G2C::Electrophysiology

Akap9 knock-out mouse

S.G.N. Grant and the G2C Consortium

Corresponding email: Seth.Grant@ed.ac.uk  

 

Overview: Effect sizes (Cohen d) for 18 electrophysiology variables

Mutant slices showed medium overall electrophysiological difference from wildtype slices, with three of nine variables significantly impacted in these mutants. In the paired pulse facilitation experiment, PPF at 50ms ampl ratio was significantly decreased in mutants. In the theta burst stimulation experiment, one electrophysiological variable, burst 1 PPF at 10ms ampl ratio, was significantly decreased in mutants. In the long term potentiation experiment, one electrophysiological variable, LTP based on amplitude, was significantly decreased in mutants. Only the basal synaptic transmission experiment was unaffected. Note that in the theta burst analysis, the tenth burst is analysed as representative of individual burst amplitude. Definitions of variables may be found below.

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The G2CMine data warehouse provides slice group summaries and individual mouse observations from the Akap9 knock-out line phenotyping.

 

Variables shown are: Max fEPSP amplitude, Maximum field excitatory postsynaptic potential (fEPSP) amplitude. PPF at 50ms, ampl ratio, Paired pulse facilitation (PPF), pulses separated by 50ms, amplitude ratio. Burst 1, PPF at 10ms, ampl ratio, Paired pulse facilitation (PPF), pulses separated by 10ms, amplitude ratio, observed during first two pulses of the first 100Hz burst during theta-burst stimulation. Burst 1, EPSP3 depr, ampl ratio, Depression observed in third fEPSP relative to the second fEPSP of the first 100Hz burst, amplitude ratio. Burst 1, ave amplitude, Average amplitude of four fEPSPs in first burst. Burst 2, ave amplitude, Average amplitude of four fEPSPs in second burst. Burst 3, ave amplitude, Average amplitude of four fEPSPs in third burst. Burst 4, ave amplitude, Average amplitude of four fEPSPs in fourth burst. Burst 5, ave amplitude, Average amplitude of four fEPSPs in fifth burst. Burst 6, ave amplitude, Average amplitude of four fEPSPs in sixth burst. Burst 7, ave amplitude, Average amplitude of four fEPSPs in seventh burst. Burst 8, ave amplitude, Average amplitude of four fEPSPs in eighth burst. Burst 9, ave amplitude, Average amplitude of four fEPSPs in ninth burst. Burst 10, ave amplitude, Average amplitude of four fEPSPs in tenth burst. Burst 2-4 ave amplitude vs Burst 1, Facilitation observed in average amplitude of bursts 2-4, relative to average amplitude of burst 1. Burst 8-10 ave amplitude vs Burst 2-4, Depression observed in average amplitude of bursts 8-10, relative to average amplitude of bursts 2-4. LTP vs PTP, amplitude ratio, Reduction in potentiation from immediately after theta-burst stimulation to one hour later, fEPSP amplitude ratio. LTP based on amplitude, Long term potentiation, ratio of amplitudes of fEPSPs in test pathway and control pathway.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
Max fEPSP amplitude µV 17 (6) 3050 (120) 26 (9) 3050 (160) 0.012 * 1
PPF at 50ms, ampl ratio % 17 (6) 178.9 (2.5) 26 (9) 171.6 (2.5) 0.71 0.048 *
Burst 1, PPF at 10ms, ampl ratio % 17 (6) 211.6 (6.3) 26 (9) 180.3 (3.6) 0.88 0.00017 ***
Burst 1, EPSP3 depr, ampl ratio % 17 (6) 66.6 (3) 26 (9) 67.1 (2.2) 0.19 0.9
Burst 1, ave amplitude µV 17 (6) 1253 (38) 26 (9) 1335 (62) 0.0087 ** 0.49
Burst 2, ave amplitude µV 17 (6) 1932 (66) 26 (9) 2011 (95) 0.0013 ** 0.69
Burst 3, ave amplitude µV 17 (6) 2223 (81) 26 (9) 2271 (106) 0.0014 ** 0.83
Burst 4, ave amplitude µV 17 (6) 2322 (86) 26 (9) 2367 (101) 0.002 ** 0.83
Burst 5, ave amplitude µV 17 (6) 2309 (80) 26 (9) 2350 (96) 0.0025 ** 0.84
Burst 6, ave amplitude µV 17 (6) 2254 (76) 26 (9) 2286 (90) 0.0025 ** 0.87
Burst 7, ave amplitude µV 17 (6) 2185 (73) 26 (9) 2222 (86) 0.0053 ** 0.84
Burst 8, ave amplitude µV 17 (6) 2110 (72) 26 (9) 2153 (86) 0.0039 ** 0.81
Burst 9, ave amplitude µV 17 (6) 2051 (76) 26 (9) 2097 (83) 0.0034 ** 0.79
Burst 10, ave amplitude µV 17 (6) 1995 (74) 26 (9) 2036 (81) 0.0056 ** 0.81
Burst 2-4 ave amplitude vs Burst 1 % 17 (6) 173.5 (3.1) 26 (9) 167.6 (3.4) 0.098 0.35
Burst 8-10 ave amplitude vs Burst 2-4 % 17 (6) 95.4 (1.6) 26 (9) 95.5 (1.4) 0.8 0.96
LTP vs PTP, amplitude ratio % 17 (6) 80.7 (1.2) 26 (9) 77.5 (1.4) 0.14 0.18
LTP based on amplitude % 17 (6) 185.7 (5.3) 26 (9) 167.4 (3.2) 0.35 0.013 *

