Claims 21
From the paper An Enhanced Red Bioluminescent Indicator for Responsive Detection of Physiological Calcium Dynamics in Cells and Mice · claims file · checked 2026-10-21: ready
- fig4b-expression-healthy
Two-photon fluorescence shows robust eBRIC expression in neuronal somata and processes in the whisker barrel cortex, and the neurons look healthy and intact three months after surgery.
Two-photon excited fluorescence revealed robust eBRIC expression in neuronal somata and processes. Neurons appeared healthy and morphologically intact even three months after surgery
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 1; Figure 4BHow it will be judged
The paper judges this by eye from images and gives no measure, so it can only be checked qualitatively. Reproduced when the dandiset's two-photon fluorescence images show labelled cell bodies and processes in the transduced region. Whether an image is from three months after surgery is reported from the file metadata if it is given; health and morphology are not judged.
- fig4c-speckle-responsive-region
Laser speckle contrast imaging shows that the region of viral expression responds with blood flow changes to electrical stimulation of the whisker pad.
Laser speckle contrast imaging was used to confirm that the viral expression was in a region that responded to electrical stimulation of the whisker pad
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 1; Figure 4C legendHow it will be judged
The paper gives no measure or test for the speckle result. Reproduced when speckle data are present for a mouse and show a change in speckle contrast (blood flow) over the eBRIC-expressing region during whisker pad stimulation compared with before it, judged from the images or traces as the paper shows them. No threshold is fixed by the paper; the size of the change is reported. Blocked if the dandiset holds no speckle data.
- fig4d-steady-baseline
Infusing DTZ at 25 uL/min through the tail vein gives a steady level of baseline bioluminescence during the recording.
Infusion at 25 μL/min generated a steady level of baseline bioluminescence
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 1; Figure 4DHow it will be judged
The paper gives no measure of steadiness. As a reading of "steady" that the analysis can apply, for each recording the pre-stimulus baseline of each trial (the mean of the signal in the 5 s before stimulus onset; 5 s matches the stimulus length, a standard choice) is computed, and the change of baseline over the recording is compared with the mean evoked response at 4 mA in the same recording (response as in fig4d-evoked-increase). Reproduced when, in the mouse or mice tested, the baseline changes from the first to the last trial by less than the mean 4 mA evoked response, so responses stand out from any drift. The baseline over time is shown for every recording. This threshold is not the paper's; the outcome is to be read with that in mind and the drift reported as numbers.
- fig4d-evoked-increase
Whisker pad stimulation (5 Hz for 5 s) at 4 mA and at 2 mA, through each of the two electrodes, gives clear increases in cortical eBRIC bioluminescence.
clear increases in bioluminescence were observed with stimulation (4 and 2 mA) at both electrodes on the whisker pad
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 1; Figure 4DHow it will be judged
From the Figure 4D legend, traces smoothed with a 1-s moving average and averaged over trials per electrode and current. The response window, the baseline window and the test are not given. The baseline is the 5 s before stimulus onset (a standard choice). The response window has two readings the text allows, as the Results describe a delayed peak for one electrode, (a) the 5-s stimulation period, (b) the stimulation period and the 5 s after it. Response per trial is the mean signal in the response window minus the baseline mean. The paper reports no test, so the test has two readings, both one-sided against zero on the trial responses, (1) a Wilcoxon signed-rank test, (2) a one-sample t-test; an increase is counted when the mean response over trials is positive and the test gives p < 0.05. With fewer than 5 trials in a condition, (1) cannot reach p < 0.05 and only (2) is applied. The windows (a, b) and tests (1, 2) are method details the paper leaves open, so the outcome is the one they all give, and methods_underspecified when they differ. Figure 4D shows one representative mouse that the paper does not identify, so there are two readings of which mice must show it, (i) at least one mouse, as in the figure, (ii) every mouse recorded with both currents and both electrodes; the claim is reproduced if it holds under either. Reproduced when all four conditions (two electrodes by two currents) show an increase; the notebook shows every combination.
