Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay.

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

Discovery of a body-wide photosensory array that matures in an adult-like animal and mediates eye-brain-independent movement and arousal.

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye-brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin-expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in "adult-like animals." Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing.

Hierarchies in light sensing and dynamic interactions between ocular and extraocular sensory networks in a flatworm.

Light sensing has independently evolved multiple times under diverse selective pressures but has been examined only in a handful among the millions of light-responsive organisms. Unsurprisingly, mechanistic insights into how differential light processing can cause distinct behavioral outputs are limited. We show how an organism can achieve complex light processing with a simple "eye" while also having independent but mutually interacting light sensing networks. Although planarian flatworms lack wavelength-specific eye photoreceptors, a 25 nm change in light wavelength is sufficient to completely switch their phototactic behavior. Quantitative photoassays, eye-brain confocal imaging, and RNA interference/knockdown studies reveal that flatworms are able to compare small differences in the amounts of light absorbed at the eyes through a single eye opsin and convert them into binary behavioral outputs. Because planarians can fully regenerate, eye-brain injury-regeneration studies showed that this acute light intensity sensing and processing are layered on simple light detection. Unlike intact worms, partially regenerated animals with eyes can sense light but cannot sense finer gradients. Planarians also show a "reflex-like," eye-independent (extraocular/whole-body) response to low ultraviolet A light, apart from the "processive" eye-brain-mediated (ocular) response. Competition experiments between ocular and extraocular sensory systems reveal dynamic interchanging hierarchies. In intact worms, cerebral ocular response can override the reflex-like extraocular response. However, injury-regeneration again offers a time window wherein both responses coexist, but the dominance of the ocular response is reversed. Overall, we demonstrate acute light intensity-based behavioral switching and two evolutionarily distinct but interacting light sensing networks in a regenerating organism.

Thymol, a monoterpene phenolic derivative of cymene, abrogates mercury-induced oxidative stress resultant cytotoxicity and genotoxicity in hepatocarcinoma cells.

Thymol (TOH) was investigated for its ability to protect against mercuric chloride (HgCl2 )-induced cytotoxicity and genotoxicity using human hepatocarcinoma (HepG2) cell line. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay confirmed the efficacy of TOH pretreatment in attenuating HgCl2 -induced cytotoxicity. Pretreatment with TOH inhibited HgCl2 -induced genotoxicity, depolarization of mitochondrial membrane, oxidative stress, and mitochondrial superoxide levels. Interestingly, TOH (100 µM) alone elevated the intracellular basal glutathione S-transferase (GST) levels and TOH pretreatment abrogated the decrease in glutathione, GST, superoxide dismutase, and catalase levels even after HgCl2 intoxication. Furthermore, TOH was also capable of inhibiting HgCl2 -induced apoptotic as well as necrotic cell death analyzed by flowcytometric analysis of cells dual stained with Annexin-FITC/propidium iodide. The present findings clearly indicate the cytoprotective potential of TOH against HgCl2 -induced toxicity, which may be attributed to its free radical scavenging ability which facilitated in reducing oxidative stress and mitochondrial damage thereby inhibiting cell death.