 

Basal Synaptic Transmission

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Variables shown are: Max fEPSP amplitude, Maximum field excitatory postsynaptic potential (fEPSP) amplitude.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
Max fEPSP amplitude µV 17 (6) 3050 (120) 26 (9) 3050 (160) 0.012 * 1

 

Paired Pulse Facilitation

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Variables shown are: PPF at 50ms, ampl ratio, Paired pulse facilitation (PPF), pulses separated by 50ms, amplitude ratio.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
PPF at 50ms, ampl ratio % 17 (6) 178.9 (2.5) 26 (9) 171.6 (2.5) 0.71 0.048 *

 

Theta Burst Stimulation

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Variables shown are: Burst 1, PPF at 10ms, ampl ratio, Paired pulse facilitation (PPF), pulses separated by 10ms, amplitude ratio, observed during first two pulses of the first 100Hz burst during theta-burst stimulation. Burst 1, EPSP3 depr, ampl ratio, Depression observed in third fEPSP relative to the second fEPSP of the first 100Hz burst, amplitude ratio. Burst 1, ave amplitude, Average amplitude of four fEPSPs in first burst. Burst 2, ave amplitude, Average amplitude of four fEPSPs in second burst. Burst 3, ave amplitude, Average amplitude of four fEPSPs in third burst. Burst 4, ave amplitude, Average amplitude of four fEPSPs in fourth burst. Burst 5, ave amplitude, Average amplitude of four fEPSPs in fifth burst. Burst 6, ave amplitude, Average amplitude of four fEPSPs in sixth burst. Burst 7, ave amplitude, Average amplitude of four fEPSPs in seventh burst. Burst 8, ave amplitude, Average amplitude of four fEPSPs in eighth burst. Burst 9, ave amplitude, Average amplitude of four fEPSPs in ninth burst. Burst 10, ave amplitude, Average amplitude of four fEPSPs in tenth burst. Burst 2-4 ave amplitude vs Burst 1, Facilitation observed in average amplitude of bursts 2-4, relative to average amplitude of burst 1. Burst 8-10 ave amplitude vs Burst 2-4, Depression observed in average amplitude of bursts 8-10, relative to average amplitude of bursts 2-4.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
Burst 1, PPF at 10ms, ampl ratio % 17 (6) 211.6 (6.3) 26 (9) 180.3 (3.6) 0.88 0.00017 ***
