for - AI and education - children will have custom tailored education program via AI

for - neuroscience - education of children - recommend no digital devices before puberty - allows learning in a 3 dimensional world

for - neuroscience - importance of maintaining curiosity - 1000 questions a day for children - 20 questions a day for adults

for - neuroscience - building design - common area to converge everyone - creates diverse social meetings - increases work efficacy - example - Steve Jobs - Pixar bathroom

for - neuroscience - validation for Stop Reset Go open source participatory system mapping for design innovation

neuroscience - validation for Stop Reset Go open source participatory system mapping for design innovation - bottom-up collective design efficacy - What Henning Beck validates for companies can also apply to using Stop Reset Go participatory system mapping within an open space to de-silo and be as inclusive as possible of many different silo'd transition actors

for - key insight - human vs artificial intelligence - humans will create the best ideas

key insight - human vs artificial intelligence - humans will create the best ideas - why? - because we are - slower - imperfect - less rational - drifting away - forgetting - and we learn from the mistakes we make and from different perspectives shared with us

for - neuroscience - example - informal diversity - increases work efficacy - via sharing diverse and novel perspectives

for - neuroscience - innovation - great ideas - mistakes and - risk

neuroscience - innovation - great ideas - mistakes and - risk - Any new idea involves taking a risk that it could be wrong - we cannot be innovators if we are not able to risk making mistakes

for - neuroscience - mistakes - and fear

for - Indyweb - Stop Reset Go participatory system mapping - benefits of open source - Henning Beck - neuroscience support

Indyweb - Stop Reset Go participatory system mapping - benefits of open source - Henning Beck validates the importance of an open source design of the Stop Reset Go participatory system mapping - By developing an open source graph for many silo'd actors to participate, they mutually desilo each other - The sharing of diverse perspectives helps to mitigate progress traps

for - neuroscience - example - diverse and low density connections beats non-diverse and high connections

neuroscience - example diverse and low density connections vs non-diverse high density connections - having access to many diverse perspectives is a key enabler of good problem-solving and innovation

for - neuroscience - efficient work - relaxation rule

neuroscience - efficient work - relaxation rule - It is necessary to build NO WORK time into effective work - 5 time units work - 1 time unit relaxation - It is necessary to step back from concentrating on a problem - for the brain to drift away from it and - relax from concentrating on the problem - so that new perspectives can develop that can be brought back to solve the problem

for - quote - neuroscience - perspective shift - benefits

for - neuroscience - ideation depends on three different brain functions and brain areas - concentration - prioritization - and drifting away

neuroscience - ideation depends on three different brain functions and brain areas - concentration<br /> - frontal area of brain - prioritization and - deep inner part of the brain - drifting away - back part of the brain

for - neuroscience - optimal working environment - varies with brain state - different phases during the day - engagement - inspiration - concentration - communication - relaxation

for - neuroscience - human abilities - example Brexit and variations

for - neuroscience - children's understanding - 3 examples is enough to consolidate new concept

for - question - AI - can AI learn to be intentionally distracted?

for - neuroscience - human understanding - what makes us excel? - forgetting and getting distracted!

for - human learning - key feature - evolutionary nature - indyweb - key feature - evolutionary nature of learning

for - comparison - human vs artificial intelligence

question - comparison - human vs artificial intelligence - Can't an AI also consider things we sit on to then generalize their classifcation algorithm?

for - comparison - brain vs machine intelligence

comparison - brain vs machine intelligence - the brain is inferior to machine in many ways - many times slower - much less accurate - network of neurons is mostly isolated in its own local environment, not connected to a global network like the internet - Yet, it is able to perform extraordinary things in spite of that - It is able to create meaning out of sensory inputs - Can we really say that a machine can do this?

for - comparison - brain vs machine intelligence - comparison - human intelligence vs artificial intelligence

comparison - human intelligence vs artificial intelligence - AI depends on feeding the AI system with huge datasets that it can - analyze and make correlations and - perform big data analysis - Humans don't operate the same way

for - key insight - claim - humans can generate new ideas by changing the way we think - AI cannot do this

for - comparison - data vs ideas - no metric for ideas

for - Henning Beck - neuroscientist - video - youtube - The Brain vs Artificial Intelligence

Case: Patient Proband SS, Female, Caucasian

DiseaseAssertion: UCD/OTCD

FamilyInfo: De novo inheritance, no family history of disease

CasePresentingHPOs: Hyperammonemia (HP:0001987), oroticaciduria (HP:0003218), Childhood onset (HP:0011463)

CaseHPOFreeText:

CaseNOTHPOs: Positive allopurinol test

CaseNOTHPOFreeText:

CasePreviousTesting: Genomic DNA isolated from peripheral blood leukocytes or cultured skin fibroblasts. Amplification by PCR used. SSCP analysis performed. Sequencing of both free and immobilized single strands carried out by dideoxy chain termination method.

SupplementalData: Table 1: Mutations in the Ornithine Transcarbamylase Gene of 17 Females

Variant: NM_000531.6:c.77+1G>A

ClinVarID: 97313

CAID: CA224773

gnomAD:

Case: Female Patient #1, Female, Japanese

DiseaseAssertion: UCD/OTCD

FamilyInfo: N/A

CasePresentingHPOs: Childhood onset (HP:0011463)

CaseHPOFreeText: Onset at 2 years

CaseNOTHPOs:

CaseNOTHPOFreeText: 16% OTC activity

CasePreviousTesting:

Variant: NM_000531.5:c.67C>T (p.Arg23*)

ClinVarID: 97292

CAID: CA224742

gnomAD:

this is happening to me right now, as I keep getting side tracked with all the other notes and highlights previously made on this article using this app.

