Genetically encoded biosensors have revolutionized the study of cell signaling by allowing the real-time monitoring of signaling activities, such as enzymatic activity or the release of second messengers, in live cells. They are therefore critical tools for uncovering the precise spatial and temporal regulation of signal transduction cascades. These biosensors can be divided into two broad classes: single-color and ratiometric. Single-color sensors, with an intensiometric activity readout, only occupy a single-color channel, allowing for more flexibility in multiplexed imaging experiments. However, they are sensitive to variations in probe concentration caused by changing expression levels or cell shape, as well as differences in imaging conditions, such as illumination intensity and focus. On the other hand, sensors with a ratiometric readout, such as those based on Förster Resonance Energy Transfer (FRET), cancel out many of these variations, enabling quantitative measurements of second messenger concentrations and better comparisons between experiments. However, the requirement for two distinct color channels limits their application in multiplexed imaging.

We therefore aimed to develop sensors that only occupy a single channel while still cancelling out the effects of varying imaging conditions and probe concentrations. Hence, rather than using the emission ratio between the FRET and donor channels, we instead used the loss of polarization of emitted light as a readout for FRET between two fluorescent proteins (FPs). Because this approach does not require the donor and acceptor to have distinct emission wavelengths, it can be used for either hetero-FRET (e.g. between different chromophores) or homo-FRET (e.g. between identical chromophores). Homo-FRET measurements have been useful for detecting protein clustering and protein oligomerization in live cells (Bader et al., 2011, Bader et al., 2009; Gautier et al., 2001), but only recently has the possibility of using homo-FRET in biosensor designs been explored (Warren et al., 2015; Cameron et al., 2016). Here, we describe the development of a panel of single-color, genetically encodable biosensors based on homo-FRET for detecting kinase activity and second messenger dynamics. We call these sensors FLuorescence Anisotropy REporters, or FLAREs.

To create our FLARE probes, we adapted existing FRET-based biosensors for homo-FRET measurements by replacing the traditional FRET pair with two FPs of the same color. The resulting biosensors include a molecular switch, which changes conformation in the presence of a particular biochemical activity, flanked by two spectrally similar FPs at the N- and C-termini. Changes in the conformation of the molecular switch, and thus the biochemical activities under study, are then monitored by observing the fluorescence anisotropy of the sensor using fluorescence polarization microscopy, with increased anisotropy corresponding to a lower-FRET state of the sensor, similar to the effect of increasing the intramolecular distance between the FRET pair (Figure 1—figure supplement 1).

To develop a Protein Kinase A (PKA) activity FLARE, the molecular switch from A Kinase Activity Reporter 4 (AKAR4) (Zhang et al., 2001; Depry et al., 2011), composed of an FHA1 domain and PKA substrate (Figure 1a), was flanked between two FPs of the same color. The FHA1 domain binds to the PKA substrate when the latter is phosphorylated, altering the conformation of the molecular switch and leading to a change in FRET between the flanking homo-FRET pair. To test the effect of FP circular permutation on these biosensors, we developed two FLARE-AKAR variants based on the yellow FP mVenus: one in which the C-terminal FP was circularly permuted at position 172 (cp172Venus), consistent with the hetero-FRET AKAR4 sensor, and one without circular permutation. We expressed mVenus-cp172Venus FLARE-AKAR in HEK293T cells and captured a time-course using fluorescence polarization microscopy. Following PKA activation using a cocktail of 50 μM forskolin (Fsk), an adenylyl cyclase activator, and 100 μM 3-isobutyl-1-methylxanthine (IBMX), a general phosphodiesterase inhibitor, the anisotropy decreased from 0.29 ± 0.003 to 0.26 ± 0.003, a decrease of 0.028 ± 0.001 (N = 44, biological replicates, unpaired, two-tailed t-test, p<0.0001), with the kinetics of the decrease matching those observed with AKAR4 (Figure 1b, Figure 1—figure supplement 2a). The yellow variant without the circular-permutation in the C-terminal FP showed slightly reduced changes in anisotropy upon stimulation with Fsk/IBMX (Figure 1c, Figure 1—figure supplement 2c), consistent with previous

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FLARE AKAR characterization.