Burst 1, EPSP3 depr, ampl ratio % 17 (6) 66.6 (3) 26 (9) 67.1 (2.2) 0.19 0.9
Burst 1, ave amplitude µV 17 (6) 1253 (38) 26 (9) 1335 (62) 0.0087 ** 0.49
Burst 2, ave amplitude µV 17 (6) 1932 (66) 26 (9) 2011 (95) 0.0013 ** 0.69
Burst 3, ave amplitude µV 17 (6) 2223 (81) 26 (9) 2271 (106) 0.0014 ** 0.83
Burst 4, ave amplitude µV 17 (6) 2322 (86) 26 (9) 2367 (101) 0.002 ** 0.83
Burst 5, ave amplitude µV 17 (6) 2309 (80) 26 (9) 2350 (96) 0.0025 ** 0.84
Burst 6, ave amplitude µV 17 (6) 2254 (76) 26 (9) 2286 (90) 0.0025 ** 0.87
Burst 7, ave amplitude µV 17 (6) 2185 (73) 26 (9) 2222 (86) 0.0053 ** 0.84
Burst 8, ave amplitude µV 17 (6) 2110 (72) 26 (9) 2153 (86) 0.0039 ** 0.81
Burst 9, ave amplitude µV 17 (6) 2051 (76) 26 (9) 2097 (83) 0.0034 ** 0.79
Burst 10, ave amplitude µV 17 (6) 1995 (74) 26 (9) 2036 (81) 0.0056 ** 0.81
Burst 2-4 ave amplitude vs Burst 1 % 17 (6) 173.5 (3.1) 26 (9) 167.6 (3.4) 0.098 0.35
Burst 8-10 ave amplitude vs Burst 2-4 % 17 (6) 95.4 (1.6) 26 (9) 95.5 (1.4) 0.8 0.96

 

Long Term Potentiation

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Variables shown are: LTP vs PTP, amplitude ratio, Reduction in potentiation from immediately after theta-burst stimulation to one hour later, fEPSP amplitude ratio. LTP based on amplitude, Long term potentiation, ratio of amplitudes of fEPSPs in test pathway and control pathway.

 

Variable Units Wildtype slices (animals) Wildtype mean (SEM) Mutant slices (animals) Mutant mean (SEM) P(animals) P(mutation)
LTP vs PTP, amplitude ratio % 17 (6) 80.7 (1.2) 26 (9) 77.5 (1.4) 0.14 0.18
LTP based on amplitude % 17 (6) 185.7 (5.3) 26 (9) 167.4 (3.2) 0.35 0.013 *

Hippocampal brain slices summarised by genotype, followed by a list of the animals from which they were prepared.

View experimental protocols.

 

Hippocampal Slice Electrophysiology Cohort Summary

GenotypeCount
WT7
mutant9

 

Individual Mice

Mouse IDGenotype
wA8dd6f00afWT
wB8ba3ede92WT
wA8ba3ede92WT
wA2b7bb2cb3WT
wBda3acbb0aWT
wAff2030dbdWT
wB2b7bb2cb3WT
mB8dd6f00afmutant
mA6e7d4b530mutant
mBc55b2b4f5mutant
mB05c85738bmutant
mBd2553e61bmutant
mAda3acbb0amutant
mA05c85738bmutant
mAd2553e61bmutant
mAc55b2b4f5mutant

Field EPSP (Maximum)

