Next generation CANCAN focusing for remote stimulation by nanosecond electric pulses (DANDI:001027)

Claims

From the paper Next generation CANCAN focusing for remote stimulation by nanosecond electric pulses

Reproducing Pakhomov et al. (2023) from dandiset 001027 3

Notebook reproduce_pakhomov_2023.ipynb

Not reproduced3

Not reproducedResults, 3.4, paragraph 2; Figure 8A
Packets 1, 2 and 3 (4 kV, 1Px4) produce the same YP uptake in the center of the array, 215 to 235 a.u.
All packets produced the same YP uptake in the center (215–235 a.u.)
Results, 3.4, paragraph 2; Figure 8A
Main reading (ROIs over +/-1.65 mm, 46 per diagonal, both diagonals, center the mean of the two ROIs flanking 0, raw intensities), center means 648.7 a.u. (Packet 1), 388.5 (Packet 2) and 383.4 (Packet 3); measured gives the lowest and highest of the three. None is within 193.5 to 258.5, and Packet 1 differs from Packets 2 and 3 by the two-sided t-test (p = 0.00044 and 0.00093). All 48 readings give not reproduced; with the background subtracted Packet 1 is 332.5 to 381.1 a.u. and its largest p-values against the others are 0.047 and 0.026. Packets 2 and 3 do not differ in any reading (smallest p 0.174) and, with the background subtracted, are 213.4 to 225.2 and 171.5 to 232.8 a.u. The Packet 1 dishes were imaged on another day with a background about twice as high (mean 308.1 a.u., against 161.8 on 17 March and 150.3 on 18 March); as a ratio to each dish's own background the centers are 2.11, 2.37 and 2.50, and only Packet 1 against Packet 3 differs (p = 0.047). Exchanging the Protocol 2 and 3 labels does not change this claim's outcome. The failure rests largely on the Packet 1 dishes and so on a difference between imaging days; a gain or exposure difference would scale all values and would not be removed by subtracting a background, and the files record neither, so this is unresolved. Dish 10 (no device named, imaged the evening after) has a center of 382.7 a.u. raw, between the other two Protocol 3 dishes (354.4 and 413.2).
How the test differed from the paper’s method
- ROIs were placed by code on images reduced to 8 x 8 pixel means, with electrodes found as the dark footprints in each image; the paper measured ROIs in MetaMorph on the full images (Section 2.4).
- The files give no pixel size; 0.65 um per pixel is taken from the ORCA-Flash4 camera (6.5-um pixels) and 10x objective the Methods (Section 2.4) and the files name. A placement that needs no scale (to 5% of the edge-to-edge diagonal short of each electrode, Section 2.4) gives the same outcome.
- The ROI count, "92 ... ROI, placed along one or both diagonals" (Section 2.4), was run as 46 and as 92 per diagonal; the ROI shape and size, which the paper does not give, is a square as wide as the spacing. The center was run as the mean of the two flanking ROIs and as one ROI at 0, and one diagonal (each) or both. Background subtraction, which the paper does not mention for monolayers, was run both ways, with the background inside the electrode footprints, the only region of the images where cells were not exposed (the files hold no cell-free region). Every reading gives the same outcome. With one ROI at 0 the ROI centers run to +/-1.65 mm, so the outermost ROIs reach about half a step past the stated span.
Not reproducedResults, 3.4, paragraph 2; Figure 8A
Packets 1 and 3 cause much more YP uptake near the electrodes (250 to 350 a.u.) than Packet 2 (about 120 a.u.), p < 0.01.
but Packets 1 and 3 caused drastically more YP uptake near the electrodes (250–350 a.u. vs ~120 a.u. for packet 1, p<0.01, Fig. 8A ).
Results, 3.4, paragraph 2; Figure 8A
What the paper reports
- Packets 1 and 3 against Packet 2, near electrodes: two-sided Student's t-test, p < 0.01 (n = 3 to 4 experiments per packet)
