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The glucose-6-phosphate dehydrogenase (G6PD) deficiency is X-linked and is the most common enzymatic deficiency disorder globally. It is a crucial enzyme for the pentose phosphate pathway and produces NADPH, which plays a vital role in regulating the oxidative stress of many cell types. The deficiency of G6PD primarily causes hemolytic anemia under oxidative stress triggered by food, drugs, or infection. G6PD-deficient patients infected with SARS-CoV-2 showed an increase in hemolysis and thrombosis. Patients also exhibited prolonged COVID-19 symptoms, ventilation support, neurological impacts, and high mortality. However, the mechanism of COVID-19 severity in G6PD deficient patients and its neurological manifestation is still ambiguous. Here, using a CRISPR-edited G6PD deficient human microglia cell culture model, we observed a significant reduction in NADPH level and an increase in basal reactive oxygen species (ROS) in microglia. Interestingly, the deficiency of the G6PD-NAPDH axis impairs induced nitric oxide synthase (iNOS) mediated nitric oxide (NO) production, which plays a fundamental role in inhibiting viral replication. Surprisingly, we also observed that the deficiency of the G6PD-NADPH axis reduced lysosomal acidification and free radical production, further abrogating the lysosomal clearance of viral particles. Thus, impairment of NO production, lysosomal functions, and redox dysregulation in G6PD deficient microglia altered innate immune response, promoting the severity of SARS-CoV-2 pathogenesis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024. Published by Elsevier B.V.)

In the pathogenesis of microglia, brain immune cells promote nitrergic stress by overproducing nitric oxide (NO), leading to neuroinflammation. Furthermore, NO has been linked to COVID-19 progression, which has caused significant morbidity and mortality. SARS-CoV-2 infection activates inflammation by releasing excess NO and causing cell death in human microglial clone 3 (HMC3). In addition, NO regulates lysosomal functions and complex machinery to neutralize pathogens through phagocytosis. Therefore, developing lysosome-specific NO probes to monitor phagocytosis in microglia during the COVID-19 infection would be a significant study. Herein, a unique synthetic strategy was adopted to develop a NO selective fluorescent probe, PDM-NO , which can discriminate activated microglia from their resting state. The nonfluorescent PDM-NO exhibits a turn-on response toward NO only at lysosomal pH (4.5-5.5). Quantum chemical calculations (DFT/TD-DFT/PCM) and photophysical study revealed that the photoinduced electron transfer (PET) process is pivotal in tuning optical properties. PDM-NO demonstrated good biocompatibility and lysosomal specificity in activated HMC3 cells. Moreover, it can effectively map the dynamics of lysosomal NO against SARS-CoV-2 RNA-induced neuroinflammation in HMC3. Thus, PDM-NO is a potential fluorescent marker for detecting RNA virus infection and monitoring phagocytosis in HMC3.

Organelle-targeted photosensitizers (PSs) having luminescence properties are potential theranostic agents for simultaneous luminescence imaging and photodynamic therapy. Herein, we report a water-soluble luminescent cyclometalated Ir III complex, Ir-Bp-Ly, as a lysosome-targeted theranostic probe. Ir-Bp-Ly exhibits exceptional photophysical properties, with good triplet-state quantum yield (0.90), singlet oxygen generation quantum yield (0.71 at pH 4), and long lifetime (1.47 μs). Interestingly, Ir-Bp-Ly localizes mostly in lysosomes due to the presence of morpholine units, suggesting its potential use as a lyso-tracker. Ir-Bp-Ly displays a notable PDT effect in C6 glioma cells, efficiently generating reactive oxygen species owing to close proximity between the energy levels of its triplet state and those of molecular oxygen ( 3 O 2 ). The mechanism of cell death has been studied through caspase-3/7 and flow cytometry analyses, which clearly established the apoptotic pathway., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The intracellular pH (pHi) in organelles, including mitochondria, endoplasmic reticulum, lysosomes, and nuclei, differs from the cytoplasmic pH, and thus maintaining the pH of these organelles is crucial for cellular homeostasis. Alterations in the intracellular pH (ΔpHi) in organelles lead to the disruption of cell proliferation, ion transportation, cellular homeostasis, and even cell death. Hence, accurately mapping the pH of organelles is crucial. Accordingly, the development of fluorescence imaging probes for targeting specific organelles and monitoring their dynamics at the molecular level has become the forefront of research in the last three decades. Among them, ratiometric fluorescent probes minimize the interference from the excitation wavelength of light, auto-fluorescence from probe concentration, environmental fluctuations, and instrument sensitivity through self-correction compared to monochromatic fluorescent probes, which are known as turn-on/off fluorescent probes. Small-molecular ratiometric fluorescent probes for detecting ΔpHi are challenging yet demanding. To date, sixty-two ratiometric pH probes have been reported for monitoring internal pH alterations in cellular organelles. However, a critical review on organelle-specific ratiometric probes for pH mapping is still lacking. Thus, in the present review, we report the most recent advances in ratiometric pH probes and the previous data on the role of mapping the ΔpHi of cellular organelles. The development strategy, including ratiometric fluorescence with one reference signal (RFRS) and ratiometric fluorescence with two reversible signals (RFRvS), is systematically illustrated. Finally, we emphasize the major challenges in developing ratiometric probes that merit further research in the future. [ABSTRACT FROM AUTHOR]