- fig4d-4ma-single-trials
At 4 mA the response is strong enough to detect individual stimulation events, in single trials without averaging.
with the 4 mA condition producing a sufficiently strong response to detect individual stimulation events
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 1; Figure 4D; Discussion, paragraph 4How it will be judged
The paper gives no detection rule. "Individual stimulation events" has two readings, (a) single 5-s stimulation trials, as the Discussion puts it ("clear neural responses to single whisker pad shocks in individual trials"), (b) single pulses of the 5 Hz train. Reading (b) cannot be resolved in traces smoothed with the 1-s moving average of the legend; it is tested on the unsmoothed signal by averaging it at each pulse time within a trial (event-triggered on the 0.2-s pulse period) and is shown as a variant. For reading (a), with windows as in fig4d-evoked-increase, a trial is detected when its response exceeds k times the standard deviation of its smoothed baseline, with k = 2 or k = 3 named as the two readings of a threshold the paper does not give. The windows and the threshold are method details the paper leaves open, so under reading (a) the outcome is the one all windows and both thresholds give, and methods_underspecified when they differ. Reproduced when most 4 mA trials (more than half) in the mouse or mice tested, as in the two readings of fig4d-evoked-increase, are detected. The paper does not say that 2 mA trials fail to be detected, so the 2 mA fraction is reported and does not decide the claim. Every fraction is reported.
- fig4d-electrode-timing
The responses to the two electrode locations differ in timing; stimulation of region 1 gives a delayed peak, and region 2 an earlier rise after stimulation onset.
Stimulation of region 1 resulted in a delayed peak response, while region 2 elicited an earlier rise in signal following stimulation onset
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 1; Figure 4DHow it will be judged
Per electrode and current, the trial-averaged trace smoothed with a 1-s moving average (Figure 4D legend), baseline the 5 s before onset. Time to peak is the time of the maximum within the stimulation period and the 5 s after it; time of rise is the first time after onset at which the averaged trace exceeds half its peak above baseline (a standard choice, not the paper's). The paper does not say which electrode is region 1, and the colours of Figure 4D could not be read without the PDF; if the dataset labels the electrodes 1 and 2, those labels are used, otherwise both assignments are shown and the claim is tested as (a) one electrode has both the later peak and the later rise. The paper does not say at which current; the readings are 4 mA, 2 mA, or both. Reproduced when region 1 has the later time to peak and region 2 the earlier time of rise at either current, in the representative mouse reading (at least one mouse) of fig4d-evoked-increase; the values for every mouse are reported.
- fig5-bla-footshock-increase
In awake head-fixed mice, footshock (0.8 mA, 1 s, every 40 s, 13 trials) gives a significant and consistent increase in BLA bioluminescence with eBRIC and sDTZ, larger than in mice expressing the Ca2+-insensitive control BREP.
As expected, we observed a significant and consistent increase in bioluminescence in the eBRIC group in response to footshocks
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 2; Figure 5B and 5CHow it will be judged
As in the Figure 5C legend, each mouse's response averaged over its 13 trials, and the eBRIC and BREP groups (4 and 3 mice) compared with an unpaired two-tailed t-test. The P value is printed only in the figure and was not read. The response measure follows "an established procedure" cited in the Methods and is not given in the text. Reproduced when eBRIC responses exceed BREP responses at p < 0.05.
- fig5-brep-minimal
Mice expressing BREP show minimal to no change in bioluminescence under footshock.
As a negative control, we generated mice expressing BREP, a Ca 2+ -insensitive bioluminescent protein, and observed minimal to no changes in bioluminescence under electric footshock stimulation
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 2; Figure 5B and 5CHow it will be judged
The paper reports no test of the BREP change on its own and no threshold for "minimal"; its only test is the eBRIC against BREP comparison of fig5-bla-footshock-increase. The BREP group mean response over 13 trials and its s.d. (Figure 5C, 3 mice) are reported. Absent a test or threshold from the paper, the claim is judged against the paper's own comparison only, reproduced when the BREP response is smaller than the eBRIC response in that test, and its size relative to zero is reported.
- fig5-10hz-imaging
eBRIC with sDTZ allowed BLI of the awake mouse brain at 10 Hz (100 ms exposures without intervals).
The combination of eBRIC and sDTZ allowed digitalization at a frequency of 10 Hz, which has not been achieved in previous studies
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 2; Methods, BLI of BLA Ca2+ Dynamics in Awake Mice - fig5-double-bric
Under the same footshock condition, the eBRIC response in the BLA is nearly twice that of BRIC in the authors' earlier experiments.
