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.

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GAF-CaMP3–sfGFP, An Enhanced Version of the Near-Infrared Genetically Encoded Positive Phytochrome-Based Calcium Indicator for the Visualization of Neuronal Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21186883 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6883

Author(s):

Oksana M. Subach ◽

Fedor V. Subach

Keyword(s):

Calcium Ions ◽

Near Infrared ◽

Fluorescent Protein ◽

Dynamic Range ◽

Fluorescent Proteins ◽

Spectral Characteristics ◽

Confocal Imaging ◽

Neuronal Cultures ◽

Decay Kinetics ◽

Calcium Indicator

The first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2–superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2–sfGFP indicator, named GAF-CaMP3–sfGFP. The GAF-CaMP3–sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger DF/F response to calcium ions. As compared to GAF-CaMP2–sfGFP, in cultured HeLa cells, GAF-CaMP3–sfGFP had similar brightness but a 1.9-fold larger DF/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3–sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3–sfGFP showed a linear DF/F response in the range of 0–20 APs and in this range demonstrated a 1.4-fold larger DF/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator.

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DDEL-12. NANOPARTICLE DELIVERY OF DOXORUBICIN FOR THE TREATMENT OF DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

Neuro-Oncology ◽

10.1093/neuonc/noaa222.047 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii286-iii286

Author(s):

Caitlin Ung ◽

Maria Tsoli ◽

Jie Liu ◽

Domenico Cassano ◽

Dannielle Upton ◽

...

Keyword(s):

Treatment Strategies ◽

Pediatric Brain Tumors ◽

Diffuse Intrinsic Pontine Glioma ◽

Confocal Imaging ◽

Cell Content ◽

Potent Effect ◽

Orthotopic Mouse Model ◽

In Vivo Testing

Abstract DIPGs are the most aggressive pediatric brain tumors. Currently, the only treatment is irradiation but due to its palliative nature patients die within 12 months. Effective delivery of chemotherapy across the blood-brain barrier (BBB) has been a key challenge for the eradication of this disease. We have developed a novel gold nanoparticle functionalised with human serum albumin (Au-NP, 98.8 ±19 nm) for the delivery of doxorubicin. In this study, we evaluated the cytotoxic efficacy of doxorubicin delivered through gold nanoparticles (Au-NP-Dox). We found that DIPG neurospheres were equally sensitive to doxorubicin and Au-NP-Dox (at equimolar concentration) by alamar blue assay. Colony formation assays demonstrated a significantly more potent effect of Au-NP-Dox compared to doxorubicin alone, while the Au-NP had no effect. Furthermore, western blot analysis indicated increased apoptotic markers cleaved Parp, caspase 3/7 and phosphorylated H2AX in Au-NP-Dox treated DIPG neurospheres. Live cell content and confocal imaging demonstrated significantly higher uptake of Au-NP-Dox compared to doxorubicin alone. Treatment of a DIPG orthotopic mouse model with Au-NP-Dox showed no signs of toxicity with stable weights being maintained during treatment. However, in contrast to the above in vitro findings the in vivo study showed no anti-tumor effect possibly due to poor penetration of Au-NP-Dox into the brain. We are currently evaluating whether efficacy can be improved using measures to open the BBB transiently. This study highlights the need for rigorous in vivo testing of new treatment strategies before clinical translation to reduce the risk of administration of ineffective treatments.

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ELISA detection of MPO-DNA complexes in human plasma is error-prone and yields limited information on neutrophil extracellular traps formed in vivo

PLoS ONE ◽

10.1371/journal.pone.0250265 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0250265

Author(s):

Hubert Hayden ◽

Nahla Ibrahim ◽

Johannes Klopf ◽

Branislav Zagrapan ◽

Lisa-Marie Mauracher ◽

...

Keyword(s):

Human Plasma ◽

Dynamic Range ◽

Neutrophil Extracellular Traps ◽

Plasma Samples ◽

Dna Complexes ◽

High Background ◽

Isotype Control ◽

Extracellular Traps

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.