Mouse IDGenotypeSlice NumberRecording ChannelFile Name
mA05c85738b mutant 1 12A 05c85738b_slice_1-2-3-x.zip
mA05c85738b mutant 2 52A 05c85738b_slice_1-2-3-x.zip
mA05c85738b mutant 3 12B 05c85738b_slice_1-2-3-x.zip
mB05c85738b mutant 4 55A 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 5 41A 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 6 55B 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 7 42B 05c85738b_slices_4-5-6-7.zip
mB8dd6f00af mutant 4 52A 8dd6f00af_slices_x-4-x-x.zip
mA6e7d4b530 mutant 2 54A 6e7d4b530_slices_1-2-3-x.zip
mA6e7d4b530 mutant 3 46B 6e7d4b530_slices_1-2-3-x.zip
mAda3acbb0a mutant 1 46A da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 2 52A da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 3 46B da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 4 46A da3acbb0a_slices_4-5-6-7.zip
mAc55b2b4f5 mutant 1 46A c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 2 42A c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 3 58B c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 4 12A c55b2b4f5_slices_4-5-6-7.zip
mBc55b2b4f5 mutant 5 73A c55b2b4f5_slices_4-5-6-7.zip
mBc55b2b4f5 mutant 6 58B c55b2b4f5_slices_4-5-6-7.zip
mAd2553e61b mutant 1 46A d2553e61b_slice_1-2-3-x.zip
mAd2553e61b mutant 2 43A d2553e61b_slice_1-2-3-x.zip
mAd2553e61b mutant 3 46B d2553e61b_slice_1-2-3-x.zip
mBd2553e61b mutant 4 42A d2553e61b_slices_x-4-5-6.zip
mBd2553e61b mutant 5 55B d2553e61b_slices_x-4-5-6.zip
mBd2553e61b mutant 6 43B d2553e61b_slices_x-4-5-6.zip
wA8ba3ede92 WT 1 55A 8ba3ede92_slices_1-2-3-x.zip
wA8ba3ede92 WT 3 55B 8ba3ede92_slices_1-2-3-x.zip
wA8ba3ede92 WT 4 38A 8ba3ede92_slices_4-5-6-7.zip
wB8ba3ede92 WT 6 12B 8ba3ede92_slices_4-5-6-7.zip
wB8ba3ede92 WT 7 52B 8ba3ede92_slices_4-5-6-7.zip
wA8dd6f00af WT 1 46A 8dd6f00af_slice_1-2-3-x.zip
wA8dd6f00af WT 2 41A 8dd6f00af_slice_1-2-3-x.zip
wA8dd6f00af WT 3 46B 8dd6f00af_slice_1-2-3-x.zip
wAff2030dbd WT 1 52A ff2030dbd_slices_x-1-x-x.zip
wBda3acbb0a WT 5 52A da3acbb0a_slices_4-5-6-7.zip
wBda3acbb0a WT 6 46B da3acbb0a_slices_4-5-6-7.zip
wBda3acbb0a WT 7 62B da3acbb0a_slices_4-5-6-7.zip
wA2b7bb2cb3 WT 2 52A 2b7bb2cb3_slices_1-2-3-x.zip
wA2b7bb2cb3 WT 3 12B 2b7bb2cb3_slices_1-2-3-x.zip
wA2b7bb2cb3 WT 4 46A 2b7bb2cb3_slices_4-5-6-7.zip
wA2b7bb2cb3 WT 5 52A 2b7bb2cb3_slices_4-5-6-7.zip
wB2b7bb2cb3 WT 6 46B 2b7bb2cb3_slices_4-5-6-7.zip
wB2b7bb2cb3 WT 7 43B 2b7bb2cb3_slices_4-5-6-7.zip

