AssayResult: 82.06

AssayResultAssertion: Indeterminate

PValue: 0.0058

Comment: Exact values reported in Table S3.

AssayResult: 76.45

AssayResultAssertion: Indeterminate

PValue: 0.0001

Comment: Exact values reported in Table S3.

AssayResult: 86.74

AssayResultAssertion: Not reported

PValue: 0.1836

Comment: Exact values reported in Table S3.

HGVS: NM_024675.3:c.104T>C p.(Leu35Pro)

Reviewer #1 (Public Review):

The authors used a CRISPR screen to investigate the basis of metastasis of ovarian cancer (OC) cells. Overall, they identified two key genes, IL20RA, one of which was studied in detail. They identify an IL20/IL20RA communication between ovarian cancer cells and peritoneal mesothelial cells to promote M1 macrophages and prevent dissemination of the cancer cells. IL-20 mediated crosstalk is blocked in metastasized OC cells by decreased expression of IL-20RA. Interestingly, IL20RA is also decreased in cells from OC patients with peritoneal metastasis, and reconstitution of IL20RA in metastatic OC cells suppresses metastasis. Moreover, OC cells induce mesothelial cells to produce IL20 and IL24.

Overall, this is a nice study. It is well-written, and the data are clear. A range of methodologies are used that support the conclusions, with both over-expression and under-expression related studies supporting some key conclusions.

The overall model is that there is crosstalk between disseminated OC cells and mesothelial cells and macrophages. OC cells when disseminated into the peritoneal cavity stimulate mesothelial cells to produced IL20 and IL24, which via IL20RA trigger STAT3 to produced OAS/RNase L and production of IL-18, to promote an M1 phenotype. The M1 phenotype lowers metastasis. Highly metastatic cells block this pathway by decreasing IL20RA expression.

These findings are interesting, with potential therapeutic ramifications.

AssayGeneralClass: BAO:0003061 reporter protein

AssayMaterialUsed: CLO:0037317 mouse embryonic stem cell line

AssayDescription: Stable expression of wild type and variant PALB2 cDNA constructs in Trp53 and Palb2-null mouse cell line containing DR-GFP reporter; I-SceI endonuclease introduces a double-stranded break in the reporter construct and efficient repair results in GFP expression, which is detected by flow cytometry

AssayReadOutDescription: Relative homologous recombination (HR) efficiency represented as mean percentages of GFP-positive cells among the mCherry-positive cells relative to wild type, which was set to 100%

AssayRange: %

AssayNormalRange: HR levels comparable to that of cells expressing wild type PALB2; no numeric threshold given

AssayAbnormalRange: HR levels ≤40% of wild type

AssayIndeterminateRange: Not reported

ValidationControlPathogenic: 12

ValidationControlBenign: 9

Replication: 2 independent experiments

StatisticalAnalysisDescription: Not reported

AssayResult: 115.71

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardErrorMean: 3.09

Comment: Exact values reported in “Source Data” file.

AssayResult: 80.95

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 0.01

StandardErrorMean: 0

Comment: Exact values reported in “Source Data” file. Discrepancy in “Supplementary Data 1” file: nucleotide reported as c.3191A>G.

AssayResult: 101.02

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 5.45

StandardErrorMean: 3.86

Comment: Exact values reported in “Source Data” file. Discrepancy in “Source Data” file: protein reported as T1064C.

AssayResult: 84.43

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 2.77

StandardErrorMean: 1.96

Comment: Exact values reported in “Source Data” file. Discrepancy in “Source Data” file: protein reported as L855P (based on matching values reported in the “Supplementary Data 1” file to values reported in the “Source Data” file.

AssayResult: 15.58

AssayResultAssertion: Abnormal

ReplicateCount: 6

StandardDeviation: 0.52

StandardErrorMean: 0.37

ControlType: Abnormal; empty vector (set 5)

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.93

AssayResultAssertion: Abnormal

ReplicateCount: 6

StandardDeviation: 0.56

StandardErrorMean: 0.39

ControlType: Abnormal; empty vector (set 4)

Comment: Exact values reported in “Source Data” file.

AssayResult: 8.71

AssayResultAssertion: Abnormal

ReplicateCount: 6

StandardDeviation: 1.75

StandardErrorMean: 1.24

ControlType: Abnormal; empty vector (set 3)

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.11

AssayResultAssertion: Abnormal

ReplicateCount: 6

StandardDeviation: 2.37

StandardErrorMean: 1.68

ControlType: Abnormal; empty vector (set 2)

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.83

AssayResultAssertion: Abnormal

ReplicateCount: 6

StandardDeviation: 1.13

StandardErrorMean: 0.8

ControlType: Abnormal; empty vector (set 1)

Comment: Exact values reported in “Source Data” file.

AssayResult: 100

AssayResultAssertion: Normal

ReplicateCount: 38

StandardDeviation: 0

StandardErrorMean: 0

ControlType: Normal; wild type PALB2 cDNA

Comment: Exact values reported in “Source Data” file.

AssayResult: 97.16

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 1.32

StandardErrorMean: 0.93

Comment: Exact values reported in “Source Data” file.

AssayResult: 30.35

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 3.64

StandardErrorMean: 2.57

Comment: Exact values reported in “Source Data” file.

AssayResult: 20.32

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 0.49

StandardErrorMean: 0.35

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.42

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 2.87

StandardErrorMean: 2.03

Comment: Exact values reported in “Source Data” file.

AssayResult: 91.47

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 8.63

StandardErrorMean: 6.1

Comment: Exact values reported in “Source Data” file.

AssayResult: 100.19

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 5.64

StandardErrorMean: 3.99

Comment: Exact values reported in “Source Data” file.

AssayResult: 10.53

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 2.79

StandardErrorMean: 1.97

Comment: Exact values reported in “Source Data” file.

AssayResult: 90.64

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 1.82

StandardErrorMean: 1.29

Comment: Exact values reported in “Source Data” file.

AssayResult: 11.37

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 0.36

StandardErrorMean: 0.26

Comment: Exact values reported in “Source Data” file.

AssayResult: 70.86

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 17.18

StandardErrorMean: 12.15

Comment: Exact values reported in “Source Data” file.

AssayResult: 81.81

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 7.45

StandardErrorMean: 5.27

Comment: Exact values reported in “Source Data” file.

AssayResult: 90.54

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 10.24

StandardErrorMean: 7.24

Comment: Exact values reported in “Source Data” file.

AssayResult: 13.45

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 2.2

StandardErrorMean: 1.55

Comment: Exact values reported in “Source Data” file.

AssayResult: 92.2

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 19.94

StandardErrorMean: 14.1

Comment: Exact values reported in “Source Data” file.