(a) Sheet 1, Figure 1b Time Course. Time course for mVenus-cp17Venus FLARE AKAR, wild type and kinase-inactive threonine-alanine (TA) mutant. (b) Sheet 2, Figure 1b Summary: Comparison of magnitude of responses for individual cells for Venus-cp172Venus FLARE AKAR, wild type and kinase-inactive threonine-alanine (TA) mutant, as well as statistics comparing the two groups. (c) Sheet 3, Figure 1c. Summary of magnitude of responses for various color variants of FLARE AKAR after stimulation with forskolin and IBMX.

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observations in hetero-FRET-based biosensors (Nagai et al., 2004; DiPilato and Zhang, 2009; Allen and Zhang, 2006). We observed a slight positive correlation between intensity and anisotropy change for Venus-cp172Venus FLARE AKAR; however, the expression level does not significantly impact the reporting ability of these sensors in general (Figure 1—figure supplement 3a). The signal-to-noise ratio (SNR) of Venus-cp172Venus FLARE-AKAR was calculated to be 32 by dividing the magnitude of the anisotropy change upon maximal PKA stimulation by the standard deviation of the baseline before stimulation.

Subsequent control experiments confirmed that this change in anisotropy is caused by a change in the FRET state due to the conformational change of the sensor upon stimulation of PKA activity. PKA inhibition using 20 μM H-89 led to an immediate slope change and increase in anisotropy (Figure 1b). On the other hand, a mutant version of the biosensor with a threonine-to-alanine (T-to-A) mutation at the phosphorylation site showed no change in anisotropy upon PKA stimulation with Fsk/IBMX or inhibition with H-89 (Figure 1b, Figure 1—figure supplement 2b), suggesting that the observed changes in anisotropy were due to phosphorylation of the PKA substrate. Furthermore, isoproterenol, a β-adrenergic agonist, induced FLARE-AKAR responses in a dose-dependent manner (Figure 1—figure supplement 4). To further demonstrate that the change in anisotropy upon PKA stimulation was due to a change in FRET, we mutated the chromophore of the C-terminal cp172Venus in Venus-cp172Venus FLARE AKAR from GYG to GGG. We observed that the magnitude of the response to Fsk/IBMX decreased to approximately one-third of that of the wild-type sensor (Figure 1—figure supplement 5). The remaining response was likely due to intermolecular FRET that occurs when the FHA1 domain of one molecule binds to the phosphorylated PKA substrate in an adjacent molecule.

In addition to the yellow sensors, we developed a panel of color variants of FLARE-AKAR, including EGFP-EGFP, mCherry-mCherry, mCerulean3-mCerulean3 (Markwardt et al., 2011), and mCerulean3-cp173Cerulean3 versions. All these variants exhibited a decrease in anisotropy in cells treated with Fsk/IBMX; however, the magnitude of the anisotropy decrease depended on the choice of FP, with the mVenus-cp172Venus variant having the largest response (Figure 1c, Figure 1—figure supplement 2). As with the Venus variants, the Cerulean3-based FLARE-AKAR showed an increased dynamic range with a circularly permutated fluorescent protein at the C-terminal position. mCherry-mCherry FLARE-AKAR, being spectrally shifted from the AKAR4 heteroFRET sensor allowed for direct comparison of FLARE and heteroFRET sensors within the same cell. As shown in Figure 1—figure supplement 6, changes in anisotropy in mCherry-mCherry FLARE-AKAR corresponded with the changes in normalized emission ratio in AKAR4, with similar kinetics.