Paired Pulse Facilitation

Mouse IDGenotypeSlice NumberRecording ChannelFile Name
mA05c85738b mutant 1 12A 05c85738b_slice_1-2-3-x.zip
mA05c85738b mutant 2 52A 05c85738b_slice_1-2-3-x.zip
mA05c85738b mutant 3 12B 05c85738b_slice_1-2-3-x.zip
mB05c85738b mutant 4 55A 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 5 41A 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 6 55B 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 7 42B 05c85738b_slices_4-5-6-7.zip
mB8dd6f00af mutant 4 52A 8dd6f00af_slices_x-4-x-x.zip
mA6e7d4b530 mutant 2 54A 6e7d4b530_slices_1-2-3-x.zip
mA6e7d4b530 mutant 3 46B 6e7d4b530_slices_1-2-3-x.zip
mAda3acbb0a mutant 1 46A da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 2 52A da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 3 46B da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 4 46A da3acbb0a_slices_4-5-6-7.zip
mAc55b2b4f5 mutant 1 46A c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 2 42A c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 3 58B c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 4 12A c55b2b4f5_slices_4-5-6-7.zip
mBc55b2b4f5 mutant 5 73A c55b2b4f5_slices_4-5-6-7.zip
mBc55b2b4f5 mutant 6 58B c55b2b4f5_slices_4-5-6-7.zip
mAd2553e61b mutant 1 46A d2553e61b_slice_1-2-3-x.zip
mAd2553e61b mutant 2 43A d2553e61b_slice_1-2-3-x.zip
mAd2553e61b mutant 3 46B d2553e61b_slice_1-2-3-x.zip
mBd2553e61b mutant 4 42A d2553e61b_slices_x-4-5-6.zip
mBd2553e61b mutant 5 55B d2553e61b_slices_x-4-5-6.zip
mBd2553e61b mutant 6 43B d2553e61b_slices_x-4-5-6.zip
wA8ba3ede92 WT 1 55A 8ba3ede92_slices_1-2-3-x.zip
wA8ba3ede92 WT 3 55B 8ba3ede92_slices_1-2-3-x.zip
wA8ba3ede92 WT 4 38A 8ba3ede92_slices_4-5-6-7.zip
wB8ba3ede92 WT 6 12B 8ba3ede92_slices_4-5-6-7.zip
wB8ba3ede92 WT 7 52B 8ba3ede92_slices_4-5-6-7.zip
wA8dd6f00af WT 1 46A 8dd6f00af_slice_1-2-3-x.zip
wA8dd6f00af WT 2 41A 8dd6f00af_slice_1-2-3-x.zip
wA8dd6f00af WT 3 46B 8dd6f00af_slice_1-2-3-x.zip
wAff2030dbd WT 1 52A ff2030dbd_slices_x-1-x-x.zip
wBda3acbb0a WT 5 52A da3acbb0a_slices_4-5-6-7.zip
wBda3acbb0a WT 6 46B da3acbb0a_slices_4-5-6-7.zip
wBda3acbb0a WT 7 62B da3acbb0a_slices_4-5-6-7.zip
wA2b7bb2cb3 WT 2 52A 2b7bb2cb3_slices_1-2-3-x.zip
wA2b7bb2cb3 WT 3 12B 2b7bb2cb3_slices_1-2-3-x.zip
wA2b7bb2cb3 WT 4 46A 2b7bb2cb3_slices_4-5-6-7.zip
wA2b7bb2cb3 WT 5 52A 2b7bb2cb3_slices_4-5-6-7.zip
wB2b7bb2cb3 WT 6 46B 2b7bb2cb3_slices_4-5-6-7.zip
wB2b7bb2cb3 WT 7 43B 2b7bb2cb3_slices_4-5-6-7.zip