AssayResult: 90.79

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 6.38

StandardErrorMean: 4.51

Comment: Exact values reported in “Source Data” file.

AssayResult: 69.83

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 5.94

StandardErrorMean: 4.2

Comment: Exact values reported in “Source Data” file.

AssayResult: 75.85

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 4.78

StandardErrorMean: 3.38

Comment: Exact values reported in “Source Data” file.

AssayResult: 94.33

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 9.99

StandardErrorMean: 7.07

Comment: Exact values reported in “Source Data” file.

AssayResult: 102.58

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 2.19

StandardErrorMean: 1.55

Comment: Exact values reported in “Source Data” file.

AssayResult: 21.79

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 1.84

StandardErrorMean: 1.3

Comment: Exact values reported in “Source Data” file.

AssayResult: 95.86

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 1.62

StandardErrorMean: 1.15

Comment: Exact values reported in “Source Data” file.

AssayResult: 91.21

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 7.32

StandardErrorMean: 5.18

Comment: Exact values reported in “Source Data” file.

AssayResult: 10.59

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 0.6

StandardErrorMean: 0.43

Comment: Exact values reported in “Source Data” file.

AssayResult: 76.97

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 3.1

StandardErrorMean: 2.19

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.92

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 1.32

StandardErrorMean: 0.94

Comment: Exact values reported in “Source Data” file.

AssayResult: 38.85

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 3.05

StandardErrorMean: 2.15

Comment: Exact values reported in “Source Data” file.

AssayResult: 16.89

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 3.12

StandardErrorMean: 2.21

Comment: Exact values reported in “Source Data” file.

AssayResult: 17.62

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 1.76

StandardErrorMean: 1.25

Comment: Exact values reported in “Source Data” file.

AssayResult: 86.41

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 17.62

StandardErrorMean: 12.46

Comment: Exact values reported in “Source Data” file.

AssayResult: 6.16

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 1.27

StandardErrorMean: 0.9

Comment: Exact values reported in “Source Data” file.

AssayResult: 95.16

AssayResultAssertion: Not reported

ReplicateCount: 3

StandardDeviation: 16.94

StandardErrorMean: 9.78

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.08

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 0.54

StandardErrorMean: 0.38

Comment: Exact values reported in “Source Data” file.

AssayResult: 14.01

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 0.53

StandardErrorMean: 0.38

Comment: Exact values reported in “Source Data” file.

AssayResult: 74.49

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 2.29

StandardErrorMean: 1.62

Comment: Exact values reported in “Source Data” file.

AssayResult: 6.53

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 0.29

StandardErrorMean: 0.21

Comment: Exact values reported in “Source Data” file.

AssayResult: 30.27

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 1.04

StandardErrorMean: 0.74

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.63

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 0.51

StandardErrorMean: 0.36

Comment: Exact values reported in “Source Data” file.

AssayResult: 63.4

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 5.92

StandardErrorMean: 4.18

Comment: Exact values reported in “Source Data” file.

AssayResult: 60.28

AssayResultAssertion: Not reported

ReplicateCount: 3

StandardDeviation: 0.14

StandardErrorMean: 0.1

Comment: Exact values reported in “Source Data” file.

AssayResult: 17.35

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 6.21

StandardErrorMean: 4.39

Comment: Exact values reported in “Source Data” file.

AssayResult: 106.23

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 14.57

StandardErrorMean: 10.3

Comment: Exact values reported in “Source Data” file.

AssayResult: 75.71

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 22.31

StandardErrorMean: 15.77

Comment: Exact values reported in “Source Data” file.

AssayResult: 6.66

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 0.28

StandardErrorMean: 0.2

Comment: Exact values reported in “Source Data” file.

AssayResult: 6.1

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 2.11

StandardErrorMean: 1.49

Comment: Exact values reported in “Source Data” file.

AssayResult: 84.07

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 2.47

StandardErrorMean: 1.75

Comment: Exact values reported in “Source Data” file.

AssayResult: 100.07

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 6.18

StandardErrorMean: 4.37

Comment: Exact values reported in “Source Data” file.

AssayResult: 91.6

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 8.2

StandardErrorMean: 5.8

Comment: Exact values reported in “Source Data” file.

AssayResult: 82.83

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 4.82

StandardErrorMean: 3.41

Comment: Exact values reported in “Source Data” file.

AssayResult: 87.35

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 11.94

StandardErrorMean: 8.44

Comment: Exact values reported in “Source Data” file.

AssayResult: 83.25

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 5.27

StandardErrorMean: 3.73

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.03

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 2.68

StandardErrorMean: 1.9

Comment: Exact values reported in “Source Data” file.

AssayResult: 77.45

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 6.2

StandardErrorMean: 4.38

Comment: Exact values reported in “Source Data” file.

AssayResult: 9.92

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 1.93

StandardErrorMean: 1.37

Comment: Exact values reported in “Source Data” file.

AssayResult: 95.02

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 0.08

StandardErrorMean: 0.06

Comment: Exact values reported in “Source Data” file.

AssayResult: 10.4

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 3.22

StandardErrorMean: 2.28

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.75

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 2.59

StandardErrorMean: 1.83

Comment: Exact values reported in “Source Data” file.

AssayResult: 75.45

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 4.03

StandardErrorMean: 2.85

Comment: Exact values reported in “Source Data” file.

AssayResult: 98.55

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 5.74

StandardErrorMean: 4.06

Comment: Exact values reported in “Source Data” file.

AssayResult: 62.31

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 11.49

StandardErrorMean: 8.13

Comment: Exact values reported in “Source Data” file.

AssayResult: 66.19

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 21.26

StandardErrorMean: 15.03

Comment: Exact values reported in “Source Data” file.

AssayResult: 105.41

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 9.45

StandardErrorMean: 6.68

Comment: Exact values reported in “Source Data” file.

AssayResult: 7.82

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 2.31

StandardErrorMean: 1.64

Comment: Exact values reported in “Source Data” file.

AssayResult: 92.32

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 2.26

StandardErrorMean: 1.6

Comment: Exact values reported in “Source Data” file.

AssayResult: 44.9

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 9.75

StandardErrorMean: 6.89

Comment: Exact values reported in “Source Data” file.

AssayResult: 97.61

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 0.97

StandardErrorMean: 0.68

Comment: Exact values reported in “Source Data” file.

AssayResult: 11.28

AssayResultAssertion: Abnormal

ReplicateCount: 2

StandardDeviation: 1.24

StandardErrorMean: 0.87

Comment: Exact values reported in “Source Data” file.

AssayResult: 86.67

AssayResultAssertion: Not reported

ReplicateCount: 2

StandardDeviation: 2.24

StandardErrorMean: 1.58

Comment: Exact values reported in “Source Data” file.