Case: Patient #38, Female, Chinese

DiseaseAssertion: UCD/OTCD

FamilyInfo: No family history of disease, mutation is inherited

CasePresentingHPOs: Hyperammonemia (HP:0001987), oroticaciduria (HP:0003218), vomiting (HP:0002013), coma (HP:0001259), lethargy (HP:0001254), seizures (HP:0001250), childhood onset (HP:0011463)

CaseHPOFreeText: Elevated plasma ammonia at 190 umol/L (Normal: 9-30 umol/L), Elevated urinary orotate at 202 mmol/mmol creatinine (Normal: 0-1.5 mmol/mmol creatinine)

CaseNOTHPOs:

CaseNOTHPOFreeText: Normal plasma glutamine at 13.5 umol/L (Normal: 6-30 umol/L), Normal plasma citrulline at 15.75 umol/L (Normal: 7-35 umol/L)

CasePreviousTesting: gDNA extracted from peripheral blood leukocytes. PCR all coding exons and exon–intron boundaries of the OTC gene using 9 pairs of synthetic oligonucleotide primers, and the primer sequences and annealing temperature. PCR products were then purified and bidirectionally sequenced. The library was sequenced using Illumina HiSeq4000 and generated 150 bp paired-end reads. Data analysis was performed as previously described [Sun Y, Hu G, Liu H, Zhang X, Huang Z, Yan H, et al. Further delineation of the phenotype of truncating KMT2A mutations: the extended Wiedemann–Steiner syndrome. Am J Med Genet A. 2017;173:510–4.]. Multiplex ligation-dependent probe amplification analysis was performed for samples in which Sanger sequencing or WES failed to detect any disease-causing mutation.

Variant: NM_000531.6:c.67C>T (p.Arg23*)

ClinVarID: 97292

CAID: CA224742

gnomAD:

Case: Patient #4, Female, Caucasian

DiseaseAssertion: UCD/OTCD

FamilyInfo: N/A

CasePresentingHPOs: Hyperammonemia (HP:0001987),

CaseHPOFreeText: Elevated plasma ammonia concentration at 123 uM/L

CaseNOTHPOs:

CaseNOTHPOFreeText: Anxiety, plasma citrulline at 2 uM/L

CasePreviousTesting: N/A

Variant: NM_000531.5:c.67C>T (p.Arg23*)

ClinVarID: 97292

CAID: CA224742

gnomAD:

H₀: μ less than or equal to 66. Hₐ: μ > 66.

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Reply to the reviewers

Reviewer #1

__Evidence, reproducibility and clarity __

The work by Przanowska et al., sought to understand the role of ORC2 in murine development and further wanted to discover its role in liver endo-reduplication. The overall methods used is sufficient enough to address its role but is not very conclusive based on their overall results and data provided as elaborated in below comments.

Major Comments:

1. The major issue of the paper is how well is ORC2 depleted in perinatal liver (Fig. 2C) and is not very clear from the data as all the western blots are at very low exposure levels and bands are very weak (still weak bands seen). There are good antibodies of ORC2 which can be used for IHC staining and can be used to address the extent of ORC2 depletion.

We have now shown that ORC2 protein is significantly decreased in the hepatocytes of the Orc2 KO and DKO livers (New Fig. 2C and 6D). The decrease is consistent, with 4-5 mice examined, and all showing the depletion. We have been unable to do immunohistochemistry on tissue sections of the mouse livers with the anti-ORC antibodies we have tried, and this could be a reflection of the low level of the proteins. On hepatocytes in culture we have obtained faint signal with the anti-ORC2 antibody in WT cells, and this is clearly absent in 100% of the hepatocytes. See Fig. R1 below.

__Reviewer Fig R1: __

A) Immunofluorescence of hepatocytes in culture from livers of WT and two DKO mice.

B) Quantitation of A) from counting 70-100 cells from each specimen.

However, the calculations in the methods and the discussion are very compelling that at least the last 6-9 cell divisions in normal development start with 2n nuclei in the livers at baseline (Fig. 3B-G and 6I).

Why in Fig 2C, the M2 mice is showing an equivalent level of ORC2 protein compared to mice M1 with NO CRE expression (compare lane1 and lane5). So, the results are based on one mouse which I do not think is significant enough to come to the conclusion. The authors need to add more data from different mice for statistical significance. Please use IHC to show the depletion of ORC2 protein in the liver sections.

We had used total liver and had pointed out that residual ORC2 protein will be seen from stromal cells (endothelia, blood vessels and blood cells). We have therefore removed the figure which measured ORC2 levels in total liver and have now shown that when hepatocytes are isolated from five animals there was a massive depletion of ORC2 in all five animals (new Fig. 3C).

As nicely demonstrated in the previous paper by Okano-Uchida et al., 2018 that ORC1 depletion in the liver shows an DNA ploidy effect from 6-week onwards. The authors need to demonstrate in this paper also when the 16N phenotype is observed starting from week1 to 12 months.

Based on the results from our previous paper (Okano-Uchida et al., 2018) we decided to measure 16N phenotype at 6 weeks of age. The endoreduplication occurs at a stage when ORC2 protein is undetectable during normal development or during regeneration.

In the double knockout experiments (ORC1 and ORC2) the authors are not even bothered to demonstrate that how much are both the proteins are actually depleted from the cells, so on the results obtained from these mice experiments are not conclusive or explanatory.

We have performed immunoblotting of isolated hepatocytes and immunohistochemistry of livers for ORC1 and ORC2. Our data shows that both proteins are depleted in all four mice tested (New Fig. 6D).

Minor points:

1. Why are scale bars missing in right panel of Fig. 2G, Fig. 6D Supp Fig. 2B KO studies. The authors need to confirm that that all the large nuclei have NO or less significant ORC2 protein through IHC H&E staining.

The scale bars are missing from the right panels to avoid redundancy. We have added “Both panels are at the same scale.” in the figure legend, according to https://doi.org/10.1371/journal.pbio.3001161.

1. Please explain why is EYFP in Fig. 5G is cytoplasmic compared to Fig 4C (nuclear). We consistently see this variability and it was there in our previous results (Okano-Uchida et al., 2018), where EYFP was cytoplasmic in tissues, but was nuclear (and some cytoplasmic) in hepatocytes in culture.

