Zhihong Lin

A Europium‐Ion‐Based Luminescent Sensing Probe for Hydrogen Peroxide

A bright idea: A 15-fold increase in fluorescence intensity occurs when the complex formed between Eu3+ and the antibiotic tetracycline binds to hydrogen peroxide at neutral pH. The complex can be used in the determination of the concentration of H2O2, the activity of oxidases, the concentration of glucose, and also in an optical sensor for H2O2.

Determination of the activity of catalase using a europium(III)-tetracycline-derived fluorescent substrate

A one-step method is described for the fluorometric determination of the activity of the enzyme catalase (EC 1.11.1.6.), based on the finding that H(2)O(2) in the europium (III)-tetracycline-hydrogen peroxide system is consumed by catalase. This is accompanied by a large decrease in both fluorescence intensity and decay time. The limit of detection (LOD; at S/N=3) for catalase at 30 degrees C for a 10-min kinetic assay is 1.0 unit/mL, with a linear range from 1.0 to 10 unit/mL. At an incubation time of 30 min at 37 degrees C for a one-point assay, the LOD is 0.046 unit/mL, with a linear range from 46 to 400 munit/mL. The assay was performed on microtiterplates and is fully compatible with existing plate readers. It is a one-step, simple, and sensitive method suitable for both continuous kinetic and one-point detections, does not require the addition of other substrates, and works best at neutral pH (with an optimum at pH 6.9). The reagent has the typical spectral features of a europium-ligand complex including a large Stokes shift (210 nm), a red line-like emission (centered at 616 nm), and a decay time in the microsecond domain. It is also the first europium-based probe that is compatible with the 405-nm diode laser. In summary, the new assay provides distinct advantages over direct ultraviolet detection and over the two-reagent (peroxidase) method.

Red fluorescent genetically encoded Ca2+ indicators for use in mitochondria and endoplasmic reticulum

Ca2+ is a key intermediary in a variety of signalling pathways and undergoes dynamic changes in its cytoplasmic concentration due to release from stores within the endoplasmic reticulum (ER) and influx from the extracellular environment. In addition to regulating cytoplasmic Ca2+ signals, these responses also affect the concentration of Ca2+ within the ER and mitochondria. Single fluorescent protein-based Ca2+ indicators, such as the GCaMP series based on GFP, are powerful tools for imaging changes in the concentration of Ca2+ associated with intracellular signalling pathways. Most GCaMP-type indicators have dissociation constants (Kd) for Ca2+ in the high nanomolar to low micromolar range and are therefore optimal for measuring cytoplasmic [Ca2+], but poorly suited for use in mitochondria and ER where [Ca2+] can reach concentrations of several hundred micromolar. We now report GCaMP-type low-affinity red fluorescent genetically encoded Ca2+ indicators for optical imaging (LAR-GECO), engineered to have Kd values of 24 μM (LAR-GECO1) and 12 μM (LAR-GECO1.2). We demonstrate that these indicators can be used to image mitochondrial and ER Ca2+ dynamics in several cell types. In addition, we perform two-colour imaging of intracellular Ca2+ dynamics in cells expressing both cytoplasmic GCaMP and ER-targeted LAR-GECO1. The development of these low-affinity intensiometric red fluorescent Ca2+ indicators enables monitoring of ER and mitochondrial Ca2+ in combination with GFP-based reporters.

Nerve Growth Factor (NGF) Regulates Activity of Nuclear Factor of Activated T-cells (NFAT) in Neurons via the Phosphatidylinositol 3-Kinase (PI3K)-Akt-Glycogen Synthase Kinase 3β (GSK3β) Pathway

Background: Neurotrophins regulate transcription factor NFAT and NFAT-mediated neuronal functions, but the underlying mechanisms are poorly defined. Results: NGF facilitated depolarization-induced NFAT activation in sensory neurons, which depended on PI3K, Akt, and GSK3β but not on PLC. Conclusion: NGF-dependent facilitation of NFAT activation is mediated by the PI3K-Akt-GSK3β pathway. Significance: This novel mechanism may represent an important component of NFAT-dependent gene regulation in neurons. The Ca2+/calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) plays an important role in regulating many neuronal functions, including excitability, axonal growth, synaptogenesis, and neuronal survival. NFAT can be activated by action potential firing or depolarization that leads to Ca2+/calcineurin-dependent dephosphorylation of NFAT and its translocation to the nucleus. Recent data suggest that NFAT and NFAT-dependent functions in neurons can also be potently regulated by NGF and other neurotrophins. However, the mechanisms of NFAT regulation by neurotrophins are not well understood. Here, we show that in dorsal root ganglion sensory neurons, NGF markedly facilitates NFAT-mediated gene expression induced by mild depolarization. The effects of NGF were not associated with changes in [Ca2+]i and were independent of phospholipase C activity. Instead, the facilitatory effect of NGF depended on activation of the PI3K/Akt pathway downstream of the TrkA receptor and on inhibition of glycogen synthase kinase 3β (GSK3β), a protein kinase known to phosphorylate NFAT and promote its nuclear export. Knockdown or knockout of NFATc3 eliminated this facilitatory effect. Simultaneous monitoring of EGFP-NFATc3 nuclear translocation and [Ca2+]i changes in dorsal root ganglion neurons indicated that NGF slowed the rate of NFATc3 nuclear export but did not affect its nuclear import rate. Collectively, our data suggest that NGF facilitates depolarization-induced NFAT activation by stimulating PI3K/Akt signaling, inactivating GSK3β, and thereby slowing NFATc3 export from the nucleus. We propose that NFAT serves as an integrator of neurotrophin action and depolarization-driven calcium signaling to regulate neuronal gene expression.