HGVS: NM_024675.3:c.1010T>C p.(L337S)

AssayGeneralClass: BAO:0000062 patch clamp

AssayMaterialUsed: CLO:0037237 HEK293-derived cell

AssayDescription: HEK293T-derived cells stably expressing wild type or variant SCN5A were patch clamped and currents elicited in response to activation, inactivation, and recovery from inactivation were recorded, as well as late current measurements.

AssayReadOutDescription: Peak current density relative to wild type, which was set to 100%

AssayRange: %

AssayNormalRange: Peak current density 75-125% of wild type

AssayAbnormalRange: Peak current density 10-50% of wildtype

AssayIndeterminateRange: Peak current density 50-75% of wildtype

ValidationControlPathogenic: 0

ValidationControlBenign: 10

Replication: At least 2 independent transfections and at least 10 replicate cells per variant (see ReplicateCount in FunctionalAssayResult annotations for each variant).

StatisticalAnalysisDescription: Two-tailed t tests or two-tailed Mann-Whitney U tests were used to compare variant parameters between groups of variants, while differences in dispersion between groups were tested with Levene’s test.

AssayResult: 100

AssayResultAssertion: Normal

ReplicateCount: 471

StandardErrorMean: 3.7

ControlType: Normal; wild type

Comment: This variant (wildtype) had normal function. All other variant parameters were normalized to the values of wildtype. (Personal communication: A. Glazer)

AssayResult: 59.3

AssayResultAssertion: Indeterminate

ReplicateCount: 30

StandardErrorMean: 8.3

Comment: This variant had mild loss of function (peak current >50% and <75% of wildtype), therefore it was considered inconclusive and neither abnormal nor normal in vitro function. (Personal communication: A. Glazer)

AssayResult: 28.4

AssayResultAssertion: Abnormal

ReplicateCount: 13

StandardErrorMean: 8.6

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 45.3

AssayResultAssertion: Abnormal

ReplicateCount: 31

StandardErrorMean: 5.1

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1).

AssayResult: 37.2

AssayResultAssertion: Abnormal

ReplicateCount: 26

StandardErrorMean: 3.8

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 23.1

AssayResultAssertion: Abnormal

ReplicateCount: 33

StandardErrorMean: 3.2

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 89.5

AssayResultAssertion: Normal

ReplicateCount: 29

StandardErrorMean: 14.6

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 0.9

AssayResultAssertion: Abnormal

ReplicateCount: 18

StandardErrorMean: 0.5

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 5.4

AssayResultAssertion: Abnormal

ReplicateCount: 19

StandardErrorMean: 1.5

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 14.8

AssayResultAssertion: Abnormal

ReplicateCount: 27

StandardErrorMean: 2.5

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 78.9

AssayResultAssertion: Normal

ReplicateCount: 38

StandardErrorMean: 7.2

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 43.3

AssayResultAssertion: Abnormal

ReplicateCount: 14

StandardErrorMean: 12.2

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 59.7

AssayResultAssertion: Indeterminate

ReplicateCount: 41

StandardErrorMean: 6.3

Comment: This variant had mild loss of function (peak current >50% and <75% of wildtype), therefore it was considered inconclusive and neither abnormal nor normal in vitro function. (Personal communication: A. Glazer)

AssayResult: 10.2

AssayResultAssertion: Abnormal

ReplicateCount: 12

StandardErrorMean: 3.4

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 0.3

AssayResultAssertion: Abnormal

ReplicateCount: 24

StandardErrorMean: 0.3

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 0

AssayResultAssertion: Abnormal

ReplicateCount: 11

StandardErrorMean: 0

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 3

AssayResultAssertion: Abnormal

ReplicateCount: 16

StandardErrorMean: 1.5

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 32.6

AssayResultAssertion: Abnormal

ReplicateCount: 10

StandardErrorMean: 6.2

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 36

AssayResultAssertion: Abnormal

ReplicateCount: 14

StandardErrorMean: 6

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 13.9

AssayResultAssertion: Abnormal

ReplicateCount: 15

StandardErrorMean: 2.8

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 3.5

AssayResultAssertion: Abnormal

ReplicateCount: 29

StandardErrorMean: 0.8

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 0.2

AssayResultAssertion: Abnormal

ReplicateCount: 25

StandardErrorMean: 0.2

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 102.6

AssayResultAssertion: Normal

ReplicateCount: 31

StandardErrorMean: 16.5

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 1.6

AssayResultAssertion: Abnormal

ReplicateCount: 15

StandardErrorMean: 0.7

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 12

AssayResultAssertion: Abnormal

ReplicateCount: 10

StandardErrorMean: 2.2

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 102.4

AssayResultAssertion: Normal

ReplicateCount: 39

StandardErrorMean: 15.5

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 47

AssayResultAssertion: Indeterminate

ReplicateCount: 10

StandardErrorMean: 15.5

Comment: This variant had a mix of multiple abnormalities: a partial loss of function of peak current (10-50% of wildtype) and a gain of function >10mV shift in activation voltage. Therefore it was considered to have inconclusive in vitro properties (neither normal nor abnormal in vitro function). (Personal communication: A. Glazer)

AssayResult: 114.7

AssayResultAssertion: Normal

ReplicateCount: 42

StandardErrorMean: 15.2

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 36

AssayResultAssertion: Abnormal

ReplicateCount: 19

StandardErrorMean: 5.9

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 121.4

AssayResultAssertion: Normal

ReplicateCount: 34

StandardErrorMean: 13.2

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 1.1

AssayResultAssertion: Abnormal

ReplicateCount: 27

StandardErrorMean: 0.8

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 29.8

AssayResultAssertion: Abnormal

ReplicateCount: 13

StandardErrorMean: 5.7

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 3.2

AssayResultAssertion: Abnormal

ReplicateCount: 16

StandardErrorMean: 0.5

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 0.8

AssayResultAssertion: Abnormal

ReplicateCount: 23

StandardErrorMean: 0.6

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 0

AssayResultAssertion: Abnormal

ReplicateCount: 43

StandardErrorMean: 0

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 16

AssayResultAssertion: Abnormal

ReplicateCount: 26

StandardErrorMean: 2.3

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 2.9

AssayResultAssertion: Abnormal

ReplicateCount: 20

StandardErrorMean: 2.1

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 117.2

AssayResultAssertion: Abnormal

ReplicateCount: 36

StandardErrorMean: 11.7

Comment: This variant had normal peak current and increased late current (>1% of peak), therefore it was considered a GOF variant (in vitro features consistent with Long QT Syndrome Type 3). (Personal communication: A. Glazer)