We do not know the reason for this difference but consistently see this difference. We now say in the text: “We did not explore why the EYFP protein is mostly nuclear in hepatocytes in culture (Fig. 4C) and mostly cytoplasmic in hepatocytes in the liver tissue (Fig. 5G, 7G), but speculate that differences in signaling pathways or fixation techniques between the two conditions contribute to this difference.”

Are authors using the same genotype of Alb-Cre mice as shown by Okano-Uchida et al., 2018 as I do not find the reference of Schuler et. al., 2004 (PMID:15282742).

We have been using two independent Alb-Cre animals. This is now described in the Methods.

Significance

The article is exactly based on their previous published paper but instead of ORC1, they were interested in dissecting the role of ORC2. Although they have discussed that CDC6 may be involved in replacing ORC1 KO mice to rescue the extensive DNA replication in endoreduplication, but instead of going to hunt the role of CDC6 in endoreduplication they checked the effect of ORC2 which actually lower the overall impact of the paper.

We studied ORC2 conditional KO mice in a similar manner to the previously published ORC1 conditional KO in order to ensure (1) that the lack of effect in the Orc1 KO was not because ORC1 can theoretically be substituted for by CDC6 and (2) to establish the double KO of Orc1 and Orc2. To the best of our knowledge this is the first description of removal of two subunits of ORC complex at once in a mouse model. Moreover, in the light of rising recognition of sex as biological variable, we report sex-dependent effects which are very intriguing.

We have not attempted knocking out CDC6 to uncover novel mechanisms of DNA replication, because we first needed to make sure that the mice can truly endo-reduplicate without two of the six subunits of ORC. Note that our published results in cancer cell lines (Shibata, 2016) show that CDC6 is still essential in the ORC KO cell lines, so a future experiment will likely reveal that CDC6 is still essential for endoreduplication in the ORC KO mice in vivo.

Reviewer #2

__Evidence, reproducibility and clarity __

It has been reported that in the absence of ORC1, liver cells can still endoreduplicate and it has been speculated that this might occur if CDC6 can replace, at least partially, the function of ORC1. Here, authors evaluate if this is also true in the absence of ORC2 and found that ORC2 is required for cell proliferation in mouse hepatocytes but not for endoreduplication. This is also the case after combining the conditional mutations of ORC1 and ORC2. They propose that a mechanism must exist to load sufficient MCM2-7 to support DNA replication in the absence of these two ORC subunits. Some of the conclusions need further experimental support. The rationale for testing the requirement of ORC2, with or without ORC1, for endoreduplication is valid. However, a key point is that the endoreduplication level seems to be higher in the absence of ORC2 or both ORC1 and ORC2, and this is not properly addressed. Also, mechanistic details on how this could be triggered are absent from this study. As indicated below almost every figure in this manuscript contains weak points (see below).

We now discuss the following: “One possible explanation of the greater endoreduplication in both our papers is that mitosis may be arrested earlier in development by G2 DNA damage checkpoints activated by incomplete licensing and replication of the genome in the absence of ORC. As a result, endoreduplication cycles could begin earlier in development resulting in greater endoreduplication.”

Major 1. Fig 1G, needs a detailed comment and justification.

We have added the following to the text: “The proliferation rate of the MEF were measured by MTT assays. Even in the Orc2+/+ MEF, the infection with adeno-Cre decreased proliferation a little (the orange line compared to the blue line in Fig. 1G). However, for Orc2f/f MEF infection with adeno-Cre impairs proliferation even further (yellow line compared to black line in Fig. 1G)..

Note that Adeno-Cre has been reported to be toxic for cell proliferation (citations 1, 2, 3), and so we included Adeno-Cre expression in ORC2+/+ (WT) as a background control.

Citation:

1. Pfeifer A, Brandon EP, Kootstra N, Gage FH, Verma IM: Delivery of the Cre recombinase by a self deleting lentiviral vector: Efficient gene targeting in vivo. Proc Natl Acad Sci USA. 2001, 98: 11450-11455. 10.1073/pnas.201415498.
2. Loonstra A, Vooijs M, Beverloo HB, Allak BA, Drunen EV, Kanaar R, Berns A, Jonkers J: Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci USA. 2001, 98: 9209-9214. 10.1073/pnas.161269798.
3. Schmidt EE, Taylor DS, Prigge JR, Barnet S, Capecchi R: Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci USA. 2000, 97: 13702-13707. 10.1073/pnas.240471297.
4. Fig 2D-F. Is this conclusion applicable to other endoreplicating tissues? Have authors consider to analyze body weight and liver weight measurements after normalization with similar data from a non-affected organ? The conditional KO was performed specifically in the liver. ORC is intact in other tissues in these animals. As a future direction our lab plans to study cardiac-specific conditional KO of ORC subunits to test whether other endo-reduplicating tissues can also synthesize DNA in the absence of ORC subunits.

Fig 3 shows inconsistent results or results that lack proper justification in the text. The 2C peak is missing in Fig 3E (yellow line, positive control). However, 2n nuclei appear in Fig 3F-H. Also, the blue and yellow peaks do not coincide in the flow cytometry profiles, in particular for 8C and 16C.

There was an error in the plotting of the former Fig. 3E. The information is better presented in the former Fig. 3F-H (now Fig. 3E-G) and so have removed the former Fig. 3E from the paper.

Fig 4. Shorter EdU pulses could be more informative of the actual amount of S-phase cells. Thus, the use of a 2h EdU pulse needs a clear justification.

The half-life of EDU incorporation differs slightly between in vivo and in vitro conditions. In vivo, slower cell proliferation requires a longer time, approximately 4 hours. However, in vitro, liver cells grow faster, and a 2-hour EDU pulse with 20 µM is sufficient for detection compared to a 3-hour pulse with 10 µM BrdU (Okano-Uchida et al., 2018). Several publications also use a 2-hour EDU incubation time (https://doi.org/10.1098/rsob.150172).