Main reading (mean of the ROIs closest to the four electrodes, raw), 806.9 a.u. (Packet 1), 480.9 (Packet 2) and 296.2 (Packet 3); measured gives the lower and higher of Packets 1 and 3, then Packet 2. Packet 3 is below Packet 2 (t = -3.88, p = 0.012), where the claim has it above. Packet 3 is below Packet 2 in all 48 readings (t from -5.3 to -1.84), so every reading gives not reproduced. Packet 1 exceeds Packet 2 in every reading (t from 1.61 to 10.35, p from 0.00015 to 0.168). Reading (b), the ROI next to e1, cannot be decided because the files do not identify e1. With the same image label as e1 in every dish, all 64 readings give not reproduced (main geometry, raw, Packet 3 against Packet 2 is 238.8 against 439.7, 317.6 against 579.6, 270.2 against 446.7 and 358.1 against 457.4 a.u.). Taking in each dish the end most favourable to the claim, all 16 readings give not reproduced (Packet 2 166.6 to 377.5 a.u., t for Packet 3 over Packet 2 from -0.5 to 0.71). Over all 1,048,576 assignments of e1 to the four electrodes of the ten dishes, none gives reproduced or partially reproduced in any reading. With the Protocol 2 and 3 labels exchanged, 43 of 48 readings still give not reproduced (with the background subtracted, Packet 1 is 498.8 a.u., above 385, and Packet 3 over Packet 2 has p = 0.029).
How the test differed from the paper’s method
- ROI placement, scale and readings as in fig8a-equal-center-uptake.
- Reading (b) of the criterion, the ROI closest to e1, cannot be decided, as the files do not say which electrode is e1 and the electrodes were labelled by angle in each image; it was run with each label as e1 in every dish, with the end most favourable to the claim in each dish, and over every assignment of e1.
Not reproducedResults, 3.4, paragraph 2; Figure 8B
Normalized to the most peripheral ROI, YP emission in the center is 183% for Packet 2 and drops to 83% for Packet 3 and 74% for Packet 1.
The superiority of Packet 2 for CANCAN targeting is highlighted by the drop of normalized YP emission in the center from 183% for Packet 2 to 83% and 74% for Packets 3 and 1, respectively ( Fig. 8B ).
Results, 3.4, paragraph 2; Figure 8B
Main reading, each dish normalized to the mean of its four most peripheral ROIs, then averaged; 82.8% (Packet 2), 129.4% (Packet 3) and 81.3% (Packet 1), against 183, 83 and 74%. Packet 3, not Packet 2, has the highest normalized center in all 96 readings (both normalizations), and Packet 2 is 52.5 to 123.7%. Per dish the Protocol 3 files exceed the Protocol 2 files (t = 4.25, p = 0.008). With the Protocol 2 and 3 labels exchanged, the main geometry gives 162.3, 76.4 and 70.2% with the background subtracted (129.4, 82.8 and 81.3% raw), and the claim would be partially reproduced in 92 of 96 readings and reproduced in 2. Reading (b), normalizing to the ROI next to e1, cannot be decided because the files do not identify e1. With the same image label as e1 in every dish, all 256 readings give not reproduced. Choosing e1 in each dish to suit the claim (the lowest end in each Packet 2 dish, the highest in each Packet 1 and 3 dish) gives partially reproduced in 27 of 64 readings, all with 92 ROIs per diagonal. Over all 1,048,576 assignments of e1, none gives reproduced, none gives partially reproduced with 46 ROIs per diagonal, and at most 1.5% do with 92. The outcome is decided by reading (a), which can be run. Every Protocol 3 dish peaks at the center (125.0 to 137.0% per dish, raw). Protocol 2 dishes 12, 13 and 14 are highest at the electrode ends (63.3 to 84.4%); Protocol 2 dish 2 is highest at one end but its other end is below its center (105.9% raw, 112.3% with the background subtracted). Every Protocol 3 dish is still above every Protocol 2 dish, so the difference does not come from one dish.
How the test differed from the paper’s method
- ROI placement, scale and readings as in fig8a-equal-center-uptake; the normalization was run per dish and on the mean profile, as the criterion names.
- Reading (b) of the most peripheral ROI, next to e1, cannot be decided, as the files do not identify e1; it was run with each label as e1, with the most favourable end in each dish, and over every assignment of e1, and does not decide the outcome.