Long Term Potentiation

Mouse IDGenotypeSlice NumberRecording ChannelFile Name
mA05c85738b mutant 1 12A 05c85738b_slice_1-2-3-x.zip
mA05c85738b mutant 2 52A 05c85738b_slice_1-2-3-x.zip
mA05c85738b mutant 3 12B 05c85738b_slice_1-2-3-x.zip
mB05c85738b mutant 4 55A 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 5 41A 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 6 55B 05c85738b_slices_4-5-6-7.zip
mB05c85738b mutant 7 42B 05c85738b_slices_4-5-6-7.zip
mB8dd6f00af mutant 4 52A 8dd6f00af_slices_x-4-x-x.zip
mA6e7d4b530 mutant 2 54A 6e7d4b530_slices_1-2-3-x.zip
mA6e7d4b530 mutant 3 46B 6e7d4b530_slices_1-2-3-x.zip
mAda3acbb0a mutant 1 46A da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 2 52A da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 3 46B da3acbb0a_slices_1-2-3-x.zip
mAda3acbb0a mutant 4 46A da3acbb0a_slices_4-5-6-7.zip
mAc55b2b4f5 mutant 1 46A c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 2 42A c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 3 58B c55b2b4f5_slices_1-2-3-x.zip
mAc55b2b4f5 mutant 4 12A c55b2b4f5_slices_4-5-6-7.zip
mBc55b2b4f5 mutant 5 73A c55b2b4f5_slices_4-5-6-7.zip
mBc55b2b4f5 mutant 6 58B c55b2b4f5_slices_4-5-6-7.zip
mAd2553e61b mutant 1 46A d2553e61b_slice_1-2-3-x.zip
mAd2553e61b mutant 2 43A d2553e61b_slice_1-2-3-x.zip
mAd2553e61b mutant 3 46B d2553e61b_slice_1-2-3-x.zip
mBd2553e61b mutant 4 42A d2553e61b_slices_x-4-5-6.zip
mBd2553e61b mutant 5 55B d2553e61b_slices_x-4-5-6.zip
mBd2553e61b mutant 6 43B d2553e61b_slices_x-4-5-6.zip
wA8ba3ede92 WT 1 55A 8ba3ede92_slices_1-2-3-x.zip
wA8ba3ede92 WT 3 55B 8ba3ede92_slices_1-2-3-x.zip
wA8ba3ede92 WT 4 38A 8ba3ede92_slices_4-5-6-7.zip
wB8ba3ede92 WT 6 12B 8ba3ede92_slices_4-5-6-7.zip
wB8ba3ede92 WT 7 52B 8ba3ede92_slices_4-5-6-7.zip
wA8dd6f00af WT 1 46A 8dd6f00af_slice_1-2-3-x.zip
wA8dd6f00af WT 2 41A 8dd6f00af_slice_1-2-3-x.zip
wA8dd6f00af WT 3 46B 8dd6f00af_slice_1-2-3-x.zip
wAff2030dbd WT 1 52A ff2030dbd_slices_x-1-x-x.zip
wBda3acbb0a WT 5 52A da3acbb0a_slices_4-5-6-7.zip
wBda3acbb0a WT 6 46B da3acbb0a_slices_4-5-6-7.zip
wBda3acbb0a WT 7 62B da3acbb0a_slices_4-5-6-7.zip
wA2b7bb2cb3 WT 2 52A 2b7bb2cb3_slices_1-2-3-x.zip
wA2b7bb2cb3 WT 3 12B 2b7bb2cb3_slices_1-2-3-x.zip
wA2b7bb2cb3 WT 4 46A 2b7bb2cb3_slices_4-5-6-7.zip
wA2b7bb2cb3 WT 5 52A 2b7bb2cb3_slices_4-5-6-7.zip
wB2b7bb2cb3 WT 6 46B 2b7bb2cb3_slices_4-5-6-7.zip
wB2b7bb2cb3 WT 7 43B 2b7bb2cb3_slices_4-5-6-7.zip

Data Files

File NameSizeChecksum (SHA256)
d2553e61b_slices_x-4-5-6.zip437006248655fd4a24a7a92ff8d35a25c165aadfa99001bf8971c7eb02eb55b653dadb4f
da3acbb0a_slices_4-5-6-7.zip55172076f177f4e9a3f1b0575249091504ce3244ad8df9f9c039b684fb3aecbd989b2fec
05c85738b_slices_4-5-6-7.zip4366802081eb567cf66b84a5a6f89dae55ecca3fcf7aaab2d7fa519c0f27409b13c6ba31
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