia retention, mainly by suppressing AOM activity. This process represents a new nitrogen management paradigm. SynopsisMoving from nitrogen removal to nitrogen recovery is critical for establishing a sustainable society. We provided proof-of-the-concept for a novel ammonia retention technology by retrofitting an activated sludge system.

microbiology↗