Akash Gulyani

The Dengue virus (DENV) like many other RNA viruses exhibits high genome sequence diversity. This poses a challenge to nucleic acid-based diagnostics, rendering them inefficient at detecting the diverse DENV strains circulating in a population. In this study, we address this challenge by developing a Toehold sensor assay that despite significant genomic diversity is able to detect ~99.4% of all strains of DENV. To this end, our custom workflow first identifies relatively conserved short stretches (36-nt) within all DENV genomes, which could potentially serve as triggers that activate Toehold RNA sensors. We then add a crucial step of mismatch-tolerance that allows related triggers with high sequence diversity and multiple mismatches in the sensor-binding region to be efficiently sensed by the same sensor. Deploying in vitro transcription translation assays, we show that the designed sensors were able to efficiently sense target RNA triggers from all the four DENV serotypes. These data demonstrate that multiple Toehold RNA sensors were able to tolerate limited mismatches in the target RNA sequences without compromising response. The sensitivity of the Toehold sensor assay is often increased by inclusion of an isothermal RNA amplification step. We show by employing multiple primer sets that diverse trigger RNAs from all four serotypes of the dengue virus can be successfully amplified and subsequently detected by the same sensor. Together, this approach has resulted in a pan-dengue Toehold sensor assay, which presents a powerful nucleic acid detection platform to detect viruses with high sequence diversity.

The Dengue virus (DENV) like many other RNA viruses exhibits high genome sequence diversity. This poses a challenge to nucleic acid-based diagnostics, rendering them inefficient at detecting the diverse DENV strains circulating in a population. In this study, we address this challenge by developing a Toehold sensor assay that despite significant genomic diversity is able to detect ∼99.4% of all strains of DENV. To this end, our workflow first identifies relatively conserved short stretches (36-nt) within all DENV genomes, which could potentially serve as triggers that activate Toehold RNA sensors. We then add a crucial step of mismatch-tolerance that allows related triggers with high sequence diversity and multiple mismatches in the sensor-binding region to be efficiently sensed by the same sensor. The sensitivity of the Toehold sensor assay is often increased by inclusion of an isothermal RNA amplification step. We show by employing multiple primer sets that diverse trigger RNAs from all four serotypes of the dengue virus can be successfully amplified and subsequently detected by the same sensor. Together, this approach has resulted in a pan-dengue Toehold sensor assay, which presents a powerful nucleic acid detection platform to detect viruses with high sequence diversity.

Monitoring dynamic changes in mitochondrial pH in live cells using pH-sensitive fluorescent probe.

Animal light sensing and processing are intimately connected to the design of the sensory apparatus, with eye-design a subject of longstanding interest. Understanding animal response to stimuli can elucidate the nature of neural-processing encoded within the photosensory system, tuned to the specific eye-design. Planarians show exquisite photosensitivity and remarkably diverse light-induced behaviors. Further, they are bilaterally-symmetric and possess prototypic cup-shaped eyes, considered an important landmark in eye evolution. Here, using quantitative analysis of behaviour, we address planarian photo-response to distinct presentation of light gradients. Notably, we find that as the nature and presentation of gradients are altered, planarians switch their navigation strategies. When subjected to side-illumination, planarians display negative-phototaxis with a predominantly angular trajectory which presumably allows the animal to maintain the intensity difference between their two eyes for orientation during taxis. However, when subjected to top illumination where light intensity difference between the two eyes would be minimal, animals are still negatively phototactic but the dominant trajectories tend to be directly away from light. Apart from spatial light-intensity sensing, planarians also appear capable of temporal light sensing; confirmed by their response to a spatially-uniform light source with intensity modified over time. Crucially data show that planarians may be using spatial and temporal light sensing to differing degrees depending on the stimuli. Further, when subjected to light illumination from below, planarians fully switch orientation and go belly-up, indicating an active sampling of light distinct from photo-aversive behaviour. Overall, our results show planarian light-sensing to be multilayered, dependent on distinct, highly plastic sensory strategies and beyond basic photo-aversive behaviour.

