A descending inhibitory mechanism of nociception mediated by an evolutionarily conserved neuropeptide system in Drosophila.

Nociception is a neural process that animals have developed to avoid potentially tissue-damaging stimuli. While nociception is triggered in the peripheral nervous system, its modulation by the central nervous system is a critical process in mammals, whose dysfunction has been extensively implicated in chronic pain pathogenesis. The peripheral mechanisms of nociception are largely conserved across the animal kingdom. However, it is unclear whether the brain-mediated modulation is also conserved in non-mammalian species. Here, we show that Drosophila has a descending inhibitory mechanism of nociception from the brain, mediated by the neuropeptide Drosulfakinin (DSK), a homolog of cholecystokinin (CCK) that plays an important role in the descending control of nociception in mammals. We found that mutants lacking dsk or its receptors are hypersensitive to noxious heat. Through a combination of genetic, behavioral, histological, and Ca2+ imaging analyses, we subsequently revealed neurons involved in DSK-mediated nociceptive regulation at a single-cell resolution and identified a DSKergic descending neuronal pathway that inhibits nociception. This study provides the first evidence for a descending modulatory mechanism of nociception from the brain in a non-mammalian species that is mediated by the evolutionarily conserved CCK system, raising the possibility that the descending inhibition is an ancient mechanism to regulate nociception.

Avoiding harm is fundamental for the survival of animals. Nerve cells called nociceptors can detect potential damage, such as extreme temperatures, sharp objects and certain chemicals. In humans, this detection ­ known as nociception ­ leads to signals travelling from nociceptors through the spinal cord to the brain, which perceives them as pain. Mammals such as humans and rodents can inhibit nociception by sending signals from the brain to the spinal cord to dampen pain. This top-down dampening process is believed to play a crucial role in regulating pain in mammals, and it has been implicated in the development of chronic pain. It was not known whether non-mammalian animals shared this inhibitory pathway. However, previous work had shown that fruit fly produce a molecule called Drosulfakinin, which is similar to the chemical that mammals use in the top-down signalling pathway which controls pain. To determine the role of Drosulfakinin in controlling fly nociception, Oikawa et al. manipulated its activity ­ and the activity of related genes ­ in specific neurons in the fruit fly nervous system. Without Drosulfakinin, fly larvae were more sensitive to heat exposure, suggesting that this molecule is required to inhibit nociception. Further experiments showed that Drosulfakinin is present only in the brain of fly larvae and activation of its signaling lowers the activity of neurons that transmit nociceptive signals in the insect equivalent of the spinal cord. This confirms that insect brains can dampen nociception via a top-down pathway, using a similar molecule to mammals. The findings provide an important foundation for pain studies using non-mammalian animals. The ability to manipulate nociception using genetic techniques in flies offers a powerful tool to understand the top-down process of controlling pain. This result also raises the possibility that this shared top-down inhibition mechanism may have developed over 550 million years ago, which could lead to further research into how nociception and pain regulation systems evolved.

Protocol to isolate temperature-sensitive SARS-CoV-2 mutants and identify associated mutations.

An inability to proliferate at high temperatures typically gives viruses an attenuated phenotype. Here, we present a protocol to obtain and isolate temperature-sensitive (TS) SARS-CoV-2 strains via 5-fluorouracile-induced mutagenesis. We describe steps for the induction of mutations in the wild-type virus and selection of TS clones. We then show how to identify the mutations associated with the TS phenotype, following forward and reverse genetics strategies. For complete details on the use and execution of this protocol, please refer to Yoshida et al. (2022).1.

A highly sensitive NanoLuc-based protease biosensor for detecting apoptosis and SARS-CoV-2 infection.

Proteases play critical roles in various biological processes, including apoptosis and viral infection. Several protease biosensors have been developed; however, obtaining a reliable signal from a very low level of endogenous protease activity remains a challenge. In this study, we developed a highly sensitive protease biosensor, named FlipNanoLuc, based on the Oplophorus gracilirostris NanoLuc luciferase. The flipped ß-strand was restored by protease activation and cleavage, resulting in the reconstitution of luciferase and enzymatic activity. By making several modifications, such as introducing NanoBiT technology and CL1-PEST1 degradation tag, the FlipNanoLuc-based protease biosensor system achieved more than 500-fold luminescence increase in the corresponding protease-overexpressing cells. We demonstrated that the FlipNanoLuc-based caspase sensor can be utilized for the detection of staurosporine-induced apoptosis with sixfold increase in luminescence. Furthermore, we also demonstrated that the FlipNanoLuc-based coronavirus 3CL-protease sensor can be used to detect human coronavirus OC43 with tenfold increase in luminescence and severe acute respiratory syndrome-coronavirus-2 infections with 20-fold increase in luminescence by introducing the stem-loop 1 sequence to prevent the virus inducing global translational shutdown.

Versatile live-attenuated SARS-CoV-2 vaccine platform applicable to variants induces protective immunity.

Live-attenuated vaccines are generally highly effective. Here, we aimed to develop one against SARS-CoV-2, based on the identification of three types of temperature-sensitive (TS) strains with mutations in nonstructural proteins (nsp), impaired proliferation at 37°C-39°C, and the capacity to induce protective immunity in Syrian hamsters. To develop a live-attenuated vaccine, we generated a virus that combined all these TS-associated mutations (rTS-all), which showed a robust TS phenotype in vitro and high attenuation in vivo. The vaccine induced an effective cross-reactive immune response and protected hamsters against homologous or heterologous viral challenges. Importantly, rTS-all rarely reverted to the wild-type phenotype. By combining these mutations with an Omicron spike protein to construct a recombinant virus, protection against the Omicron strain was obtained. We show that immediate and effective live-attenuated vaccine candidates against SARS-CoV-2 variants may be developed using rTS-all as a backbone to incorporate the spike protein of the variants.