AssayResult: 21

AssayResultAssertion: Abnormal

ReplicateCount: 12

StandardErrorMean: 5.1

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 38.9

AssayResultAssertion: Abnormal

ReplicateCount: 27

StandardErrorMean: 7.2

Comment: This variant had partial loss of function of peak current (10-50% of wildtype) and a >10mV loss of function shift in Vhalf activation, therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 120.5

AssayResultAssertion: Normal

ReplicateCount: 41

StandardErrorMean: 10.5

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 105.3

AssayResultAssertion: Normal

ReplicateCount: 41

StandardErrorMean: 10.8

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 77.5

AssayResultAssertion: Normal

ReplicateCount: 30

StandardErrorMean: 8.6

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 41.7

AssayResultAssertion: Abnormal

ReplicateCount: 15

StandardErrorMean: 10.8

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 63.8

AssayResultAssertion: Indeterminate

ReplicateCount: 25

StandardErrorMean: 10.1

Comment: This variant had mild loss of function (peak current >50% and <75% of wildtype), therefore it was considered inconclusive and neither abnormal nor normal in vitro function. (Personal communication: A. Glazer)

AssayResult: 0.9

AssayResultAssertion: Abnormal

ReplicateCount: 12

StandardErrorMean: 0.6

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 68.1

AssayResultAssertion: Indeterminate

ReplicateCount: 18

StandardErrorMean: 8.7

Comment: This variant had mild loss of function (peak current >50% and <75% of wildtype), therefore it was considered inconclusive and neither abnormal nor normal in vitro function. (Personal communication: A. Glazer)

AssayResult: 32

AssayResultAssertion: Abnormal

ReplicateCount: 31

StandardErrorMean: 5

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 1.2

AssayResultAssertion: Abnormal

ReplicateCount: 11

StandardErrorMean: 0.7

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 3.4

AssayResultAssertion: Abnormal

ReplicateCount: 22

StandardErrorMean: 0.8

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 0

AssayResultAssertion: Abnormal

ReplicateCount: 39

StandardErrorMean: 0

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 0.6

AssayResultAssertion: Abnormal

ReplicateCount: 25

StandardErrorMean: 0.4

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 28.5

AssayResultAssertion: Abnormal

ReplicateCount: 21

StandardErrorMean: 7.6

Comment: This variant had partial loss of function of peak current (10-50% of wildtype) and a >10mV loss of function shift in Vhalf activation, therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 113.2

AssayResultAssertion: Normal

ReplicateCount: 30

StandardErrorMean: 13.9

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 0

AssayResultAssertion: Abnormal

ReplicateCount: 24

StandardErrorMean: 0

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 1.3

AssayResultAssertion: Abnormal

ReplicateCount: 67

StandardErrorMean: 0.3

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 0.8

AssayResultAssertion: Abnormal

ReplicateCount: 14

StandardErrorMean: 0.6

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 34.2

AssayResultAssertion: Abnormal

ReplicateCount: 14

StandardErrorMean: 6.7

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 109.6

AssayResultAssertion: Normal

ReplicateCount: 11

StandardErrorMean: 19.8

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 117.8

AssayResultAssertion: Normal

ReplicateCount: 15

StandardErrorMean: 14.5

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 39

AssayResultAssertion: Abnormal

ReplicateCount: 16

StandardErrorMean: 6.4

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 119.6

AssayResultAssertion: Normal

ReplicateCount: 22

StandardErrorMean: 19.5

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 0.2

AssayResultAssertion: Abnormal

ReplicateCount: 15

StandardErrorMean: 0.2

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 32.8

AssayResultAssertion: Abnormal

ReplicateCount: 16

StandardErrorMean: 5

Comment: This variant had partial loss of function of peak current (10-50% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 89.4

AssayResultAssertion: Normal

ReplicateCount: 26

StandardErrorMean: 12.7

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 85.1

AssayResultAssertion: Normal

ReplicateCount: 35

StandardErrorMean: 10.6

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 103.2

AssayResultAssertion: Normal

ReplicateCount: 33

StandardErrorMean: 12.7

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 120.5

AssayResultAssertion: Normal

ReplicateCount: 33

StandardErrorMean: 13.6

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 94.8

AssayResultAssertion: Normal

ReplicateCount: 33

StandardErrorMean: 12.6

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 109.1

AssayResultAssertion: Normal

ReplicateCount: 26

StandardErrorMean: 14.8

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 101

AssayResultAssertion: Normal

ReplicateCount: 41

StandardErrorMean: 8.9

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 104.3

AssayResultAssertion: Normal

ReplicateCount: 30

StandardErrorMean: 16.3

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 105.8

AssayResultAssertion: Normal

ReplicateCount: 36

StandardErrorMean: 12.7

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 103.2

AssayResultAssertion: Normal

ReplicateCount: 37

StandardErrorMean: 21.8

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 51.9

AssayResultAssertion: Indeterminate

ReplicateCount: 12

StandardErrorMean: 18.8

Comment: This variant had a mild loss of function in peak current (50-75% of wildtype). It had unmeasured late current, but has been previously reported to have high late current (GOF feature). Therefore it was considered to meet neither the abnormal or normal functional parameter. (Personal communication: A. Glazer)

AssayResult: 64.8

AssayResultAssertion: Abnormal

ReplicateCount: 31

StandardErrorMean: 11.1

Comment: This variant had a mild loss of function in peak current (50-75% of wildtype). It also had a very large increase in recovery from inactivation (>10-fold slower). Therefore it was considered to have a partial loss of function (in vitro function consistent with Brugada Syndrome). (Personal communication: A. Glazer)

AssayResult: 2.2

AssayResultAssertion: Abnormal

ReplicateCount: 16

StandardErrorMean: 1

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1. (Personal communication: A. Glazer)

AssayResult: 114.3

AssayResultAssertion: Abnormal

ReplicateCount: 16

StandardErrorMean: 22.4

Comment: This variant had normal peak current and increased late current (>1% of peak), therefore it was considered a GOF variant (in vitro features consistent with Long QT Syndrome Type 3). (Personal communication: A. Glazer)

AssayResult: 23.2

AssayResultAssertion: Abnormal

ReplicateCount: 14

StandardErrorMean: 7.1

Comment: This variant had partial loss of function of peak current (10-50% of wildtype) and a >10mV loss of function shift in Vhalf activation, therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 113

AssayResultAssertion: Normal

ReplicateCount: 17

StandardErrorMean: 28.6

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 0.1

AssayResultAssertion: Abnormal

ReplicateCount: 19

StandardErrorMean: 0.1

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1. (Personal communication: A. Glazer)