Fibrosis results from excessive extracellular matrix (ECM) deposition, which causes tissue stiffening and organ dysfunction. Activated fibroblasts, central to fibrosis, exhibit increased migration, proliferation, contraction, and ECM production. However, it remains unclear if the same fibroblast performs all of the processes that fall under the umbrella term of “activation.” Owing to fibroblast heterogeneity in connective tissues, subpopulations with specific functions may operate under distinct regulatory controls. Using a transgenic mouse model of skin fibrosis, we found that Mindin (also known as spondin-2), secreted by Snail- transgenic keratinocytes, differentially regulates fibroblast subpopulations. Mindin promotes migration and inflammatory gene expression in SCA1+ dermal fibroblasts via Fyn kinase. In contrast, it enhances contractility and collagen production in papillary CD26 ⁺ fibroblasts through c-Src signaling. Moreover, in the context of the fibrotic microenvironment of the tumor stroma, we found that differential responses of resident fibroblast subpopulations to Mindin extend to the generation of functionally heterogeneous cancer-associated fibroblasts. This study identifies Mindin as a key orchestrator of dermal fibroblast heterogeneity, reshaping cellular dynamics and signaling diversity in the complex landscapes of skin fibrosis and cancer.

PH dynamically regulates diverse cellular functions and processes. At the Inner Mitochondrial Membrane (IMM), nanoscale pH gradients generated by the Electron Transport Chain (ETC) play a critical role in contributing to mitochondrial membrane potential that drives ATP synthesis and thermogenesis. However, tools to decouple pH gradients from the overall IMM potential in living cells are limited. This study integrates a fluorescent “benzo-indole” chromophore with a pH-sensitive “phenol” moiety into a single covalent skeleton to build a sensitive, red-shifted, cell-permeable pH probe (Mito-pH2). Mito-pH2 localizes inside mitochondria with high specificity presumably to the mitochondrial inner membrane by virtue of being an amphiphilic cation and can report dynamic changes in mitochondrial pH in living cells. Our design ensures that Mito-pH2 exhibits pH-sensitive dual-excitation and dual-emission peaks enabling ratiometric pH-sensing. Further, Mito-pH2 reports an increase in pH in the pH range of 3-9 through a striking colour change from yellow to purple making it a sensitive all-purpose colorimetric pH probe. A combination of DFT calculations and spectroscopy shed light on likely sensing mechanisms including photophysics. Quantitative live-cell fluorescence imaging reveals that Mito-pH2 can detect dynamic changes in mitochondrial pH upon extracellular pH modulation with little or no measurable cytotoxicity during live imaging. Red-emitting Mito-pH2 opens new avenues of quantitative mapping of physiological mitochondrial membrane pH and significantly enhances the repertoire of environment-sensitive and low-toxicity mitochondrial probes that link mitochondrial state and micro-environment.

The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM ‘microviscosity’/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

Extracellular matrix (ECM) is an important component of stem cell niche. Remodeling of ECM mediated by ECM regulators, such as matrix metalloproteinases (MMPs) plays a vital role in stem cell function. However, the mechanisms that modulate the function of ECM regulators in the stem cell niche are understudied. Here, we explored the role of the transcription factor (TF) ETS-1, which is expressed in the cathepsin-positive cell population, in regulating the expression of the ECM regulator, mt-mmpA, thereby modulating basement membrane thickness. In planarians, the basement membrane around the gut/inner parenchyma is thought to act as a niche for pluripotent stem cells. It has been shown that the early epidermal progenitors migrate outwards from this region and progressively differentiate to maintain the terminal epidermis. Our data shows that thickening of the basement membrane in the absence of ets-1 results in defective migration of stem cell progeny. Furthermore, the absence of ets-1 leads to a defective epidermal progenitor landscape, despite its lack of expression in those cell types. Together, our results demonstrate the active role of ECM remodeling in regulating tissue homeostasis and regeneration in the planarian Schmidtea mediterranea. This article has an associated First Person interview with one of the co-first authors of the paper.

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.

Significance: This study highlights the breathtaking sophistication of form and function possible with eye-independent light-sensory systems. We have discovered a body-wide sensory organization of unique photoreceptor cells that allows even head-removed flatworms to move like intact animals, revealing the mechanistic framework underpinning one of the most sensitive eye–brain-independent photoresponses known. Distinct from the ocular system, the body-wide sensory array matures in adult-like animals, can trigger arousal of intact animals from a “resting-state” and employs “noncanonical” opsins. Our discovery of a body-wide network of photoreceptor cells triggering coordinated movement is intriguing and conceptualizes how “dispersed”-sensory nodes may network to control outputs typically through a centralized brain. Our work illustrates how eye-independent systems can deeply influence animal physiology and behavior.