We furthermore demonstrated the ability of FLARE-AKAR sensors to monitor kinase activity at particular subcellular compartments. By fusing Venus-cp172Venus FLARE-AKAR to targeting motifs from Lyn kinase and DAKAP1, we were able to detect PKA activity at the plasma membrane and outer mitochondrial membrane, respectively (Figure 1—figure supplement 7). Moreover, we used untargeted FLARE sensors to detect differential PKA activity dynamics in different compartments; diffusable Venus-cp172Venus FLARE AKAR in HeLa showed that PKA activity has slower kinetics and a lower magnitude in the nucleus than the cytosol (Figure 1—figure supplement 8).

To demonstrate the generalizability of FLAREs, we developed a family of single-color kinase activity or activation reporters in various colors (Figure 2). To construct a single-color Erk activity biosensor, we replaced the PKA sensor domain from Venus-cp172Venus FLARE-AKAR with the sensor domain from EKAR-EV, composed of a WW domain (PAABD), a flexible EV linker, and an Erk substrate peptide (Figure 2a) (Harvey et al., 2008; Vandame et al., 2014). When expressed in HEK293T cells, Venus-cp172Venus FLARE-EKAR-EV exhibited a decrease in anisotropy of 0.02 ± 0.001 (N = 13) after treatment with 100 ng/mL epidermal growth factor (EGF) to activate the MAPK pathway (Figure 2a, Figure 2—figure supplement 1). This response was reversed upon MEK inhibition using 20 μM U0126, and no change in anisotropy was observed with a T-to-A mutant sensor. Likewise, we developed a panel of PKC activity reporters, called FLARE-CKARs, by flanking a PKC sensor domain composed of an FHA1 domain and a PKC substrate (Herbst et al., 2011) from a CKAR2 construct (Figure 2—figure supplement 2) with mVenus-cp172Venus. We observed an anisotropy decrease of 0.02 ± 0.001 (N = 26) upon activation of PKC with 100 ng/mL phorbol 12-myristate 13-acetate (PMA) (Figure 2b, Figure 2—figure supplement 3). For both FLARE-EKAR (Figure 2—figure supplement 1) and FLARE CKAR (Figure 2—figure supplement 3), we likewise repeated this process for various color variants, and like the FLARE-AKARs, the sensors based on mVenus exhibited the largest responses. Furthermore, a myosin light chain kinase (MLCK) sensor was converted to a FLARE by exchanging the cyan FP for mVenus (Isotani et al., 2004). Calmodulin (CaM) association with the MLCK-CaM binding domain in between the FPs decreases FRET, leading to an increase in fluorescence anisotropy upon forced calcium (Ca²⁺) entry with 30 mM KCl (N = 13) (Figure 2c, Figure 2—figure supplement 4).

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FLARE kinase biosensor panel.

(a) Sheet 1, Figure 2a. Time course for Venus-cp172Venus FLARE EKAR, both wild type and kinase-inactive (TA) mutant. (b) Sheet 2, Figure 2a. Comparison of magnitudes of anisotropy changes for Venus-cp172Venus FLARE EKAR, both wild type and kinase-inactive (TA) mutant, of upon EGF addition, and relevant statistical tests to compare the two variants. (c) Sheet 3, Figure 2—figure supplement 1c. Changes of magnitudes of anisotropy change for various FLARE EKAR variants upon EGF addition. (d) Sheet 4, Figure 2b. Time course for Venus-cp172Venus FLARE CKAR, both wild type and kinase-inactive (TA) mutant. (e) Sheet 5, Figure 2b. Comparison of magnitudes of anisotropy changes for Venus-cp172Venus FLARE EKAR, both wild type and kinase-inactive (TA) mutant, of upon EGF addition, and relevant statistical tests to compare the two variants. (f) Sheet 6, Figure 2—figure supplement 2. Time courses for CKAR1 and CKAR2. (g) Sheet 7, Figure 2—figure supplement 3c. Changes of magnitudes of anisotropy change for various FLARE CKAR variants upon PMA addition. (h) Sheet 8, Figure 2c. Time course for FLARE MLCK, with either addition of KCl and vehicle only control. (i) Sheet 9, Figure 2c. Summary for magnitude of responses for FLARE MLCK.