Fig 5. EYFP is cytoplasmic, in contrast with results shown in Fig 4C

We consistently see this variability and it was there in our previous results (Okano-Uchida et al., 2018), where EYFP was cytoplasmic in tissues, but was nuclear (and some cytoplasmic) in hepatocytes in culture.

We do not know the reason for this difference but consistently see this difference. We now say in the text: “We did not explore why the EYFP protein is mostly nuclear in hepatocytes in culture (Fig. 4C) and mostly cytoplasmic in hepatocytes in the liver tissue (Fig. 5G, 7G), but speculate that differences in signaling pathways or fixation techniques between the two conditions contribute to this difference.”

Fig 6. Results obtained with the double mutant are poorly described.

We have split the figure into two figures (New Fig. 6 and 7) edited the results section to ensure that they are easily comprehended by the readers. We have also included Westerns from hepatocyte cell lysates of four DKO mice to show that ORC1 and ORC2 proteins are reproducible decreased (New Fig. 6D).

What are the level of other pre-RC components in the mutants used in this study. This could be easily evaluated by Western blotting

Despite the technical difficulty of not having antibodies that recognize all the mouse initiation proteins, we have now measured mouse ORC1, ORC2, ORC3, ORC5, ORC6, CDC6 and the MCM2 and MCM3 subunits of MCM2-7. The results do not show a consistent decrease or increase of any of these proteins in individual mice of the two genotypes, Orc2-/- or DKO (New Fig. 2D and 6E)

How do authors justify their claim that a very limited amount of ORC are sufficient to load a vast excess of MCM2-7 hexamers?

The rationale is stated in the introduction from data from cancer cell lines: “Given that WT cells have about 150,000 molecules of ORC2, even if this truncated protein is functional ORC2, ~150 molecules of the protein would be expected to load MCM2-7 double hexamers on at least 50,000 origins of replication. Experimentally, we show in Shibata, 2020 (Fig. 7C), that although ORC subunits are undetectable on Westerns, MCM2-7 association with the chromatin is unchanged. By the way, we do not say “vast excess” of MCM2-7, just sufficient MCM2-7 to fire 50,000 origins.

Minor 1. The titles of the Results section could be more informative of the main conclusion rather than simply descriptive

We updated our Results titles to be more informative.

The Discussion is too long

We have shortened the discussion by removing our calculations to the Results section and abbreviating some of the discussion on endoreduplication. However we had to insert new items brough forth by the reviewers. Due to the controversy of this topic in our field, we had to include extensive discussion of current literature and put our results in their proper context.

Significance

The topic is relevant and the hypothesis tested is reasonable, although the conceptual advance is limited (see also below). The major limitation is the absence of mechanistic details addressing the occurrence of extra endoreduplication cycles (compared to controls) in the ORC1 and ORC2 mutants.

Reviewer #3

__Evidence, reproducibility and clarity: __

The origin recognition complex (ORC) is an essential loading factor for the replicative Mcm2-7 helicase complex. Despite ORC's critical role in DNA replication, there have been instances where the loss of specific ORC subunits has still seemingly supported DNA replication in cancer cells, endocycling hepatocytes, and Drosophila polyploid cells. Critically, all tested ORC subunits are essential for development and proliferation in normal cells. This presents a challenge, as conditional knockouts need to be generated, and a skeptic can always claim that there were limiting but sufficient ORC levels for helicase loading and replication in polyploid or transformed cells. That being said, the authors have consistently pushed the system to demonstrate replication in the absence or extreme depletion of ORC subunits.

Here, the authors generate conditional ORC2 mutants to counter a potential argument with prior conditional ORC1 mutants that Cdc6 may substitute for ORC1 function based on homology. They also generate a double ORC1 and ORC2 mutant, which is still capable of DNA replication in polyploid hepatocytes. While this manuscript provides significantly more support for the ability of select cells to replicate in the absence or near absence of select ORC subunits, it does not shed light on a potential mechanism. While a mechanistic understanding of how these cells proliferate in the absence or extreme depletion of ORC subunits is outside the scope of the current manuscript, it would have been beneficial to see more functional analyses to help guide the field. For example, is there a delay or impairment in Mcm2-7 loading in G1 (FACs-based loading assay from the Cook Lab (Matson et al., eLife. 2017)) in primary hepatocytes with the ORC2 conditional deletion? Is copy number maintained as cells increase polyploidy in the absence of ORC subunits, or are some regions of the genome more sensitive to ORC depletion (CGH arrays or sequencing of the flow-sorted polyploid cells)?

We thank the reviewer for recognizing the main point of these experiments: to dispel the argument that CDC6 can substitute for ORC1 in the six-subunit ORC (although no one has demonstrated this, the argument is made on the basis of close sequence homology between CDC6 and ORC1). The second point, also appreciated by the reviewer is to show that it is possible to find cells that replicate in the absence or near absence of two ORC subunits.

The mechanistic questions raised are important, and we will address them here:

Is there a delay or impairment of MCM2-7 loading in G1? The hepatocytes in culture are fragile and not immortalized and thus, this issue can be much more easily addressed in the cancer cell lines we have made that are missing several ORC subunits and will do that in a later paper. Note however, the surprising lack of change in MCM2-7 association in cell lines where both ORC2 and ORC5 are deleted (Shibata, 2020, Fig. 7C).

Are some regions of the genome more sensitive to ORC deletion during the polyploidization? We could not find any paper where people have investigated whether the whole genome is uniformly polyploidized in livers. In other words, the baseline conditions in WT livers have not been established. We therefore have postponed experiments to answer this question for a later paper. Note that in unpublished data from mapping SNS-seq origins in WT and ORC deletion cell lines there does not appear to be selective firing of certain origins over others in the deletion cell lines.

Additional points: I didn't understand how the numbers were derived in Table 2. Was there really a 20-fold decrease in nuclear density for female ORC1 and ORC2 double-deletion hepatocytes? The differences in Figure S2 are dramatic, but not 20-fold dramatic.