AssayResult: 86.7

AssayResultAssertion: Normal

ReplicateCount: 28

StandardErrorMean: 8.6

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

AssayResult: 0.7

AssayResultAssertion: Abnormal

ReplicateCount: 17

StandardErrorMean: 0.6

Comment: This variant had loss of function of peak current (<10% of wildtype), therefore it was considered abnormal (in vitro features consistent with Brugada Syndrome Type 1). (Personal communication: A. Glazer)

AssayResult: 115.6

AssayResultAssertion: Normal

ReplicateCount: 19

StandardErrorMean: 24.7

Comment: This variant had normal function (75-125% of wildtype peak current, <1% late current, no large perturbations to other parameters). These in vitro features are consistent with non-disease causing variants. (Personal communication: A. Glazer)

HGVS: NM_198056.2:c.1003T>C p.(Cys335Arg)

AssayGeneralClass: BAO:0010044 targeted transcriptional assay

AssayMaterialUsed: BTO:0000773 lymphoblastoid cell line derived from control individuals or individuals with germline TP53 variants

AssayDescription: Comparative transcriptomic analysis using RNA-Seq to compare EBV cell lines of wild type and pathogenic TP53 in the context of genotoxic stress induced by doxorubicin treatment. 10 biomarkers corresponding to p53 targets were measured to determine a functionality score.

AdditionalDocument: PMID: 23172776

AssayReadOutDescription: In the treated condition, the peak height of each of the 10  p53 target genes was measured and divided by the sum of the heights of the three control genes. This value was then divided by the same ratio calculated in the untreated condition. In the assay, the mean of the 10 values defines the p53 functionality score. The final p53 functionality score is the mean of the scores obtained in RT-MLPA and RT-QMPSF assays.

AssayRange: An arbitrary functionality score was calculated from the induction score of the 10 p53 targets.

AssayNormalRange: N/A

AssayAbnormalRange: N/A

AssayIndeterminateRange: N/A

AssayNormalControl: wild type TP53

AssayAbnormalControl: LFS patient cells

ValidationControlPathogenic: 8 Individuals with dominant-negative TP53 missense variants, 10 Individuals with null TP53 variants, and 13 Individuals with other TP53 missense variants

ValidationControlBenign: 3 patients with wild type TP53

Replication: experiments were performed in triplicates.

StatisticalAnalysisDescription: Differentially expressed genes between doxorubicin-treated and untreated cells were arbitrarily defined using, as filters, a P<0.01 and fold-change cutoffs >2 or <2, for up and down regulation, respectively. The resultant signal information was analyzed using one-way analysis of variance (ANOVA, P= 0.001), assuming normality but not equal variances with a Benjamani–Hochberg correction for multiple comparisons using three groups: controls, null, and missense mutations.

SignificanceThreshold: P=0.001

Comment: statistical analysis and P value from previous publication.

Reviewer #1 (Public Review):

In this work, Panigrahi et. al. develop a powerful deep-learning-based cell segmentation platform (MiSiC) capable of accurately segmenting bacteria cells densely packed within both homogenous and heterogeneous cell populations. Notably, MiSiC can be easily implemented by a researcher without the need for high-computational power. The authors first demonstrate MiSiC's ability to accurately segment cells with a variety of shapes including rods, crescents and long filaments. They then demonstrate that MiSiC is able to segment and classify dividing and non-dividing Myxococcus cells present in a heterogenous population of E. coli and Myxococcus. Lastly, the authors outline a training workflow with which MiSiC can be trained to identify two different cell types present in a mixed population using Myxococcus and E. coli as examples.

While we believe that MiSiC is a very powerful and exciting tool that will have a large impact on the bacterial cell biological community, we feel explanations of how to use the algorithm should be more greatly emphasized. To help other scientists use MiSiC to its fullest potential, the range of applications should be clarified. Furthermore, any inherent biases in MiSiC should be discussed so that users can avoid them.

Major Concerns:

1) It is unclear to us how a MiSiC user should choose/tune the value for the noise variance parameter. What exactly should be considered when choosing the noise variance parameter? Some possibilities include input image size, cell size (in pixels), cell density, and variance in cell size. Is there a recommended range for the parameter? These questions along with our second minor correction can be addressed with a paragraph in the Discussion section.

2) Could the authors expand on using algorithms like watershed, conditional random fields, or snake segmentation to segment bacteria when there is not enough edge information to properly separate them? How accurate are these methods at segmenting the cells? Should other MiSiC parameters be tuned to increase the accuracy when implementing these methods?

3) Can the MiSiC's ability to accurately segment phase and brightfield images be quantitatively compared against each other and against fluorescent images for overall accuracy? A figure similar to Fig. 2C, with the three image modalities instead of species would nicely complement Fig. 2A. If the segmentation accuracy varies significantly between image modalities, a researcher might want to consider the segmentation accuracy when planning their experiments. If the accuracy does not vary significantly, that would be equally useful to know.

4) The ability of MiSiC to segment dense clusters of cells is an exciting advancement for cell segmentation algorithms. However, is there a minimum cell density required for robust segmentation with MiSiC? The algorithm should be applied to a set of sparsely populated images in a supplemental figure. Is the algorithm less accurate for sparse images (perhaps reflected by an increase in false-positive cell identifications)? Any possible biases related to cell density should be noted.

5) It is exciting to see the ability of MiSiC to segment single cells of M. xanthus and E. coli species in densely packed colonies (Fig. 4b). Although three morphological parameters after segmentation were compared with ground truth, the comparison was conducted at the ensemble level (Fig. 4c). Could the authors use the Mx-GFP and Ec-mCherry fluorescence as a ground truth at the single cell level to verify the results of segmentation? For example, for any Ec cells identified by MiSiC in Fig. 4b, provide an index of whether its fluorescence is red or green. This single-cell level comparison is most important for the community.

Reviewer #1 (Public Review):

In this paper, authors did a fine job of combining phylogenetics and molecular methods to demonstrate the parallel evolution across vRNA segments in two seasonal influenza A virus subtypes. They first estimated phylogenetic relationships between vRNA segments using Robinson-Foulds distance and identified the possibility of parallel evolution of RNA-RNA interactions driving the genomic assembly. This is indeed an interesting mechanism in addition to the traditional role for proteins for the same. Subsequently, they used molecular biology to validate such RNA-RNA driven interaction by demonstrating co-localization of vRNA segments in infected cells. They also showed that the parallel evolution between vRNA segments might vary across subtypes and virus lineages isolated from distinct host origins. Overall, I find this to be excellent work with major implications for genome evolution of infectious viruses; emergence of new strains with altered genome combination.

Comments:

I am wondering if leaving out sequences (not resolving well) in the phylogenic analysis interferes with the true picture of the proposed associations. What if they reflect the evolutionary intermediates, with important implications for the pathogen evolution which is lost in the analyses?

