Near-Infrared Genetically Encoded Positive Calcium Indicator Based on GAF-FP Bacterial Phytochrome

A variety of genetically encoded calcium indicators are currently available for visualization of calcium dynamics in cultured cells and in vivo. Only one of them, called NIR-GECO1, exhibits fluorescence in the near-infrared region of the spectrum. NIR-GECO1 is engineered based on the near-infrared fluorescent protein mIFP derived from bacterial phytochromes. However, NIR-GECO1 has an inverted response to calcium ions and its excitation spectrum is not optimal for the commonly used 640 nm lasers. Using small near-infrared bacterial phytochrome GAF-FP and calmodulin/M13-peptide pair, we developed a near-infrared calcium indicator called GAF-CaMP2. In vitro, GAF-CaMP2 showed a positive response of 78% and high affinity (Kd of 466 nM) to the calcium ions. It had excitation and emission maxima at 642 and 674 nm, respectively. GAF-CaMP2 had a 2.0-fold lower brightness, 5.5-fold faster maturation and lower pH stability compared to GAF-FP in vitro. GAF-CaMP2 showed 2.9-fold higher photostability than smURFP protein. The GAF-CaMP2 fusion with sfGFP demonstrated a ratiometric response with a dynamic range of 169% when expressed in the cytosol of mammalian cells in culture. Finally, we successfully applied the ratiometric version of GAF-CaMP2 for the simultaneous visualization of calcium transients in three organelles of mammalian cells using four-color fluorescence microscopy

Hydration of the Carboxylate Group in Anti-Inflammatory Drugs: ATR-IR and Computational Studies of Aqueous Solution of Sodium Diclofenac

Diclofenac (active ingredient of Voltaren) has a significant, multifaceted role in medicine, pharmacy, and biochemistry. Its physical properties and impact on biomolecular structures still attract essential scientific interest. However, its interaction with water has not been described yet at the molecular level. In the present study, we shed light on the interaction between the steric hindrance (the intramolecular N–H···O bond, etc.) carboxylate group (−CO₂^–) with water. Aqueous solution of sodium declofenac is investigated using attenuated total reflection-infrared (ATR-IR) and computational approaches, i.e., classical molecular dynamics (MD) simulations and density functional theory (DFT). Our coupled classical MD simulations, DFT calculations, and ATR-IR spectroscopy results indicated that the −CO₂^– group of the diclofenac anion undergoes strong specific interactions with the water molecules. The combined experimental and theoretical techniques provide significant insights into the spectroscopic manifestation of these interactions and the structure of the hydration shell of the −CO₂^– group. Moreover, the developed methodology for the theoretical analysis of the ATR-IR spectrum could serve as a template for the future IR/Raman studies of the strong interaction between the steric hindrance −CO₂^– group of bioactive molecules with the water molecules in dilute aqueous solutions

Suppressed neurogenesis without cognitive deficits: effects of fast neutron irradiation in mice.

This study assessed the effects of combined low-dose neutron and γ-ray irradiation on hippocampal neurogenesis and hippocampal-dependent memory. Neural progenitor cell division and survival were evaluated in brain sections and whole hippocampal preparations following head irradiation at a dose of 0.34 Gy for neutron radiation and 0.36 Gy for γ-ray radiation. Hippocampal-dependent memory formation was tested in a contextual fear conditioning task following irradiation at doses of 0.4 Gy for neutron radiation and 0.42 Gy for γ-ray radiation. Cell division was suppressed consistently along the entire dorsoventral axis of the hippocampus 24 h after the irradiation, but quiescent stem cells remained unaffected. The control and irradiated mice showed no differences in terms of exploratory behavior or anxiety 6 weeks after the irradiation. The ability to form hippocampus-dependent memory was also unaffected. The data may be indicative of a negligible effect of the low-dose of fast neutron irradiation and the neurogenesis suppression on animal behavior at 6 weeks after irradiation

Slowly Reducible Genetically Encoded Green Fluorescent Indicator for In Vivo and Ex Vivo Visualization of Hydrogen Peroxide

Hydrogen peroxide (H2O2) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H2O2 dynamics in live cells within a limited field of view. The visualization of H2O2 within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H2O2 concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with H2O2, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from E. coli. When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (pKa of 5.9 vs. ≥7.6) and oxidized states (pKa of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to H2O2 and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to H2O2 when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to H2O2 and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to H2O2 upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of H2O2 with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H2O2 indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging

Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi

<div><p>Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator <i>in vitro</i>, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP <i>in vivo</i> to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.</p></div