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Vascular-dependent effects of elevated glucose on postganglionic sympathetic neurons

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00300.2010 ◽

2011 ◽

Vol 300 (4) ◽

pp. H1386-H1392 ◽

Cited By ~ 6

Author(s):

Deborah H. Damon

Keyword(s):

Vascular Function ◽

Sympathetic Neurons ◽

Vascular Complications ◽

Sympathetic Nerves ◽

T Test ◽

Neuronal Cultures ◽

Elevated Glucose ◽

Low Glucose

Perivascular sympathetic nerves are important determinants of vascular function that are likely to contribute to vascular complications associated with hyperglycemia and diabetes. The present study tested the hypothesis that glucose modulates perivascular sympathetic nerves by studying the effects of 7 days of hyperglycemia on norepinephrine (NE) synthesis [tyrosine hydroxylase (TH)], release, and uptake. Direct and vascular-dependent effects were studied in vitro in neuronal and neurovascular cultures. Effects were also studied in vivo in rats made hyperglycemic (blood glucose >296 mg/dl) with streptozotocin (50 mg/kg). In neuronal cultures, TH and NE uptake measured in neurons grown in high glucose (HG; 25 mM) were less than that in neurons grown in low glucose (LG; 5 mM) ( P < 0.05; n = 4 and 6, respectively). In neurovascular cultures, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release from neurovascular cultures grown in HG (1.8 ± 0.2%; n = 5) was greater than that from cultures grown in LG (0.37 ± 0.28%; n = 5; P < 0.05; unpaired t-test). In vivo, elevated glucose did not affect TH or NE uptake, but it increased NE release. Release in hyperglycemic animals (9.4 + 1.1%; n = 6) was greater than that in control animals (5.39 + 1.1%; n = 6; P < 0.05; unpaired t-test). These data identify a novel vascular-dependent effect of elevated glucose on postganglionic sympathetic neurons that is likely to affect the function of perivascular sympathetic nerves and thereby affect vascular function.

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Abstract 418: Caveolar Disruption of L-type Calcium Channel and Ryanodine Receptor Facilitates Atrial Ectopy and Arrhythmogenesis in Heart Failure Mice

Circulation Research ◽

10.1161/res.127.suppl_1.418 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Di Lang ◽

Lucas ratajczyk ◽

Leonid Tyan ◽

Daniel Turner ◽

Francisco Alvarado ◽

...

Keyword(s):

Heart Failure ◽

Optical Mapping ◽

Ryanodine Receptors ◽

Tissue Level ◽

Confocal Imaging ◽

Right Atrial ◽

Isolated Atria ◽

The Right

Atrial fibrillation (AF) often occurs during heart failure (HF). Ectopic foci that trigger AF, are linked to discrete atrial regions that experience the highest remodeling and clinically used for AF ablation; however, mechanisms of their arrhythmogenic propensity remain elusive. We employed in vivo ECG telemetry, in vitro optical mapping and confocal imaging of Ca 2+ transients (CaT) from myocytes isolated from the right atrial appendage (RAA) and inter-caval region (ICR) of wild type (WT, n=10), caveolin-3 knockout (KO, n=6) and 8-weeks post-myocardial infarction HF (n=8) mice. HF and KO mice showed an increased susceptibility to pacing-induced AF and enhanced ectopy originated exclusively from ICR. Optical mapping in isolated atria showed prolongation of CaT rise up time (CaT-RT) in HF ICR, which suggested a remodeled coupling between L-type Ca 2+ channels (LTCCs) and ryanodine receptors (RyRs) in this specific region. In WT mice, RAA consists of structured myocytes with a prominent transverse-axial tubular system (TATS) while ICR myocytes don’t have TATS. In RAA, CaT-RT depends on LTCCs in TATS triggering RyR, while in ICR, all the LTCCs are localized in surface caveolae where they can activate subsarcolemmal RyRs and lead to a slow diffusion of Ca 2+ inside the cell interior. Downregulation of caveolae was observed specifically in HF ICR. To mimic this, we used cav3-KO mice. Triggered activities were observed in myocytes isolated from HF and KO ICR, which presumably underlie the ectopic activities in tissue level. These myocytes presented significantly unsynchronized sarcoplasmic reticulum (SR) Ca 2+ releases (synchronization index: 10.8±0.9 in WT vs 38.3±4.1 in HF vs 21.5±2.1 in KO, p <0.01 for HF and KO vs WT respectively) especially at the subsarcolemmal space that prolongs CaT-RT (62.2±4.1 ms in WT vs 122.5±12.8 ms in KO, p <0.01). In addition, failing ICR myocytes showed a higher occurrence and size of spontaneous Ca 2+ sparks which were linked to CaMKII activity and associated phosphorylation of RyR. Our findings demonstrate that in HF, caveolar disruption creates “hot spots” for arrhythmogenic ectopic activity emanated from discrete vulnerable regions of the right atrium which are associated with desynchronized SR Ca 2+ release and elevated fibrosis.

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Effects of a human-based mixture of persistent organic pollutants on the in vivo exposed cerebellum and cerebellar neuronal cultures exposed in vitro

Environment International ◽

10.1016/j.envint.2020.106240 ◽

2021 ◽

Vol 146 ◽

pp. 106240

Author(s):

Hanne Friis Berntsen ◽

Nur Duale ◽

Cesilie Granum Bjørklund ◽

Oscar Daniel Rangel-Huerta ◽

Kine Dyrberg ◽

...

