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Esta definición queda mejor en la sección 2.1.

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Ok, very good. An important subject treated with a good amount of research. Some of the writing could be revised for claritv and better grammatical formation, but all in all a good use of the platform and the sources. The images should be captioned completely in-line: artist, title, medium, dimensions, date, repository etc.

SPELLING

for - Medium article - The nyam of non-duality - Aleander Vezhnevets - Apr. 12, 2023

for - adjacency - thinking of life as death - we are always dying - Deep Humanity - living is dying - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - Tibetan - Thukdam - can end suddenly and dramatically - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - Tibetan - Tukdam - ordinary people with no training also go into Tukdam - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - Tibetan - Kagyu and Nyingma schools don't make a big deal out of Tukdam - nondual awareness can emerge with other techniques - key insight - Buddhism - Tibetan - Clear light meditation at time of death - Tukdam - a physiological technique - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - key insight - Buddhism - Tibetan - Clear light meditation at time of death - Tukdam - a physiological technique to get to the same place as philosophical analysis - recognizing nondual, ultimate nature of reality - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - key insight - Buddhism - TIbetan - Clear light meditation - Tukdam at time of death - no longer capable of thinking, seeing, hearing, etc - all that's left is naked consciousness without even subject-object from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - book - Death, Intermediate State and Rebirth - Jeffrey Hopkins

for - Buddhism - Tibetan - meditation - Perfection stage - bring the winds into the central channel to the heart chakra - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - Tibetan - Mandala - is a 2 dimensional representation that the practitioner must imagine as a 3 dimensional object - This is the generation stage practice - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - TIbetan - Clear light meditation - purpose of - deity visualization - become the deity to practice giving up your ordinary thoughts and feelings - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - definition - unexcelled yoga tantra - the ultimate practice of simulating clear light meditation while still alive, in the Gelupa tradition of Tibetan Buddhism - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - TIbetan - clear light meditation - practice - how to practice simulation of Tukdam while still alive? - Stop ordinary thoughts and feelings - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - key insight - one's ordinary identity IS the pattern of the flow of the winds - this makes practice of Tukdam very difficult - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne - a tendency towards lust, aversion, etc is accompanied by a flow of wind. - to practice this during life, we have to get out of the deep patterns we identify with in life

for - Buddhism - Tibetan - clear light meditation practice - 5 primary and 5 secondary flows of energy in channels in the body - meditators practice a desired flow configuration at time of death - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - Tibetan - clear light meditation at time of death - can practice while alive a simulated version meditation - requires mastery of the internal "winds" - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - Tibetan - clear light meditation at time of death - can practice while alive a simulated version meditation - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - meditation - clear light meditation at time of death - Tukdam - why? The most fundamental state of mind occurs at the time of our death - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - relationship between - ultimate nature of reality - ultimate nature of mind - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for\ - Buddhism - relationship - Gelupa and Yogacara - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Buddhism - Tantric logic - Become a buddha - to experience the ultimate nature of reality - to relieve suffering of others - cultivate wisdom - experience ultimate nature of mind - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - definition - avidya (Sanskrit) or Ma Ri Pa (Tibetan) - Fundamental misunderstanding (both intellectual and affective) about the (ultimate) nature of reality itself - from Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

for - Mahayana Buddhism - Lay description - Helping others to help themselves - Youtube - Between Life and Death: Understanding Tukdam - John D. Dunne

Ok, Very good. The images are excellent and the StoryMap is well presented and written. The bibliography is not as deep as one might hope...more reading ( books and periodicals) and thesis development would be an improvement.

I would start right away with "Robert Adam" section rather than the intro to neoclassical arch. The mapping part of the platform might have been more focussed on the Grand tour elements...maybe what specific influences and examples inspired Adam... but really very nice to see a project on important architecture!

I haven't researched where the color-coding thing started, though I suspect content creators/influencers online in the last decades as a means of making their content "pretty" rather than necessarily functional.