Lines 50-51: Can you please elaborate? I think this might be useful for the reader to better understand the context. Also, a brief description on functional association between different known fragments might instigate curiosity among the readers from the very beginning. At present, it largely caters to people already familiar with the biology of influenza virus.

Lines 95-96 Were these strains all swine-origin? More details on these lineages will be useful for the readers.

Lines 128-132: I think it will be nice to talk about these hypotheses well in advance, may be in the Introduction, with more functional details of viral segments.

Lines 134-136: Please rephrase this sentence to make it more direct and explain the why. E.g. "... parallel evolution between PB1 and HA is likely to be weaker than that of PB1 and PA" .

Lines 222-223: Please include a set of hypotheses to explain you results? Please add a perspective in the discussion on how this contribute might to the pandemic potential of H1N!?.

Lines 287-288: I am wondering how likely is this to be true for H1N1.

Reviewer #1 (Public Review):

In this paper, the authors tried to investigate complex roles of immune cells during acute myocardial infarction (AMI) by examining immune cells in blood samples from acute coronary syndrome (ACS) patients. They found an increase in the circulating levels of CD14+HLA-DRneg/low monocytes and CD16+CD66b+CD10neg neutrophils in the blood of ACS patients compared to healthy people, all of which were correlated with elevated levels of inflammatory markers in serum. Those findings were then further explored at a mechanistic level by using in vitro and in vivo experiments. Interestingly, the researchers also found that high cytomegalovirus (CMV) antibody titers could affect the immunoregulatory mechanisms in AMI patients. Taken together, the findings of the researchers could potentially contribute to the development of a more effective strategy to prevent cardiac deterioration and cardiovascular adverse events after AMI.

Strengths:

This paper contains novel insight regarding role of neutrophil and monocyte subset in pathophysiology of AMI. Although the increased level of CD10neg subsets of neutrophils in AMI patients has recently been reported (Marechal, P., et al. 2020. Neutrophil phenotypes in coronary artery disease. Journal of Clinical Medicine), the current paper aptly complemented the previous findings obtained by using its in vitro and in vivo mice model. This study also has robust methods to support their conclusion.

Weakness:

To further improve the strength of their conclusion, the experiments investigating the effects of immunoregulatory function of immature neutrophils and HLA-DRneg/low monocytes subsets would be advised.

Reviewer #1 (Public Review):

The manuscript by He et al. reveals a novel role for PKC-theta, following T cell receptor (TCR) stimulation, in regulating the nuclear translocation of several key activation-dependent transcription factors by regulating the assembly of key components of the nuclear pore complex (NPC). The authors make use of T cell lines and primary T cells to show that following TCR stimulation, PKC-theta phosphorylates RanGAP1 to promote its interaction with Ubc9 and increase the sumoylation of RanGAP1, which, in turn, enhances assembly of the RanBP2 subcomplex of the NPC that then promotes the nuclear import of AP-1, NFAT and NFB. These conclusions are well supported by a rigorous experimental approach, which included the use of PKC-theta deficient, sumoyltion-defective, kinase-dead, and constitutively active mutants, and RanGAP1-deficient cells.

Reviewer #1 (Public Review):

This paper uses a large breeding colony of guppies to measure genetic correlations between hormonal stress responses and behavior in an open-field test. Although we know a lot about the mechanisms of hormone-mediated behavior, we know less about variation in hormonal systems, particularly genetic variation. Understanding how hormones relate genetically to the behaviors they mediate is particularly important because it helps us understand how the entire hormone-behavior system evolves. A priori, we would expect genetic correlations between hormones and the behavior they underlie, such that selection on the hormone would lead to a response in the behavior and vice versa. However, evidence for this pattern is rare.

Here, the authors show that stress-induced levels of cortisol are repeatable and heritable. Interestingly, they also show that individuals show a lower stress response to later stress and slightly less variation, indicating a G X E interaction. There was a significant genetic correlation between the hormonal response and one of the behaviors measured in the open field test, and the hormone loaded positively in the first genetic principal component along with all the behaviors. This is evidence of an correlated suite of traits that would evolve together in response to selection.

This is an important study, because evidence of genetic variation in hormonal systems, not to mention genetic covariation with hormone-mediated traits, is rare. The results presented here provide insight into how a hormone-behavior complex might adapt to a changing environment. They are also relevant to ideas about the maintenance of variation in coping styles in natural populations.

Reviewer #1 (Public Review):

Using various voltage and concentration protocols in a heterologous expression system, the authors provide compelling evidence for strong block of GluR1 AMPA receptors by intracellular NASPM, and unlike spermine, the block is independent of auxiliary subunit expression. The authors also show that intracellular NASPM provides a more complete block than spermine of synaptic currents in GluR2-KO neurons.

Overall the manuscript contains high quality data that is clearly presented. It seems likely that this approach will be useful for assessing the contribution of CP-AMPARs in various scenarios. However, currently the authors have fallen short of providing a comprehensive analysis of the use of NASPM to differentiate between CP and CI AMPARs in intact systems containing multiple AMPAR subunits and auxiliary proteins.

Reviewer #1 (Public Review):

In "Asymptomatic Bordetella pertussis infections in a longitudinal cohort of young African infants and their mothers", the authors analyze longitudinal data from a cohort in Zambia of infant/mother pairs to investigate the evidence for subclinical and asymptomatic infections in both pairs as well as the use of IS481 qPCR cycle threshold (CT) values in providing evidence for pertussis infection. Overall, the manuscript lacks substantial statistical support or clear evidence of some of the patterns they are stating and would require a substantial revision to justify their conclusions. The majority of the manuscript relies on 8 infant/mother pairs where they have evidence of pertussis infection and rely on the dense sampling to investigate infection dynamics. However, this is a very small sample size and further, based on the results displayed in Figure 1, it is not obvious that the data has a very pattern that warrant their assertions.

Major comments:

The main results and conclusions are highly reliant on details from eight mother/infant pairs. However, Figure 1 does not show a clear picture of the fade-in/fade-out. The authors go into great detail describing each of these 8 pairs, however based on the figure and text there does not appear to be clear evidence of an underlying pattern. While there are some instances with a combination of higher/lower IS481 CT values, it does not appear to have a clear pattern. For example, what are possible explanations for time periods between samples with evidence of IS481 and those without (such as pair A, C, D, E, F and H)? There also does not appear to be a clear pattern of symptoms in any of these samples (aside from having fewer symptoms in the mothers than infants). Further, it is not obvious how similar these observed (such as a mixture of times of high or low values often preceded or followed by times when IS481 was not detected) is similar to different to the rest of the cohort (in contrast to those who have a definitive positive NP sample during a symptomatic visit). The main results are primarily a descriptive analysis of these 8 mother/infant pairs with little statistical analyses or additional support.