Compared to prior experiments with BRIC under the same stimulation condition, the response magnitude of eBRIC has nearly doubled
Results, In Vivo BLI of Ca2+ Dynamics in Live Mice, paragraph 2; Discussion, paragraph 2How it will be judged
The BRIC values come from "prior experiments with BRIC under the same stimulation condition", which the text does not identify at this sentence. The candidates the paper names are the BRIC paper, Tian et al. 2022, Nat. Commun. 13 3967 (doi:10.1038/s41467-022-31673-x), and the bioRxiv preprint on water-soluble luciferins (doi:10.1101/2024.04.30.591933) listed in the paper's notes; which of them, if either, holds BRIC footshock BLA data is for the author to establish. The eBRIC value comes from Figure 5C. The ratio of the eBRIC group mean response to the BRIC group mean response, each as the mean over mice of the 13-trial average. "Nearly doubled" is read as the expected value 2 with 10% tolerance.
- fig1a-lysate-18fold
After three rounds of directed evolution, eBRIC shows an 18.2-fold Ca2+-induced increase in bioluminescence (39 uM Ca2+ against no Ca2+) in bacterial cell lysates.
Through three rounds of directed evolution, we successfully generated a drastically enhanced BRIC (eBRIC) variant with an 18.2-fold Ca 2+ -included bioluminescence increase in cell lysates
Results, Engineering and Characterization of eBRIC In Vitro, paragraph 2; Figure 1A - fig1b-mutations
eBRIC carries five mutations from BRIC, two in teLuc, one in mScarlet-I and two in the CaM-M13 region, and the two CaM-M13 mutations are mainly responsible for the larger response.
During this process, five mutations were identified ... Notably, the two mutations in the CaM-M13 region are primarily responsible for the observed response enhancement
Results, Engineering and Characterization of eBRIC In Vitro, paragraph 3; Figure 1A to 1C - fig1d-purified-response
In purified protein, the response between 65 nM and 1.35 uM Ca2+ rises from 1.7-fold (BRIC) to 4.6-fold (eBRIC), and the change between 0 and 39 uM Ca2+ from 6.5-fold to about 17-fold.
From BRIC to eBRIC, the response between 65 nM and 1.35 μM of Ca 2+ increased from 1.7-fold to 4.6-fold, while the overall Ca 2+ -induced change between 0 and 39 μM of free Ca 2+ increased from 6.5-fold to ∼17-fold
Results, Engineering and Characterization of eBRIC In Vitro, paragraph 4; Figure 1D - fig1e-kd
The apparent Kd of eBRIC for Ca2+ is 2.3 uM with a Hill coefficient of 1.3, from a four-parameter Hill fit to bioluminescence at 600 nm (3 technical replicates); BRIC's was 133 nM.
A four-parameter Hill equation was used to fit the data to determine the dissociation constant ( K d = 2.3 μM and Hill coefficient = 1.3)
Figure 1E legend; Results, Engineering and Characterization of eBRIC In Vitro, paragraph 4 - lysate-brightness
In bacterial lysates, eBRIC is dimmer than BRIC in the Ca2+-free state and brighter in the Ca2+-bound state.
Compared to BRIC, eBRIC displayed reduced brightness in the Ca 2+ -free state but enhanced brightness in the Ca 2+ -bound state
Results, Engineering and Characterization of eBRIC In Vitro, paragraph 2; Figure S2How it will be judged
Figure S2 is in the Supporting Information, which was not in the text read, so its values and any test are unknown. Judged on the direction of the two differences only.
- fig2b-hela-5fold
In HeLa cells stimulated with 20 uM histamine, eBRIC reports Ca2+ oscillations, and its maximal change (dBL/BL0) is nearly 5-fold that of BRIC (33 eBRIC and 34 BRIC cells, unpaired two-tailed t-test).