Esterases enzymes regulate the body's homeostasis by catalyzing the hydrolysis of various esters. These are also involved in protein metabolism, detoxification, and signal transmission. Most importantly, esterase plays a significant role in cell viability and cytotoxicity assays. Hence, developing an efficient chemical probe is essential for monitoring the esterase activity. Several fluorescent probes for esterase have also been reported targeting cytosol and lysosomes. However, the ability to create efficient probes is constrained due to a lack of understanding of the esterase's active site for hydrolyzing the substrate. In addition, the fluorescent turn‐on may limit efficient monitoring. Herein, we have developed a unique fluorescent probe, PM‐OAc, to monitor mitochondrial esterase enzyme activity ratiometrically. This probe exhibited a bathochromic wavelength shift with esterase enzyme in alkaline pH (pH∼8.0) due to an intramolecular charge transfer (ICT) process. The phenomenon is well supported by TD‐DFT calculation. Moreover, the substrate (PM‐OAc) binding at the active site of esterase and its catalytic mechanism to hydrolyze the ester bond are elucidated by molecular dynamics (MD) simulation and QM/MM (Quantum mechanics/molecular mechanics) calculations, respectively. Fluorescent image‐based analysis of the cellular environment reveals that our probe can distinguish between live and dead cells based on esterase enzyme activity. [ABSTRACT FROM AUTHOR]

Mitochondria are valuable subcellular organelles and play crucial roles in redox signaling in living cells. Substantial evidence proved that mitochondria are one of the critical sources of reactive oxygen species (ROS), and overproduction of ROS accompanies redox imbalance and cell immunity. Among ROS, hydrogen peroxide (H2O2) is the foremost redox regulator, which reacts with chloride ions in the presence of myeloperoxidase (MPO) to generate another biogenic redox molecule, hypochlorous acid (HOCl). These highly reactive ROS are the primary cause of damage to DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and proteins, leading to various neuronal diseases and cell death. Cellular damage, related cell death, and oxidative stress are also associated with lysosomes which act as recycling units in the cytoplasm. Hence, simultaneous monitoring of multiple organelles using simple molecular probes is an exciting area of research that is yet to be explored. Significant evidence also suggests that oxidative stress induces the accumulation of lipid droplets in cells. Hence, monitoring redox biomolecules in mitochondria and lipid droplets in cells may give a new insight into cell damage, leading to cell death and related disease progressions. Herein, we developed simple hemicyanine-based small molecular probes with a boronic acid trigger. A fluorescent probe AB that could efficiently detect mitochondrial ROS, especially HOCl, and viscosity simultaneously. When the AB probe released phenylboronic acid after reacting with ROS, the product AB–OH exhibited ratiometric emissions depending on excitation. This AB–OH nicely translocates to lysosomes and efficiently monitors the lysosomal lipid droplets. Photoluminescence and confocal fluorescence imaging analysis suggest that AB and corresponding AB–OH molecules are potential chemical probes for studying oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: Organelle‐targeted photosensitizers (PSs) having luminescence properties are potential theranostic agents for simultaneous luminescence imaging and photodynamic therapy. Herein, we report a water‐soluble luminescent cyclometalated IrIII complex, Ir‐Bp‐Ly, as a lysosome‐targeted theranostic probe. Ir‐Bp‐Ly exhibits exceptional photophysical properties, with good triplet‐state quantum yield (0.90), singlet oxygen generation quantum yield (0.71 at pH 4), and long lifetime (1.47 μs). Interestingly, Ir‐Bp‐Ly localizes mostly in lysosomes due to the presence of morpholine units, suggesting its potential use as a lyso‐tracker. Ir‐Bp‐Ly displays a notable PDT effect in C6 glioma cells, efficiently generating reactive oxygen species owing to close proximity between the energy levels of its triplet state and those of molecular oxygen (3O2). The mechanism of cell death has been studied through caspase‐3/7 and flow cytometry analyses, which clearly established the apoptotic pathway. [ABSTRACT FROM AUTHOR]