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In addition to biosensors for monitoring enzymes, we also developed FLAREs for monitoring second messenger dynamics (Figure 3a). We developed a Ca²⁺ FLARE by utilizing the sensor domain from the Cameleon family of biosensors, composed of CaM and the Ca²⁺/CaM-binding peptide M13 (Nagai et al., 2004; Miyawaki et al., 1999). When expressed in HEK293T cells, Venus-cp172Venus FLARE-Cameleon exhibited a decrease in anisotropy of 0.03 ± 0.002 (N = 10) upon addition of 1 μM ionomycin and 5 mM CaCl₂, with the Venus-Venus, mCerulean3-mCerulean3, and mCherry-mCherry versions also showing detectable responses (Figure 3b, Figure 3—figure supplement 1a,b). The mVenus-based FLARE sensors tend to show larger dynamic ranges for a variety of

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FLARE second messenger biosensor panel.

(a) Sheet 1, Figure 3b. Time course for Venus-cp172Venus FLARE Cameleon. (b) Sheet 2, Figure 3b. Summary of magnitude of responses for Venus-cp172Venus FLARE Cameleon upon addition of calcium chloride and ionomycin. (c) Sheet 3, Figure 3—figure supplement 1b. Summary of magnitudes of responses for various FLARE Cameleon variants upon addition of calcium chloride and ionomycin. (d) Sheet 4, Figure 3—figure supplement 2. In vitro calibration of Venus-cp172Venus FLARE Cameleon, both raw data and sigmoidal curve fits. (e) Sheet 5, Figure 3—figure supplement 3. Summary of magnitude of anisotropy changes for CFP FLARE D1ER upon addition of ionomycin and three different doses of calcium. (f) Sheet 6, Figure 3c. Time course for Venus-cp172Venus FLARE ICUE. (g) Sheet 7, Figure 3c Summary of magnitudes of changes in anisotropy for Venus-cp172Venus FLARE ICUE upon addition of forskolin and IBMX.

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 FLARE sensors than mCerulean3 or mCherry variants, likely due to the superior extinction coefficient and quantum yield of mVenus, which make it a good FRET donor and acceptor. We further demonstrated the ability of Venus-cp172Venus FLARE Cameleon to detect submaximal responses by monitoring calcium transients in histamine-stimulated HeLa cells (Figure 3—figure supplement 1c). In order to determine the dissociation constant and Hill coefficients, we purified Venus-cp172Venus Cameleon and measured the fluorescence anisotropy in solutions of known free Ca²⁺ concentration at different temperatures (Figure 3—figure supplement 2). The resulting parameters are in good agreement with other Cameleon sensors (Nagai et al., 2004). Furthermore, we developed another calcium FLARE sensor, based on D1-ER (Palmer et al., 2004), with a sensitivity appropriate for calcium monitoring in the ER. The anisotropy decreases upon increasing calcium concentration and increases when calcium is depleted from the ER upon treatment with thapsigargin (Figure 3—figure supplement 3). In addition to Ca²⁺, we developed a FLARE to detect intracellular cAMP based on the ICUE family of sensors, in which a conformational change in a truncated form of the cAMP effector Epac leads to a decrease in FRET efficiency in the presence of cAMP (DiPilato and Zhang, 2009) (Figure 3a). When expressed in HEK293T cells, the fluorescence anisotropy of Venus-cp172Venus FLARE-ICUE increased by 0.02 ± 0.001 (N = 40) upon stimulation with Fsk/IBMX (Figure 3c, Figure 3—figure supplement 4).