Comparing this to BRIC at identical conditions, the magnitude of the change (ΔBL/BL 0 ) was nearly 5-fold higher
Results, Characterization of eBRIC in Cultured Mammalian Cells and Primary Neurons, paragraph 1; Figure 2A and 2BHow it will be judged
As in the Figure 2B legend, the maximal dBL/BL0 of each cell after histamine, with baselines corrected for monoexponential decay, compared between 33 eBRIC and 34 BRIC cells by an unpaired two-tailed t-test. The P value is printed only in the figure and was not read, and the text claims only the fold difference, so the test is reported and does not decide the claim. Reproduced when the ratio of means is within tolerance.
- fig2c-hek-3p5fold
In HEK 293T cells, 10 uM acetylcholine gives a 3.5-fold (dBL/BL0) eBRIC bioluminescence increase (20 cells).
We observed a robust 3.5-fold (ΔBL/BL 0 ) bioluminescence increase
Results, Characterization of eBRIC in Cultured Mammalian Cells and Primary Neurons, paragraph 1; Figure 2CHow it will be judged
The Figure 2C legend shows traces of 20 cells as mean and s.d. and does not say how the 3.5-fold value is taken, so two readings, (a) the peak dBL/BL0 of the mean trace, (b) the mean over cells of each cell's maximal dBL/BL0, as in Figures 2B and 3B. The claim is reproduced if either is within tolerance.
- fig3-neurons-7p3fold
In cultured primary mouse neurons, high K+ depolarization gives a 7.3-fold (dBL/BL0) increase in eBRIC bioluminescence, 5.6 times the response of BRIC (24 eBRIC and 17 BRIC cells, unpaired two-tailed t-test).
high K + depolarization induced a 7.3-fold increase (ΔBL/BL 0 ) in eBRIC bioluminescence, resulting in a 5.6-fold increase in responsiveness from BRIC
Results, Characterization of eBRIC in Cultured Mammalian Cells and Primary Neurons, paragraph 1; Figure 3BHow it will be judged
As in the Figure 3B legend, the maximal dBL/BL0 of each cell, 24 eBRIC and 17 BRIC cells, compared by an unpaired two-tailed t-test (P value only in the figure, not read; the text claims only the fold values, so the test is reported and does not decide the claim). The 7.3-fold value is read as the mean over eBRIC cells of the maximal dBL/BL0. Reproduced when both values are within tolerance.
- cell-brightness
In HeLa cells eBRIC is dimmer than BRIC in the Ca2+-free state and about twice as bright after histamine stimulation, with a similar trend in primary neurons.
eBRIC exhibited reduced brightness in the Ca 2+ -free state but demonstrated approximately twice the brightness of BRIC after stimulation. A similar trend was observed in primary neurons
Results, Characterization of eBRIC in Cultured Mammalian Cells and Primary Neurons, paragraph 2; Figure S2How it will be judged
Figure S2 was not in the text read, so how brightness was measured is unknown. Judged on the ratio and the direction of the Ca2+-free difference.
- hek-spectrum
The emission spectrum of eBRIC in HEK 293T cells closely resembles that in lysates and as purified protein, so expression in mammalian cells does not change BRET efficiency.
The spectral profile closely resembled that of eBRIC in lysates and as a purified protein, suggesting that expression in mammalian cells does not alter the BRET efficiency
Results, Characterization of eBRIC in Cultured Mammalian Cells and Primary Neurons, paragraph 2; Figure S3How it will be judged
Figure S3 was not in the text read; the paper's measure of resemblance is unknown.
- table-s1-fold-gains
In physiologically stimulated live cells, eBRIC is 4.5- to 5.6-fold more responsive than BRIC and 7.4- to 18-fold more responsive than Orange CaMBIs.
Compared to Orange CaMBIs, we gained 7.4- to 18-fold increase in Ca 2+ responsiveness using the new eBRIC Ca 2+ sensor in physiologically stimulated live cells ( Table S1 ). Compared to our previously reported BRIC sensor, eBRIC is 4.5- to 5.6-fold more responsive
Discussion, paragraph 1; Table S1How it will be judged
Table S1 is in the Supporting Information, which was not in the text read. The Orange CaMBI values come from the literature (Oh et al. 2019, Nat. Chem. Biol. 15 433, doi:10.1038/s41589-019-0256-z); which cells and stimuli enter each ratio is given only in Table S1. The range holds when each bound is within the range widened by 10% of each bound.
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