The authors do not provide evidence or detail about what is known about the variability in IS481 CT values, amongst individuals, or over time, or pre/post vaccination. Without this information, it is not clear how informative some of this variability is versus how much variability in these values is expected. I think particularly in Figure 1, how many of the individuals have periods between times when IS481 evidence was observed when it was not observed, is concerning that these data (at this granular a level) are measuring true infection dynamics. Adding in additional information about the distribution and patterns of these values for the other cohort members would also provide valuable insight into how Figure 1 should be interpreted in this context. As it stands, the authors do not provide sufficient interpretation and evidence for having relevant infection arcs.

It appears that Figure 2A was created using only 8 data points (from the infant data values). If so, this level of extrapolation from such few data points does not provide enough evidence to support to the results in the text (particularly about evidence for fade-in/fade-out population-level dynamics). Also, in Figure 2, it is not clear to me the added value of Figure 2C and the main goal of this figure.

The authors have created a measure called, evidence for infection (EFI), which is a summary measure of their IS481 CT values across the study. However, it is not clear why the authors are only considering an aggregated (sum) value which loses any temporality or relationship with symptoms/antibiotic use. For example, the values may have been high earlier in the study, but symptoms were unrelated to that evidence for infection - or visa versa. This seems to be an important factor - were these possible undiagnosed, asymptomatic, or mild symptomatic pertussis infections? It is not clear why the authors only focus on a sum value for EFI versus other measures (such as multiple values above or below certain thresholds, etc.) to provide additional support and evidence for their results.

It is not clear why the authors have emphasized the novelty and large proportion of asymptomatic infections observed in these data. For example, there have been household studies of pertussis (see https://academic.oup.com/cid/article-abstract/70/1/152/5525423?redirectedFrom=PDF which performed a systematic review that included this topic) that have also found such evidence. While cross-sectional surveys may be commonly used in practice, it is not clear that there is no other type of study that provides any evidence for asymptomatic infections. Further, it is not clear why the authors refer to widespread asymptomatic pertussis when a large proportion of individuals with evidence for pertussis infection had symptoms. Would it not be undiagnosed pertussis if it is associated with clinical symptomatology?

Reviewer #1 (Public Review):

Galdadas et al. applied a combinatorial approach of equilibrium and nonequilibrium molecular dynamics methods to study two important members of the Class A β-lactamase enzyme family in detail. Authors carefully chose two representative enzymes from this family, TEM-1 and KPC-2 in this study. Understanding of the nature of the communication pathways between allosteric ligand binding site and the active site has been the main focus of this study. Another very interesting finding of this study was the position of clinical variants that was precisely mapped along the allosteric communication pathway. This approach certainly has broad utility as it can be applied to study long-range communications in enzymes that are triggered by binding of a ligand (drug candidate) to an alternative/remote site, and also in cases where certain mutations occur far away from the active site but lead to drug resistance.

Overall, the manuscript is well written, and the conclusions are mostly well supported by data.

Reviewer #1 (Public Review):

The authors aimed to survey a large transfusion database in Sweden to catalog associations between ABO/RhD blood group antigens and a wide variety of clinical phenotypes in a systematic, unbiased and comprehensive manor. They succeed at surveying over 1200 phenotypes in over 5 million people and identify 49 statistically significant associations for ABO blood group and point out a couple novel associations. Their statistical methods are appropriate and help eliminate potential false positive associations. The strengths of this study are the unbiased survey of a large database and the appropriate corrections for multiple observations which allow the authors to explore a large number of associations without loosing site of what is really a significant association.

This study sheds light on a topic of interest to many scientists. The ABO gene encodes a glycosyltransferase enzyme that has 4 major haplotypes in human populations and results in a specific pattern of posttranslational modification of plasma proteins and blood cells including erythrocytes. Proteins decorated with an H antigen can receive additional carbohydrate antigens from ABO transferase intracellularly. The common A allele transfers UDP-GalNAc while the B allele transfers UDP-Gal. The A2 allele is hypomorphic compared to the A allele and transfers lower amounts of UDP-GalNAc and the common O allele is a null resulting in no transferase activity.

The allele frequencies of these common alleles varies by ancestry and has geographic differences. Variation at ABO is unconstrained with many rare variants contributing to the four common haplotypes at ABO. Interestingly, geographically specific selective pressures may have led to allele frequency differences. For example. ~40-50% of individuals are homozygous for the null (type O) allele. These null haplotypes are more common in individuals of Latino or African ancestry while 'A' haplotypes are slightly more common in individuals of European origin and 'B' alleles are more common in individuals of Asian and African ancestry. Overall, O is more common than A or B alleles. An unbiased survey of phenotype frequencies by blood type allows for confirmation of previous associations and discovery of novel associations. In this largely European ancestry cohort, blood type A is the most common (45-47%) while blood type O is second most common at 38-39%.

Limitations of Phenome-wide Association Studies (PheWAS) like the one presented in this manuscript should be noted. Associations with complex phenotypes or those with small effect size will not be detected even in a large cohort such as the SCANDAT. This study is also biased toward associations with phenotypes more common in the Scandinavian population. This may present associations related to the population substructure and not a direct association with ABO. In genome-wide association studies this can be addressed through multiple methods but it is not clear how the authors correct for population structure in this study. Likewise, the insight into the mechanistic reasons for ABO associations is not a strength of this study and will await subsequent studies for many phenotypes. Mechanistic insight might be particularly interesting for the novel associations uncovered by this study.

Reviewer #1 (Public Review):

In this manuscript, the authors use single cell RNA sequencing to investigate cell-type specific eQTL within C. elegans. This relies on the well known ability to genotype individuals via their transcriptome allowing the authors to generate both phenotypes and genotypes from single cell transcriptomes. This identifies a blend of cis and trans-eQTL that are cell type specific and starts to provide numerical observations to the communities expectation of cell type specificity.

The use of simultaneous single cell sequencing on a diversity of individuals is a unique method that is absolutely essential to get around the vast scale issues that are presented when contemplating single cell eQTL within multicellular organisms. However, an unfortunate outcome of this approach that the cell-autonomy of the eQTL cannot be studied. Instead the cell types have to be considered completely independent of each other.

The authors conduct an analysis of eQTL per each cell type to get at specificity. This identifies a number of eQTL found in only a single cell type but these binary tests can have an ascertainment issue that may be over-estimating the cell type specificity. Optimally, this would be conducted by incorporating the different cell types as different environments within a single eQTL model but given the different sample sizes, this may not be feasible. Alternatively an investigation of how eQTLs specific to one cell type are or are not found by shifting the detection threshold in the other tissues could test this possibility.