Not tested in a notebook 15

15 data missing from the dataset

Blocked15

BlockedResults, 3.1, paragraph 1; Figure 4· Data missing from the dataset
A single unipolar 600-ns pulse at 15 kV/cm causes the strongest YP uptake of all treatments in single CHO cells, reaching 126 +/- 14 a.u. (n = 25) by the end of recording.
A single 600-ns pulse at 15 kV/cm caused by far the strongest YP uptake, reaching 126±14 a.u. (n=25) by the end of recording.
Results, 3.1, paragraph 1; Figure 4
The single-cell time-lapse of Figure 4 (confocal images every 10 s for 600 s, Section 2.2) is not in the dandiset, whose ten files each hold one stitched monolayer image of the Figure 8 experiment. The paper names no other archive for its data.
BlockedResults, 3.1, paragraph 1; Figure 4· Data missing from the dataset
Bipolar signals starting with the same pulse cause significantly weaker uptake than the single pulse (p < 0.001), reaching 20.8 +/- 2.0 a.u. (4-phase), 6.9 +/- 1.6 a.u. (8-phase) and 25.4 +/- 2.8 a.u. (8-phase with 5-us delays) by the end of recording.
Bipolar signals started with exactly the same pulse (600 ns, 15 kV/cm) but caused significantly weaker effects (p<0.001). By the end of recording, YP signal reached only 20.8±2.0 a.u. for the 4-phasic signal; 6.9±1.6 a.u. for the 8-phasic signal; and 25.4±2.8 a.u. for the 8-phasic signal with 5-μs delays between phases.
Results, 3.1, paragraph 1; Figure 4
What the paper reports
- each bipolar signal against the single unipolar pulse: not named for Fig. 4; the only test in the Methods is a two-sided Student's t-test, p < 0.001 (n = 21 to 30 cells per group)
The single-cell time-lapse of Figure 4 is not in the dandiset (see fig4-unipolar-strongest).
BlockedResults, 3.1, paragraph 1; Figure 4· Data missing from the dataset
Sham-exposed cells show no gain in YP fluorescence over the 10 min of observation.
Sham-exposed cells showed no gain in YP fluorescence during the 10 min of observation.
Results, 3.1, paragraph 1; Figure 4
The single-cell time-lapse of Figure 4, sham group included, is not in the dandiset.
BlockedResults, 3.1, paragraph 1, and 3.4, paragraph 1; Figure 4 legend· Data missing from the dataset
The 8-phase bipolar signal causes about 20-fold weaker electroporation than a single unipolar pulse identical to its first phase, while the 4-phase and the delayed 8-phase signals cancel only 4 to 5-fold.
the other two types of bipolar signals which were less efficient at cancellation (4–5-fold, Fig. 4 ). ... Note that applying the 8-phasic bipolar signal with no inter-phase delays caused about 20-fold weaker electroporation than a single unipolar pulse
Results, 3.1, paragraph 1, and 3.4, paragraph 1; Figure 4 legend
The single-cell time-lapse of Figure 4 is not in the dandiset.
BlockedResults, 3.2, paragraph 1; Figure 5A· Data missing from the dataset
Without rotation, the unipolar control trains from e1 cause much stronger electroporation throughout the array than 40 Packet 2 applications from e1, and the uptake with trains follows the electric field strength.
The lack of cancellation caused a much stronger electroporation throughout the array, and the distribution of YP uptake expectedly followed the electric field strength distribution in Fig. 3C.
Results, 3.2, paragraph 1; Figure 5A
The Figure 5 dishes (Packet 2 at 6.4 kV, 40 packets or 40 trains) are not in the dandiset. Every file's session description names a first pulse of 4.0 kV and one packet from each electrode. Dandiset 000686 from the same group holds monolayer images of the Figure 5 conditions and is tested in its own registry entry (000686-pakhomov-2023); it is outside the dataset the claims file names.
BlockedResults, 3.2, paragraph 1; Figure 5A· Data missing from the dataset
Packet 2 without rotation enhances electroporation in the center of the array despite the weakening field, but uptake near e1 remains stronger than in the center.
In contrast, Packet 2 enhanced electroporation in the center of the array, despite gradual weakening of the electric field with distance from e1. However, the bipolar cancellation only partially offset the electric field weakening, and electroporation near e1 remained stronger than in the center.
Results, 3.2, paragraph 1; Figure 5A
The Figure 5A dishes (40 packets from e1 at 6.4 kV) are not in the dandiset.
BlockedResults, 3.2, paragraph 2; Figure 5B and 5C· Data missing from the dataset
Rotating the first-energized electrode with Packet 2 (1Px40) makes electroporation in the center exceed that at the perimeter, while the same rotation of control trains leaves a void of electroporation in the center.
Rotation with Packet 2 “smeared” the electroporation effect at the perimeter and emphasized the remote effect in the center. This remote effect contrasted with a void of electroporation in the center when the pulse train was applied in the same manner instead of the packet.
Results, 3.2, paragraph 2; Figure 5B and 5C
The Figure 5B and 5C dishes (1Px40 and 1T x 40 at 6.4 kV) are not in the dandiset.
BlockedResults, 3.3, paragraph 1; Figure 6A· Data missing from the dataset