We measure the relative nuclear density by counting the number of plump nuclei (hepatocytes) per field as described for Fig. 5F and 7F now in the Methods section. The reviewer is correct in that we overestimated the decrease of nuclear density in the female DKO mice by two-fold. The revised calculations suggest that 6 cell divisions occur in the female DKO mice after the ORC proteins have decreased to at least __Significance: __

The strengths of this manuscript are the mouse genetics and the generation of conditional alleles of Orc2 and the rigorous assessment of phenotypes resulting from limiting amounts of specific ORC subunits. It also builds on prior work with ORC1 to rule out Cdc6 complementing the loss of ORC1. The weakness is that it is a very hard task to resolve the fundamental question of how much ORC is enough for replication in cancer cells or hepatocytes. Clearly, there is a marked reduction in specific ORC subunits that is sufficient to impact replication during development and in fibroblasts, but the devil's advocate can always claim limiting levels of ORC remaining in these specialized cells. The significance of the work is that the authors keep improving their conditional alleles (and combining them), thus making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC. At this point, the investigators and the field are well-positioned to attempt future functional CRISPR screens to identify other factors that may modulate the response to the loss of ORC subunits. This work will be of interest to the DNA replication, polyploidy, and genome stability communities.

We thank the reviewer for getting the important point of this paper: “making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC….” In other words, either ORC is completely dispensable for loading MCM2-7 in certain cancer cell lines and hepatocytes or it is highly catalytic and one molecule of ORC can load a few hundred MCM2-7 doublets so that most origins in the genome are licensed and capable of firing. We are trying the CRISPR screens in cancer cell lines that the reviewer envisages

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Referee #3

Evidence, reproducibility and clarity

The origin recognition complex (ORC) is an essential loading factor for the replicative Mcm2-7 helicase complex. Despite ORC's critical role in DNA replication, there have been instances where the loss of specific ORC subunits has still seemingly supported DNA replication in cancer cells, endocycling hepatocytes, and Drosophila polyploid cells. Critically, all tested ORC subunits are essential for development and proliferation in normal cells. This presents a challenge, as conditional knockouts need to be generated, and a skeptic can always claim that there were limiting but sufficient ORC levels for helicase loading and replication in polyploid or transformed cells. That being said, the authors have consistently pushed the system to demonstrate replication in the absence or extreme depletion of ORC subunits.

Here, the authors generate conditional ORC2 mutants to counter a potential argument with prior conditional ORC1 mutants that Cdc6 may substitute for ORC1 function based on homology. They also generate a double ORC1 and ORC2 mutant, which is still capable of DNA replication in polyploid hepatocytes. While this manuscript provides significantly more support for the ability of select cells to replicate in the absence or near absence of select ORC subunits, it does not shed light on a potential mechanism. While a mechanistic understanding of how these cells proliferate in the absence or extreme depletion of ORC subunits is outside the scope of the current manuscript, it would have been beneficial to see more functional analyses to help guide the field. For example, is there a delay or impairment in Mcm2-7 loading in G1 (FACs-based loading assay from the Cook Lab (Matson et al., eLife. 2017)) in primary hepatocytes with the ORC2 conditional deletion? Is copy number maintained as cells increase polyploidy in the absence of ORC subunits, or are some regions of the genome more sensitive to ORC depletion (CGH arrays or sequencing of the flow-sorted polyploid cells)?

Additional points: I didn't understand how the numbers were derived in Table 2. Was there really a 20-fold decrease in nuclear density for female ORC1 and ORC2 double-deletion hepatocytes? The differences in Figure S2 are dramatic, but not 20-fold dramatic.

Significance

The strengths of this manuscript are the mouse genetics and the generation of conditional alleles of Orc2 and the rigorous assessment of phenotypes resulting from limiting amounts of specific ORC subunits. It also builds on prior work with ORC1 to rule out Cdc6 complementing the loss of ORC1. The weakness is that it is a very hard task to resolve the fundamental question of how much ORC is enough for replication in cancer cells or hepatocytes. Clearly, there is a marked reduction in specific ORC subunits that is sufficient to impact replication during development and in fibroblasts, but the devil's advocate can always claim limiting levels of ORC remaining in these specialized cells. The significance of the work is that the authors keep improving their conditional alleles (and combining them), thus making it harder and harder (but not impossible) to invoke limiting but sufficient levels of ORC. At this point, the investigators and the field are well-positioned to attempt future functional CRISPR screens to identify other factors that may modulate the response to the loss of ORC subunits. This work will be of interest to the DNA replication, polyploidy, and genome stability communities.

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Referee #2

Evidence, reproducibility and clarity

It has been reported that in the absence of ORC1, liver cells can still endoreduplicate and it has been speculated that this might occur if CDC6 can replace, at least partially, the function of ORC1. Here, authors evaluate if this is also true in the absence of ORC2 and found that ORC2 is required for cell proliferation in mouse hepatocytes but not for endoreduplication. This is also the case after combining the conditional mutations of ORC1 and ORC2. They propose that a mechanism must exist to load sufficient MCM2-7 to support DNA replication in the absence of these two ORC subunits.

Some of the conclusions need further experimental support. The rationale for testing the requirement of ORC2, with or without ORC1, for endoreduplication is valid. However, a key point is that the endoreduplication level seems to be higher in the absence of ORC2 or both ORC1 and ORC2, and this is not properly addressed. Also, mechanistic details on how this could be triggered are absent from this study. As indicated below almost every figure in this manuscript contains weak points (see below).

Major

1. Fig 1G, needs a detailed comment and justification.
2. Fig 2D-F. Is this conclusion applicable to other endoreplicating tissues? Have authors consider to analyze body weight and liver weight measurements after normalization with similar data from a non-affected organ?
3. Fig 3 shows inconsistent results or results that lack proper justification in the text. The 2C peak is missing in Fig 3E (yellow line, positive control). However, 2n nuclei appear in Fig 3F-H. Also, the blue and yellow peaks do not coincide in the flow cytometry profiles, in particular for 8C and 16C.
4. Fig 4. Shorter EdU pulses could be more informative of the actual amount of S-phase cells. Thus, the use of a 2h EdU pulse needs a clear justification.
5. Fig 5. EYFP is cytoplasmic, in contrast with results shown in Fig 4C
6. Fig 6. Results obtained with the double mutant are poorly described.
7. What are the level of other pre-RC components in the mutants used in this study. This could be easily evaluated by Western blotting
8. How do authors justify their claim that a very limited amount of ORC are sufficient to load a vast excess of MCM2-7 hexamers?