Reviewer #1 (Public Review):

This manuscript describes the curation of a training dataset, CEM500K, of cellular electron microscope (EM) data including STEM, TEM of sections, electron tomography, serial section and array tomography SEM, block-face and focused-ion beam SEM. Using CEM500K to train an unsupervised deep learning algorithm, MoCoV2, the authors present segmentation results on a number of publically available benchmark datasets. They show that the standard Intersection-over-Union scores obtained with the CEM500K-trained MoCoV2 model, referred to as CEM500K-moco, equal or exceed the scores of benchmark segmentation results. They also demonstrate the robustness of CEM500K-moco's performance with respect to input image transformations, including rotation, Gaussian blur and noise, brightness, contrast and scale. The authors make the remarkable discovery that MoCoV2 spontaneously learned to use organelles as "landmarks" to identify important features in images, simulating human behavior to some degree.

Reviewer #1 (Public Review):

The submitted manuscript 'Distinct higher-order representations of natural sounds in human and ferret auditory cortex' by Landemard and colleagues seeks to investigate the neural representations of sound in the ferret auditory cortex. Specifically, they examine the stages of processing via manipulating the complexity and sound structure of stimuli. The authors create synthetic auditory stimuli that are statistically equivalent to natural sounds in their cochlear representation, temporal modulation structure, spectral modulation structure, and spectro-temporal modulation structure. The authors use functional ultrasound imaging (fUS) which allowed for the measurement of the hemodynamic signal at much finer spatial scales than fMRI, making it particularly suitable for the ferret. The authors then compare their results to work done in humans that has previously been published (e.g. Norman-Haignere and McDermott, 2018) and find that: 1. While human non-primary auditory cortex demonstrates a significant difference between natural speech/music sounds and their synthetic counterparts, the ferret non-primary auditory cortex does not. 2. For each sound manipulation in humans, the dissimilarity increases as the distance from the primary auditory cortex increases, whereas for ferrets it does not. 3. While ferrets behaviorally respond to con-specific vocalizations, the ferret auditory cortex does not demonstrate the same hierarchical processing stream as humans do.

Overall, I find the approach (especially the sound manipulations) excellent and the overall finding quite intriguing. My only concern, is that it is essentially a null-result. While this result will be useful to the literature, there is always the concern that a lack of finding could also be due to other factors.

Major points:

1) What if the stages in the ferret are wrong? The authors use 4 different manipulations thought to reflect key elements of sound structure and/or the relevant hierarchy of the processing stages of the auditory cortex, but it's possible that the dimensions in the ferret auditory cortex are along a different axis than spectro/temporal modulations. While I do not expect the authors to attempt every possible axis, it would be beneficial to discuss.

2) For the ferret vocalizations, it is possible that a greater N would allow for a clearer picture of whether or not the activation is greater than speech/music? While it is clear that any difference would be subtle and probably require a group analysis, this would help settle this result/issue (at least at the group level).

3) Relatedly, did the magnitude of this effect increase outside the auditory cortex?

4) It would be useful to have a measure of the noise floor for each plot and/or species for NSE analyses. This would make it easier to distinguish whether, for instance, in 2A-D, an NSE of 0.1 (human primary) vs. an NSE of 0.042 (ferret primary) should be interpreted as a bit more than double, or both close to the noise floor (which is what I presume).

Reviewer #1 (Public Review):

In this study, the authors set out to address the interesting question of how activating septal cholinergic neurons affects learning and memory of reward locations. The work provides compelling evidence showing that activation of septal cholinergic cells at reward zones suppresses sharp wave-ripples and impairs memory performance in freely behaving animals. The data are properly controlled and analyzed, and the results support the conclusions. The results shed new light on the functional significance of cholinergic projections in reward learning. Future follow-up studies designed to selectively activate cholinergic projections specifically at times when sharp wave-ripples occur will be interesting to determine the importance of cholinergic sharp wave-ripple suppression for these effects.

Reviewer #1 (Public Review):

Here, Houri-Ze'evi, et al. treated progeny of parents that had inherited small RNA response (silencing of an artificial, single-copy GL-expressed gfp with anti-gfp dsRNA) with 3 stresses – heat shock, hyperosmolarity, or starvation – starting at L1, and examined gfp silencing. All three treatments reduced silencing (visible as increased GFP fluorescence) in subsequent generations (F1-F3). The authors tested resetting of endogenous (endo-siRNA) and piRNAs using an endo-siRNA sensor target sequence and piRNA recognition sites, respectively. Again, all 3 stressors reset both in the same generation, but did not reset the effect transgenerationally, suggesting that exogenous RNAi resetting functions through a different mechanism than endogenous.

Next, they tested adults, which also led to resetting. However, only the F1 generation, not F2, is susceptible to resetting (how? Why?), revealing a critical period for resetting susceptibility. Reversal of the stress with RNAi treatment does not result in resetting, nor does simpy changing conditions. The authors then went on to examine mutants that might be defective in stress responses or in resetting; MAPK genes and skn-1 are required for resetting. Small RNA-seq from stressed worms and their progeny showed a decrease overall with stresses, and reveals some potential classes of genes, including targets of the mutator genes, and overlap with classic stress response pathways (dauer, IIS). Overall, this work presents some interesting phenomena and moves towards explaining how it might work through the identification of a critical period and some genes that are required.

In this version, the authors have added more information regarding the relationship between MAPK and SKN-1, and transcriptional targets. Most importantly, they have performed tissue-specific rescue of sek-1; in neurons, this rescues, but intestine did not.

These data add to prior work from the Rechavi lab and others in the field, which together address the interplay of small RNAs, response to stress, and transgenerational inheritance.

Reviewer #1:

Single molecule localization microscopy (SMLM) has become an important method for understanding the subcellular distribution of fluorescently labelled biomolecules at length scales of a few tens of nanometers. A critical challenge has been to find out, whether and to what extent biomolecular clustering occurs. While methods have been published which address the problem of identifying biomolecular clusters in SMLM images, they still suffer from many user-defined parameters, which - if selected inappropriately - influence the obtained results substantially. The StormGraph-3D method proposed here addresses these issues, based on a comprehensive mathematical framework which reduces the number of user-defined input parameters. The method was evaluated using comprehensive simulations of data, which show its robustness compared to alternative approaches.

The methods part of the paper would benefit, however, from more realistic data of single molecule blinking behavior, and the evaluation of the consequences on the performance of the method. As the authors acknowledge, overcounting due to blinking has challenged data analysis previously, and gave rise to artifactual localization clusters that do not represent the underlying protein distribution. It would be of particular interest, which results in the method yielded for a random biomolecular distribution.