The fact that FLAREs only occupy a single-color channel and are highly generalizable for different biosensors, as well as color variants, highlights their utility for multiplexed imaging applications. While multiplexing of hetero-FRET sensors is generally limited to two probes (Depry et al., 2013; Shcherbakova et al., 2012), we demonstrate that FLAREs can facilitate co-imaging of three biosensors simultaneously. We co-expressed mCherry-mCherry FLARE-AKAR, Venus-cpVenus FLARE-EKAR-EV, and mCerulean3-mCerulean3 FLARE-Cameleon in HEK293T cells and acquired a time-course with sequential treatment using Fsk/IBMX, EGF, and thapsigargin. Clear and distinct decreases in anisotropy were observed in the red channel after Fsk/IBMX treatment, in the yellow channel after EGF stimulation, and in the cyan channel after thapsigargin treatment, corresponding to an increase in PKA activity, Erk activity, and intracellular Ca²⁺, respectively (N = 17) (Figure 4a, Figure 4—figure supplement 1).

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Multiparameter imaging of FLAREs.

(a) Sheet 1, Figure 4a. Time course for multiplexed imaging of mCherry-mCherry FLARE AKAR, Venus-cp172Venus FLARE EKAR, and mCer3-mCer3 FLARE Cameleon, expressed in HEK293T cells and treated with forskolin and IBMX, EGF, and thapsigargin at t = 0 min, t = 7.5 min, and t = 32.5 min, respectively. (b) Sheet 2, Figure 4b. Time course for Venus-cp172Venus FLARE ICUE and mCer3-mCer3 FLARE Cameleon in Min6 cells, treated with TEA at t = 0 min. (c) Sheet 3, Figure 4c. Time course for Venus-cp172Venus FLARE Cameleon co-expressed with mCherry tagged hChR2-ER or mCherry alone. (d) Sheet 4, Figure 4e. Summary data of 2-photon in vivo imaging of Venus-cp172Venus FLARE Cameleon and mCherry-mCherry FLARE AKAR, in the muscle cells in the feet of live mice.

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We further aimed to show that FLARE sensors can be used to monitor multiple signaling activities simultaneously in different cellular contexts. For example, mCer3-mCer3 FLARE Cameleon, co-imaged with other FLARE sensors in HeLa cells, can detect calcium responses to physiologically relevant stimulation conditions, such as histamine (Figure 4—figure supplement 2). Additionally, when mCer3-mCer3 FLARE AKAR was co-imaged with Venus-cp172Venus cameleon in HEK293T cells, activation of the β-adrenergic with isoproterenol led to a transient decrease in anisotropy in the cyan-channel (Figure 4—figure supplement 3). Furthermore, we used FLAREs to study the cAMP-Ca²⁺ oscillatory circuit in pancreatic β-cells. MIN6 β-cells were transiently transfected with Venus-cp172Venus FLARE-ICUE and mCerulean3-mCerulean3 FLARE-Cameleon to simultaneously monitor cAMP and Ca²⁺ dynamics, respectively (N = 19). We observed clear fluorescence anisotropy oscillations in both channels following stimulation with 20 mM tetraethylammonium chloride (TEA) (Figure 4b, Figure 4—figure supplement 4). Ca²⁺ and cAMP exhibit synchronized oscillations, with Ca²⁺ increases corresponding to cAMP decreases, consistent with previous findings (Landa et al., 2005; Ni et al., 2011). These data demonstrate that even the lower signal mCerulean3-based FLARE sensors can be used under sub-maximal and physiologically relevant stimulation conditions in multiplexed imaging experiments.

In addition to multiplexed biosensor imaging, the fact that FLARE sensors occupy only a single color channel also permits simultaneously perturbing and monitoring biochemical activities using optogenetics and FLAREs, respectively. We coexpressed an mCherry-tagged,

ER-targeted channelrhodopsin2 (hChR2) (Nagel et al., 2003; Lin et al., 2009; Markwardt et al., 2016), a light-gated calcium ion channel, with Venus-cp172 FLARE-Cameleon in REF52 cells, a rat embryonic fibroblast cell line. Illumination with blue light produced an immediate decrease in anisotropy, corresponding to an increase in intracellular Ca²⁺ (Figure 4c, Figure 4—figure supplement 5). Control cells lacking hChR2 expression showed no change in anisotropy in the yellow channel.