Minor

1. The titles of the Results section could be more informative of the main conclusion rather than simply descriptive
2. The Discussion is too long

Significance

The topic is relevant and the hypothesis tested is reasonable, although the conceptual advance is limited (see also below). The major limitation is the absence of mechanistic details addressing the occurrence of extra endoreduplication cycles (compared to controls) in the ORC1 and ORC2 mutants

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Referee #1

Evidence, reproducibility and clarity

The work by Przanowska et al., sought to understand the role of ORC2 in murine development and further wanted to discover its role in liver endo-reduplication. The overall methods used is sufficient enough to address its role but is not very conclusive based on their overall results and data provided as elaborated in below comments.

Major Comments:

1. The major issue of the paper is how well is ORC2 depleted in perinatal liver (Fig. 2C) and is not very clear from the data as all the western blots are at very low exposure levels and bands are very weak (still weak bands seen). There are good antibodies of ORC2 which can be used for IHC staining and can be used to address the extent of ORC2 depletion.
2. Why in Fig 2C, the M2 mice is showing an equivalent level of ORC2 protein compared to mice M1 with NO CRE expression (compare lane1 and lane5). So, the results are based on one mouse which I do not think is significant enough to come to the conclusion. The authors need to add more data from different mice for statistical significance. Please use IHC to show the depletion of ORC2 protein in the liver sections.
3. As nicely demonstrated in the previous paper by Okano-Uchida et al., 2018 that ORC1 depletion in the liver shows an DNA ploidy effect from 6-week onwards. The authors need to demonstrate in this paper also when the 16N phenotype is observed starting from week1 to 12 months.
4. In the double knockout experiments (ORC1 and ORC2) the authors are not even bothered to demonstrate that how much are both the proteins are actually depleted from the cells, so on the results obtained from these mice experiments are not conclusive or explanatory.

Minor points:

1. Why are scale bars missing in right panel of Fig. 2G, Fig. 6D Supp Fig. 2B KO studies. The authors need to confirm that that all the large nuclei have NO or less significant ORC2 protein through IHC H&E staining.
2. Please explain why is EYFP in Fig. 5G is cytoplasmic compared to Fig 4C (nuclear).
3. Are authors using the same genotype of Alb-Cre mice as shown by Okano-Uchida et al., 2018 as I do not find the reference of Schuler et. al., 2004 (PMID:15282742).

Significance

The article is exactly based on their previous published paper but instead of ORC1, they were interested in dissecting the role of ORC2. Although they have discussed that CDC6 may be involved in replacing ORC1 KO mice to rescue the extensive DNA replication in endoreduplication, but instead of going to hunt the role of CDC6 in endoreduplication they checked the effect of ORC2 which actually lower the overall impact of the paper.

de cet art

capturé à l'improviste

par l'horreur

terreurs nocturnes

garder

a way to summarise quantitative data;

describes what values are present in the data, and how often those values appear

Red arrow icon is needed

lmao

bran's a bit annoying

why not ask your dad for help

i thought he was like 9

yeah sansa

:(( lady killed by lannisters and soon robb too

bloodraven?

YEAH NO

ewww

STOPPP

suprised no one in westeros has mentioned them yet

the prophecy

oh im sure

barristan selmy?

the dance..

:(

YESS

omg they can fly now

there is some truth to this :(

the beginning kinda reminds me of robb and jeyne but red is such a specific color..

hmm interesting

oh sweet

oh he's a wildling? HOW DID BRAIME GET TO THE WALL WHAT

he still keeps the watch with him?

yup had a feeling

kinda reminds me sansa meeting the tyrells like metting a king while she met a queen, a blonde women and a pregnant one

yeah him and theon are polar opposites

a dutiful traitor

a good man in westeros? now thats rare

oo foreshadowing

interestingg

ok but WHY

FUCKK SHES SO IN LOVE :((

ok so thats what she was after

YUP

now what is he scheming...

aww lmao

HIT AFTER HIT BRO

lmao

the way she was engaged to a gay targ and is now talking about another one now lmao

sureee

erryk and arryk lmao

ICK

YES IVE BEEN WAITING FOR HIS GRIEF

:( sorry girl not this guy

oo yes more siblings

can't even blame her cuz he sounds so good

wait yess

for - new term - curiosity trap - When distractions take us out of the concentration and focusing zone

for - time sense - more new events gives a longer sense of time

for - neuroscience - why brains forget - active forgetting

neuroscience -active forgetting - leaves behind a small set of salient ideas

for - neuroscience - memories - reconstructed in the present - with new information - Indyweb - talking to our old selves - memories

for - neuroscience - perspectival knowing - why it's important to know other perspectives - perspectival knowing - Abraham Lincoln quote - I don't know that man - I better get to know his perspective

for - neuroscience - time estimation - non-experts are better at providing time budgets - neuroscience - non-experts give better time estimates than consultants

for - Henning Beck - neuroscience

for - neuroscience - how to - create perception of a long life - increase new activities

https://youtu.be/D5in5EdjhD0?si=oc7InD9Cj24GYmfz

好み～好む　の重複が気になりました。

代案

好みであれば、この手法が気に入るかもしれません

ちなみに command と入力しても同じ画面になります

Click New to add a new path to the current list of paths. の訳が抜けてます。

訳案

続いて新規ボタンを押して現在のパスに新しいパスを追加します。

わかりにくいです。

代案

XAMPPインストーラーを実行したときにデフォルト設定のままにしていたのであれば、表示されるボックスにC:\xamp\phpを追加してOKをクリックします。

typo 編集

訳が違うかも。

代案

WSL環境のセットアップについてはここでは説明しきれません。

訳は合っていると思うのですがややぶつ切り感があります。

代案

好きなOSで開発でき、好きなホストにプッシュできます。また、プロジェクトをプッシュするツールは十分に開発されており、リモートシステムで正しく稼働するように必要に応じてプロジェクトを修正してくれます。