Increasing the total number of rotated Packet 2 applications from 4 to 20 and 40 strongly increases YP uptake throughout the array.
Increasing the total number of applied packets from 4 to 20 and 40 profoundly enhanced the electroporative YP dye uptake throughout the electrode array
Results, 3.3, paragraph 1; Figure 6A
The Figure 6 dishes (Packet 2, 1Px4, 5Px4 and 10Px4) are not in the dandiset. Its Packet 2 dishes at 4 kV, 1Px4, could be the 1Px4 group of Figure 6 only if Figure 6 used 4 kV, which the paper does not say, and the 5Px4 and 10Px4 groups are absent in any case. Other dandisets from the same group (000875, on the number of packets) may hold these data; they are outside the dataset the claims file names.
BlockedResults, 3.3, paragraph 1; Figure 6B· Data missing from the dataset
The central peak is almost twofold higher than the periphery for 1Px4, and about equal to the periphery for 5Px4 and 10Px4; the least affected area between center and electrodes is about 2-fold lower than the center for all treatments.
Its brightness was almost twofold higher than at the periphery for a single packet was applied 4 times from different directions, or about the same as at the periphery for the rotation of 5 and 10 packets ( Fig. 6B ). Interestingly, the least affected area of the cell monolayer (between the center and the electrodes) showed about 2-fold lower fluorescence intensity than in the center for all the treatments.
Results, 3.3, paragraph 1; Figure 6B
The 5Px4 and 10Px4 dishes of Figure 6 are not in the dandiset.
BlockedResults, 3.3, paragraph 1; Figure 6· Data missing from the dataset
The central peak becomes narrower (sharper) as the number of packets increases.
Also, the central peak appeared narrower (sharper) with increasing the number of packets.
Results, 3.3, paragraph 1; Figure 6
The 5Px4 and 10Px4 dishes of Figure 6 are not in the dandiset.
BlockedResults, 3.3, paragraph 2; Figure 7A· Data missing from the dataset
Raising the first-pulse amplitude from 2.4 to 5.6 kV (small array, 1Px4) increases YP fluorescence about 16-fold at all locations, 15.5 times at the most peripheral point, 16.2 times half-way to the center and 16.8 times in the center.
when the first pulse amplitude was increased from 2.4 to 5.6 kV ... YP fluorescence increased 15.5 times at the most peripheral point; 16.2 times half-way to the center; and 16.8 times in the center
Results, 3.3, paragraph 2; Figure 7A
The Figure 7 dishes (first pulse 2.4 to 5.6 kV) are not in the dandiset; every file is at 4.0 kV. Other dandisets from the same group (001025, on voltage and array size) may hold these data; they are outside the dataset the claims file names.
BlockedResults, 3.3, paragraph 2; Figure 7B and 7D· Data missing from the dataset
Normalized to the most peripheral ROI, the profiles for different amplitudes fully overlap, in the small array with 1Px4 and in the large array with 10Px4, so targeting does not depend on nsEP amplitude within the range studied.
When the YP emission was normalized to the most peripheral ROI, the data for different nsEP amplitudes fully overlapped ( Fig. 7B ). ... The findings held true for rotating multiple packets and when using the larger array ( Fig. 7C and D ).
Results, 3.3, paragraph 2; Figure 7B and 7D
The Figure 7 dishes at several amplitudes, in either array, are not in the dandiset.
BlockedDiscussion, paragraph 5; Figure 7· Data missing from the dataset
Remote targeting does not depend on array size, so findings in small arrays can be extrapolated to larger inter-electrode distances.
the remote targeting, at least within studied limits, did not depend on either the electric pulse amplitude or array size ( Fig. 7 ).
Discussion, paragraph 5; Figure 7
Depends on fig7bd-targeting-independent-of-amplitude, whose data are not in the dandiset; all ten files are from the small array.
BlockedResults, 3.5, paragraph 1; Figure 9A· Data missing from the dataset
Raising the large array 1, 2 and 3 mm above the monolayer reduces YP emission near the electrodes 2.4, 6.4 and 9 times, but in the center only 1.02, 1.4 and 3.8 times.
Specifically, YP emission near the electrodes decreased 2.4, 6.4, and 9 times with 1-, 2- and 3-mm gaps (as compared to electrodes being in the same plane with cells); in the center, it decreased just 1.02, 1.4, and 3.8 times, respectively.
Results, 3.5, paragraph 1; Figure 9A
The Figure 9 dishes (large array 0 to 3 mm above the monolayer, 6.4 kV, 10Px4) are not in the dandiset. Other dandisets from the same group (001028, 3D) may hold related data; they are outside the dataset the claims file names.
BlockedResults, 3.5, paragraph 1; Figure 9B and 9C· Data missing from the dataset
The gap boosts targeting efficiency, the ratio of the center to the periphery, to as much as 600% at 2 mm.
Such disproportionally faster fading of the effect near electrodes boosted the CANCAN targeting efficiency (measured as a ratio of the effect in the center and at the periphery) to as much as 600% for the 2-mm gap ( Fig. 9B ).
Results, 3.5, paragraph 1; Figure 9B and 9C
The Figure 9 dishes are not in the dandiset.