Keyword(s):

Organic Pollutants ◽

Persistent Organic Pollutants ◽

Neuronal Cultures

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Properties of Fluorescent Far-Red Anti-TNF Nanobodies

Antibodies ◽

10.3390/antib7040043 ◽

2018 ◽

Vol 7 (4) ◽

pp. 43 ◽

Cited By ~ 2

Author(s):

Ekaterina Gorshkova ◽

Grigory Efimov ◽

Ksenia Ermakova ◽

Ekaterina Vasilenko ◽

Diana Yuzhakova ◽

...

Keyword(s):

Autoimmune Diseases ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Humanized Mice ◽

Whole Body ◽

Whole Body Imaging ◽

Theranostic Agent ◽

Variable Heavy

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases.

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Repair of the UL21 Locus in Pseudorabies Virus Bartha Enhances the Kinetics of Retrograde, Transneuronal Infection In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.02102-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1173-1183 ◽

Cited By ~ 28

Author(s):

D. Curanović ◽

M. G. Lyman ◽

C. Bou-Abboud ◽

J. P. Card ◽

L. W. Enquist

Keyword(s):

Pseudorabies Virus ◽

Particle Production ◽

Neural Circuit ◽

Neuronal Cultures ◽

Wild Type ◽

Infectious Particle ◽

Viral Spread ◽

Kinetics Of

ABSTRACT The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among neurons is diminished. Repair of the UL21 locus with wild-type sequence restored efficient transneuronal spread both in vitro and in vivo. It is likely that mutations in the Bartha UL21 gene confer defects that affect infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection. These events manifest as slower kinetics of retrograde viral spread in a neural circuit.

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C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽

10.1098/rsob.200010 ◽

2020 ◽

Vol 10 (5) ◽

pp. 200010

Author(s):

Navaneethan Palanisamy ◽

Mehmet Ali Öztürk ◽

Emir Bora Akmeriç ◽

Barbara Di Ventura

Keyword(s):

Escherichia Coli ◽

In Silico ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Time Lapse ◽

Enhanced Yellow Fluorescent Protein ◽

Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

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Ratiometric BRET Measurements of ATP with a Genetically-Encoded Luminescent Sensor

Sensors ◽

10.3390/s19163502 ◽

2019 ◽

Vol 19 (16) ◽

pp. 3502 ◽

Cited By ~ 4

Author(s):

Se-Hong Min ◽

Alexander R. French ◽

Keelan J. Trull ◽

Kiet Tat ◽

S. Ashley Varney ◽

...

Keyword(s):

Luminescence Intensity ◽

High Throughput Screening ◽

Dynamic Range ◽

Expression Level ◽

Mouse Tissue ◽

Photon Flux ◽

Color Palette ◽

Animal Imaging

Luciferase-based reporters provide a key measurement approach in a broad range of applications, from in vitro high-throughput screening to whole animal imaging. For example, luminescence intensity is widely used to measure promoter activity, protein expression levels, and cell growth. However, luminescence intensity measurements are subject to quantitative irregularities caused by luminescence decay and variation in reporter expression level. In contrast, bioluminescence resonance energy transfer (BRET) sensors provide the advantages of luciferase-based reporters but overcome the aforementioned irregularities because of the inherently ratiometric readout. Here, we generated a new ratiometric BRET sensor of ATP (ARSeNL—ATP detection with a Ratiometric mScarlet-NanoLuc sensor), and we demonstrated that it provides a stable and robust readout across protein, cell, and whole animal tissue contexts. The ARSeNL sensor was engineered by screening a color palette of sensors utilizing variants of the high photon flux NanoLuc luciferase as donors and a panel of red fluorescent proteins as acceptors. We found that the novel combination of NanoLuc and mScarlet exhibited the largest dynamic range, with a 5-fold change in the BRET ratio upon saturation with ATP. Importantly, the NanoLuc-mScarlet BRET pair provided a large spectral separation between luminescence emission channels that is compatible with green and red filter sets extensively used in typical biological microscopes and animal imaging systems. Using this new sensor, we showed that the BRET ratio was independent of luminescence intensity decay and sensor expression level, and the BRET ratio faithfully reported differences in live-cell energy metabolism whether in culture or within mouse tissue. In particular, BRET analyte sensors have not been used broadly in tissue contexts, and thus, in principle, our sensor could provide a new tool for in vivo imaging of metabolic status.

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