typo？　を

リソースにはいろんな意味があるので、ここでは日本語に訳したほうがよいのでは

代案

情報源

Platform.sh上の　としたほうがPaaSの一種なんだな、と伝わりやすいと思います

日本語としてやや不自然に感じました。

代案

アプリケーションのデプロイがうまくいくというのはとても満足感が高いものです。一度もデプロイしたことがなければなおさらです。

typo:トル

「ログのアウトプット」が良いかなと思いましたが

「の」が3連続するので、いっそ

「以下ログの抜粋を読み、」とアウトプットを削るのはどうでしょうか

typo: 「リモート接続を」

最後句点がないのが今までの文と比較して違和感がありました。

デベロッパー？

トル？

「か」が一つ多いのでトル

「なにが発生しているかが」

typo トル

typo: ローカルシステム

助詞「は」が連続しているので、どちらかをとったほうが良いと思います。

Ex1) この付録にある追加情報でデプロイメントのプロセス全体が成功しない場合は、

Ex2) この付録にある追加情報ではデプロイメントのプロセス全体が成功しない場合、

https://www.theguardian.com/world/article/2024/aug/04/adriatic-warming-sea-italy-fishers-mucilage-microalgae

*See Video Library "Additional Functions screen functions"

See Video Library "iCombi Classic Settings screen functions"

See Video Library "iCombi Classic Settings screen functions"

See Video Library "iCombi Classic Initial screen functions"

See Video Library "iCombi Classic First Screen after pressing "menu" button demo"

See Video Library "iCombi Classic "Start Screen with Menu Button"

See Video Libray "iCombi Classic On/Off switching procedure"

See Video Libray "iCombi Classic On/Off switching procedure"

It is no longer possible to enter the iCombi Pro Service menu unless you have downloaded the Rational PIN Creator App and have a valid log in and password.

Attempts to enter the Service Menu will generate a QR code on the screen.

Scanning the QR code with the Rational PIN Creator app will generate a PIN which if used on the screen will enable entry to the Service Menu for technicians.

See Video Library "Access to Service menu for Technicians"

If you have undergone technical training by Rational UK or its approved training partners you can obtain a log in and password from Rational UK.

See Video Library " iCombi Pro Start screen function"

Please note: When the on/off switch LED illuminates amber, immediately release the on/off switch.

Failure to do so will result in the LED blinking amber/green/amber/green continuously and the machine will not switch off.

See Video Library "iComb Pro switch off procedure"

Please note: When the on/off switch LED illuminates green, immediately release the on/off switch.

Failure to do so will result in the LED blinking green/amber/green/amber continuously and the machine will not switch on.

See Video Library "iCombi Pro switch on precedure"

See video Library "Drain Connection iCombi"

Table of Contents.

X51 is a 12 pole female connector from which a 12 core cable carries data to and from A10 and the CPU (A12).

The 12 wire cable also caries a 5V "stand by" voltage from I/O board A10 to on/off switch on CPU (A12) .

The "stand by" voltage enables operation of the on/off switch and the CPU as soon as power is supplied to the oven (as the isolator is switched on).

When the on/off switch is operated, ESTL completes its safety checks and providing no high temperature errors or component errors exist, contactor K1 energises and a 12 V supply is also sent to the CPU via this connector and its associated cable.

Core probe with single thermocouple situated at the end of the probe. It is important when using the probe to ensure the end of the probe is in the centre of the procduct to be cooked.

Connection X19 has 6 pin terminals but only 1, 2, & 5 are used to supply 3 solenoid valves in the water distruibution block as required by the process selected. Each valve operates at 240V ac.

Terminal Pin no 1 (White cable) drives solenoid valve Y2 (Control nozzle) and is ndicated with a small triangle printed onto the pcb.

Terminal Pin no 2 (Yellow cable) drives solenoid valve Y1 (Water box / steam generator filling).

Terminal Pin no 5 (Black cable) drives solenoid valve Y4 (Care box fill).

Terminal Pin no 6 (Blue cable) is the Neutral to all solenoid valves in the block.

As all connections to the pcb this connection is also coded (poke yoke principal) However during covid, some Combi Steamers were issued with uncoded connectors for X19.

This raisese the possibility of the connector becoming out of algnment!, resulting in no solenoid valve functions or incorrect solenoid valve functions.

Always check for the correct the alignment of this connector

過去データの期間を変更できるようにする * 1年 * 3年 * 5年 * 10年

https://www.theatlantic.com/science/archive/2018/06/toolmaking-language-brain/562385/?fbclid=IwAR0fqdjBn1m6t6yu3xsD2fLnBPXOBS6UJjwCB_L0mB237QQbzwxifMQMd18

https://youtu.be/ycC5oZqNXsk?si=TOC9tWYErsrQAbjs

for - climate crisis - psychology - wrong approach

summary - Climate scientist professor Mojib Latif explores why our best efforts at rapid intervention to deal with the climate crisis are failing - Near the end of the program, he interviews professor Henning Beck, a neuroscientist who suggests that human brains have evolved to be rewarded for securing more. - Dopamine is released when we get more and we have not designed our intervention strategies aligned with this basic property of our brains

for - neuroscience - Henning Beck - more education - can lead to - more stubbornness

Test

( ~19:15 )

Johannes Schmidt calls Luhmann: "Without a doubt the most important German sociologist of the 20th century."

https://www.theguardian.com/global-development/article/2024/aug/01/uruguay-anger-environmental-cost-google-datacentre-carbon-emissions-toxic-waste-water

https://www.theguardian.com/us-news/article/2024/aug/02/great-salt-lake-shrinking-greenhouse-gas-emissions

by whom?