Findings about the data

What attempts found about the dataset itself, for its owner and the archive.

Data missingThe dandiset holds only the Figure 8 experiment, ten monolayer images (Packet 1, 3 dishes; Packet 2, 4; Packet 3, 3; first pulse 4 kV, one rotation, small array). The single-cell time-lapse of Figure 4 and the monolayer experiments of Figures 5, 6, 7 and 9 are not in it, and the paper names no other archive, so 15 of the 18 claims cannot be tested.
Metadata errorThe packet labels of the files labelled Protocol 2 and Protocol 3 appear exchanged with respect to the paper. The files labelled Protocol 3 (8 phases with 5-us delays) show the strongest central focusing (center 125 to 137% of the most peripheral ROIs per dish, raw) and the least uptake next to the electrodes, which the paper reports for Packet 2 (8 phases without delays), and the files labelled Protocol 2 show 63 to 106%, as the paper reports for Packet 3. With the two labels exchanged the order of Figure 8B appears. The dandiset alone cannot say whether its labels or the paper's figure are exchanged; each file's description defines the protocols as the paper defines the packets, so the data were tested as labelled and the question is unresolved.
Description missingThe images have no pixel size (ImageSeries resolution is 96 in nine files and 1.0 in one, unit "n.a."), so distances, which the paper's ROIs are defined by (+/-1.65 mm), rest on the camera's pixel and the objective. The files do not record where the electrodes are or which is e1, so they were found in the images and a reading that needs e1 could not be run. Exposure and gain are not recorded; the three Packet 1 dishes, imaged on 24 March 2021, have about twice the background of the seven others (mean 308 a.u., against 162 and 150 on 17 and 18 March), so a.u. values may not be comparable between imaging days.
Metadata errorThe dandiset's species summary is "Cricetulus griseus - Cricetulus aureus" for one cell line (CHO, Chinese hamster); each subject is a dish with age P1D, which does not apply to a cell line. The file of dish 10 names no microscope, camera or objective, where the other nine name the ORCA-Flash4.0 and a 10x objective, and its session starts on 18 March 2021 at 18:50, the evening after the other Packet 2 and 3 dishes.

Notebooks

Every registered entry that uses this dataset. An entry can hold several notebooks, and can use more than one dataset.