In vivo, two-photon imaging of FLAREs was tested using a skeletal muscle preparation (Figure 4d,e). Exclusive excitation of mVenus (855 nm) or mCherry (1200 nm) was verified by imaging capillary tubes filled with recombinant proteins (Figure 4d). Plasmids encoding Venus-cp172Venus FLARE-Cameleon and mCherry FLARE-AKAR sensors were electroporated into the flexor digitorum brevis muscle of a live mouse (DiFranco et al., 2009; Kerr et al., 2015). Administration of an electrical current stimulated a rise in intracellular Ca²⁺ concentration, as indicated by a decrease in FLARE-Cameleon anisotropy, independent of changes in FLARE-AKAR anisotropy (Figure 4e). Activation of AKAR was then induced by intraperitoneal injection of isoproterenol (0.5 mg/kg). Thus, FLAREs enable in vivo multiparametric biosensor measurements.

We have demonstrated that FLAREs are a highly generalizable, accessible platform for creating single-color sensors to detect biochemical activities in individual cells in real time. Their ratiometric readout allows for fluctuations in light intensity and probe concentration to be cancelled out, permitting quantitative measurements of intracellular concentrations. We showed that these sensors have an SNR of 3–32 (Table 1) and a dynamic range comparable with first-generation FRET sensors (Newman and Zhang, 2008; Zhou et al., 2015). Future development and optimization should further enhance their performance. We demonstrated that current FLAREs are already useful for multiplexed imaging applications. They can also be used in conjunction with optogenetic tools to enable all-optical interrogation of cellular regulation, and for intravital two-photon imaging to facilitate studies in tissues and living animals. FLAREs, by allowing researchers to monitor multiple activities within the same cell, as well as to both monitor and optogenetically perturb activities in the same cell, could be used to study the how the spatiotemporal regulation of biochemical activities in highly integrated pathways allows a small number of signals to produce diverse cellular behaviors.

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Author details

1. Brian L Ross

   1. Department of Pharmacology, University of California, San Diego, San Diego, United States
   2. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9020-627X

2. Brian Tenner

   1. Department of Pharmacology, University of California, San Diego, San Diego, United States
   2. Program in Molecular Biophysics, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation

   Competing interests

   No competing interests declared

3. Michele L Markwardt

   Department of Physiology, University of Maryland Baltimore, Baltimore, United States

   Contribution

   Resources, Data curation, Validation, Investigation

   Competing interests

   No competing interests declared

4. Adam Zviman

   Department of Physiology, University of Maryland Baltimore, Baltimore, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Guoli Shi

   Department of Orthopaedics, University of Maryland Baltimore, Baltimore, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Jaclyn P Kerr

   Department of Orthopaedics, University of Maryland Baltimore, Baltimore, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Nicole E Snell

   Department of Physiology, University of Maryland Baltimore, Baltimore, United States

   Contribution

   Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

8. Jennifer J McFarland

   Department of Physiology, University of Maryland Baltimore, Baltimore, United States

   Contribution

   Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

9. Joseph R Mauban

   Department of Physiology, University of Maryland Baltimore, Baltimore, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

10. Christopher W Ward

    Department of Orthopaedics, University of Maryland Baltimore, Baltimore, United States

    Contribution

    Resources, Supervision, Investigation, Methodology, Project administration

    Competing interests

    No competing interests declared

11. Megan A Rizzo

    Department of Physiology, University of Maryland Baltimore, Baltimore, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    mrizz001@umaryland.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6528-7768

12. Jin Zhang

    1. Department of Pharmacology, University of California, San Diego, San Diego, United States
    2. Program in Molecular Biophysics, Johns Hopkins University School of Medicine, Baltimore, United States
    3. Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, United States

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing—review and editing

    For correspondence

    jzhang32@ucsd.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-7145-7823

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