French text can be found here: http://1libertaire.free.fr/MFoucault248.html

https://www.youtube.com/watch?v=OFyvkcMygC0

https://www.youtube.com/watch?v=RfdY3FjN25Q

https://www.youtube.com/watch?v=mmz778N12_E

https://www.youtube.com/watch?v=jlTRDytQLT8

https://www.youtube.com/watch?v=4I4cOkA5SPg

https://www.youtube.com/watch?v=LeG1f6jzPpI

https://www.youtube.com/watch?v=GQWgFRSQUq8

https://www.youtube.com/watch?v=0-gJrAAZLxM

what each object can do

the properties or what each object knows about itself

objestcs

provide a blueprint for creating objects of a certain kind

kind of value that combines data and the code that operates on that data into a single unit.

(~1:20)

Apparently Markus Krajewski is the one with "perhaps the most intimate knowledge of the Zettelkasten process alive"...

Why?

https://www.youtube.com/watch?v=8w-sthcusIQ

https://www.youtube.com/watch?v=nR6veVRme4M

https://www.youtube.com/watch?v=MA1jzKaeSM4

https://www.youtube.com/watch?v=b-favkyWZDE

https://www.youtube.com/watch?v=GBm46sZLUIo

https://www.youtube.com/watch?v=Blr59QrACHI

https://www.youtube.com/watch?v=3xMQRcs59O4

https://www.youtube.com/watch?v=CaUb3m4-Eco

https://www.youtube.com/watch?v=Cq1-Y5pU9XU

https://www.youtube.com/watch?v=NYYK9fv2P2E

https://www.youtube.com/watch?v=qwXTxYTKeiw

The lecture "Propositions as Types" by Philip Wadler covers several significant concepts linking logic to computation, influencing the design of theorem provers and programming languages. Here are the key points:

1. Concept Overview: The principle of "Propositions as Types" connects logical propositions with computational types, an idea that initially seems coincidental but is fundamentally robust and impactful.

2. Historical Context: Various historical figures and concepts are touched upon, including:

   • David Hilbert: His contributions to formalizing mathematics.
   • Kurt Godel's Incompleteness Theorems.
   • Alonzo Church: Known for his work on Lambda Calculus.
   • Alan Turing: Contributions to the theory of computation and Turing Machines.

3. Lambda Calculus: Introduced by Alonzo Church, this is essential for understanding function abstraction and application in programming languages.

4. Natural Deduction and Formal Proofs: These concepts illustrate how logical reasoning can be systematically formalized, which is integral to both mathematics and computer science.

5. Type Theory: A framework in which every "term" has a "type," which is analogous to a logical proposition. This theory underpins many modern programming languages, especially functional ones.

6. Evaluation of Programs: Discusses how computational processes can be seen as logical deductions, further solidifying the Propositions as Types link.

7. Influence on Programming Languages: The principle has heavily inspired the design and development of various languages and theorem provers, stressing its importance in computational theory.

8. Philosophical and Practical Implications: The theory’s depth extends into philosophical debates about the nature of mathematics and computation, questioning whether mathematics is discovered or invented.

9. Polymorphic Lambda Calculus: Examines how types can be generalized, adding flexibility and power to programming languages.

10. Multiverses and Logic: Explores the implications of type theory and Propositions as Types in broader logical systems and multiple contexts.

These points together underscore the significance of Propositions as Types in both theoretical and practical realms of computer science.

https://www.youtube.com/watch?v=xSzolhEbZyc

https://www.youtube.com/watch?v=xykJKvTz-3c

https://www.youtube.com/watch?v=Sg62nTcGFJo

https://www.youtube.com/watch?v=bKK6a9B6iOE

https://www.youtube.com/watch?v=Z6B83swhhDo

( ~8:00 )

This explanation of why to read books in a certain order in dependency of each other is analogous to why a Zettelkasten (in Luhmannian sense) cannot be used collaboratively.

In order for someone else to understand your notes (not meant to be published), they would have to understand both the source text you are referencing and the implicit references you make. Things you understand instinctively and do not need to write down.

Because others do not have your experiences and worldview, it is more difficult for them, perhaps impossible, to completely comprehend your Zettels, your notes.

( ~5:00 ) Reading Aids should be used after initial interpretation. This is to prevent framing bias.

( ~1:40)

Suggested to train analytical reading in high school.

New Punch List: Olympia SG1 by [[maryech]]

Big Blue Gets Renewed by [[Joe Van Cleave]]

I like the aftermarket upgrades you've made.

The Cheers reference reminds me that I've been trying to pair drinks with machines as I write. I had a blue cocktail on a cruise to Alaska last year that used glacier ice (which is blue), but since that's not easy to source in ABQ, maybe try an "Electric Iced Tea" which is a variation on the Long Island iced tea but which swaps blue curaçao for the triple sec and a clear lemon-lime based soda for the cola.

This is a surprising fact because with TV shows they make it seem like eye witnesses know for sure who they see so these statistics for eyewitness misidentification is very surprising information

This is a tricky fact because that can make it very difficult for the police to make sure they are getting the correct details. I know from peoples stories that my memories can alter in my brain so it is very tricky with suggestibility.

بعبع

This is a very interesting fact. I never knew this was what it was called or why momentous events can be so easily memorized because everyone talks about when a moment big enough like 9/11 happens people will forget specific details.

yeah

Gratitude to the machine: Clarice Lispector on typewriters by [[Richard Polt]]

p155 image of Harrison's Arca Studiorum

arca studiorum appears on p 124

p90

The chart on this page appears to be that of John Locke's indexing system from 1685 and is here being re-published in 1689.

https://archive.org/details/bub_gb_IgMVAAAAQAAJ/page/n105/mode/2up

Gessner in Placcius

Students cannot succeed it is critical that a teacher establishes a clear learning goal for students to strive for. They also need to understand the steps to get there. If a teacher does this successfully, students will be set up for success in all they do.

I believe that students will do much better in a class when they are DOING something, as said in the article. When students are actively using their hands, brains, and all other senses, they will learn better. Arts integration is a great way to get kids moving.