Reproduction Next generation CANCAN focusing by packets of unipolar nanosecond pulses (Pakhomov et al. 2023)

Tests the findings of Pakhomov, Gudvangen, Mangalanathan, Kondratiev, Redondo and Semenov (2023) on dandiset 001027, which holds the YO-PRO-1 images of the Figure 8 experiment, ten CHO monolayer dishes treated with Packets 1, 2 and 3 in a quadrupole electrode array. It finds the electrodes in each stitched image and measures ROI profiles along the diagonals, then compares the uptake in the center and near the electrodes between packets. The single-cell and other monolayer experiments of Figures 4 to 7 and 9 are not in the dandiset. All files

Reproducing Pakhomov et al. (2023) from dandiset 001027
reproduce_pakhomov_2023.ipynbExecuted, with outputs · Source

Verification

Round 1
2026-11-01Agreed with 18 of 18 outcomes. Independent agent run by CatalystNeuro (same harness, separate context, did not write the notebook). Reviewed: The handover of the first run, the executed notebook with the conclusions and summary removed, the helper module and the claims file. The verifier also read the paper and queried the archive for the dandiset's assets.
Round 2
2026-11-01Agreed with 18 of 18 outcomes. Independent agent run by CatalystNeuro (same harness, separate context, did not write the notebook). Reviewed: The full executed notebook of the revised run, with its conclusions and summary, the helper module, the claims file, the README and the attempt record with the round-1 responses. The verifier also read the paper.

Method differences

How the tests differed from the paper’s method or data, and the claims each applies to.

ROIs were placed by code on images reduced to 8 x 8 pixel means, with electrodes found as the dark footprints in each image; the paper measured ROIs in MetaMorph on the full images (Section 2.4).fig8a-equal-center-uptake
The files give no pixel size; 0.65 um per pixel is taken from the ORCA-Flash4 camera (6.5-um pixels) and 10x objective the Methods (Section 2.4) and the files name. A placement that needs no scale (to 5% of the edge-to-edge diagonal short of each electrode, Section 2.4) gives the same outcome.fig8a-equal-center-uptake
The ROI count, "92 ... ROI, placed along one or both diagonals" (Section 2.4), was run as 46 and as 92 per diagonal; the ROI shape and size, which the paper does not give, is a square as wide as the spacing. The center was run as the mean of the two flanking ROIs and as one ROI at 0, and one diagonal (each) or both. Background subtraction, which the paper does not mention for monolayers, was run both ways, with the background inside the electrode footprints, the only region of the images where cells were not exposed (the files hold no cell-free region). Every reading gives the same outcome. With one ROI at 0 the ROI centers run to +/-1.65 mm, so the outermost ROIs reach about half a step past the stated span.fig8a-equal-center-uptake
ROI placement, scale and readings as in fig8a-equal-center-uptake.fig8a-more-uptake-near-electrodes
Reading (b) of the criterion, the ROI closest to e1, cannot be decided, as the files do not say which electrode is e1 and the electrodes were labelled by angle in each image; it was run with each label as e1 in every dish, with the end most favourable to the claim in each dish, and over every assignment of e1.fig8a-more-uptake-near-electrodes
ROI placement, scale and readings as in fig8a-equal-center-uptake; the normalization was run per dish and on the mean profile, as the criterion names.fig8b-packet2-best-targeting
Reading (b) of the most peripheral ROI, next to e1, cannot be decided, as the files do not identify e1; it was run with each label as e1, with the most favourable end in each dish, and over every assignment of e1, and does not decide the outcome.fig8b-packet2-best-targeting

Provenance

Attempt record: 001027-pakhomov-2023-01
Paper: Next generation CANCAN focusing for remote stimulation by nanosecond electric pulses
Date: 2026-11-01
Made by: An agent, run by CatalystNeuro
Dataset: DANDI:001027
Version: 0.240605.1859 (doi:10.48324/dandi.001027/0.240605.1859)
Run on: GitHub Actions
Harness: Claude Code
Model: claude-opus-5-5
Protocol: pilot-13

Transcripts of the agent runs

Each run’s prompt, messages and tool calls, with long output cut and the text of the paper left out, and its final report.

Other datasets that name the same paper

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