Historically commonplaces were based on huge varieties of topics/subject headings, so colors and symbols were not frequently used. Most who needed greater organization or search capabilities indexed their commonplaces. One of the most popular means was detailed by philosopher John Locke in 1685. Here's some pointers to his work in this area in my own digital commonplace using Hypothesis: https://hypothes.is/users/chrisaldrich?q=tag%3A%22commonplace+books%22+tag%3A%22John+Locke%22

---

reply to u/_cold_one at https://old.reddit.com/r/commonplacebook/comments/1hhavye/20_topics_colour_coding/

See also my implementation at https://pigeon.fly

históricos, de las variables

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Not a Type-oh! Last Minute Christmas Gift for the Typewriter Lover in Your Life. by [[Mary Beth Klatt]]

Gendersegregatie is de scheiding van mannen en vrouwen in verschillende rollen, taken of activiteiten, vaak gebaseerd op traditionele gendernormen.

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La revue Prescrire n'est pas "intransigeante sur les médicaments", elle est simplement indépendante. Je trouve que ces précisions sur la position de Prescrire et de la HAS auraient du être explicitées. S'il n'y a aucun problème, comme le laisse entendre le ton général de l'article, pourquoi des recommdations aussi strictes et prudentes ??

Cette mention est trompeuse car elle suggère que la France serait épargnée par les risques. Or le coup d'arrêt aux THM en 2002 a été suivi par une baisse rapide et spectaculaire des cancers du sein chez les Françaises de 50-64 ans : https://gco.iarc.fr/overtime/en/dataviz/trends?populations=250&sexes=1_2&types=0&multiple_populations=1&cancers=14&age_start=10&age_end=12

En seulement 7 ans, l'incidence standardisée des cancers du sein dans cette tranche d'âge est passée de 346 cas à 295 cas. Soit une réduction de 16% sur l'ensemble de cette classe d'âge. Soit une baisse de 32% pour les 50% de femmes de cet âge suivant une THM. Cela représente un surrisque de cancer du sein de 50% environ pour les femmes traitées, cohérent avec les résultats des études épidémiologiques.

L'attribution de cette chute à la fin des traitements est sans équivoque. Dans la classe d'âge 35-50 ans, aucune baisse de l'incidence n'est observable en 2003:

https://gco.iarc.fr/overtime/en/dataviz/trends?populations=250&sexes=1_2&types=0&multiple_populations=1&cancers=14&age_start=7&age_end=9

eLife Assessment

This important work advances our understanding of parabrachial CGRP threat function. The evidence supporting CGRP aversive outcome signaling is solid, while the evidence for cue signaling and fear behavior generation is incomplete. The work will be of interest to neuroscientists studying defensive behaviors.

Reviewer #1 (Public Review):

Summary

The authors asked if parabrachial CGRP neurons were only necessary for a threat alarm to promote freezing or were necessary for a threat alarm to promote a wider range of defensive behaviors, most prominently flight.

Major Strengths of Methods and Results

The authors performed careful single-unit recording and applied rigorous methodologies to optogenetically tag CGRP neurons within the PBN. Careful analyses show that single-units and the wider CGRP neuron population increases firing to a range of unconditioned stimuli. The optogenetic stimulation of experiment 2 was comparatively simpler but achieved its aim of determining the consequence of activating CGRP neurons in the absence of other stimuli. Experiment 3 used a very clever behavioral approach to reveal a setting in which both cue-evoked freezing and flight could be observed. This was done by having the unconditioned stimulus be a "robot" traveling along a circular path at a given speed. Subsequent cue presentation elicited mild flight in controls and optogenetic activation of CGRP neurons significantly boosted this flight response. This demonstrated for the first time that CGRP neuron activation does more than promote freezing. The authors conclude by demonstrating that bidirectional modulation of CGRP neuron activity bidirectionally affects freezing in a traditional fear conditioning setting and affects both freezing and flight in a setting in which the robot served as the unconditioned stimulus. Altogether, this is a very strong set of experiments that greatly expand the role of parabrachial CGRP neurons in threat alarm.

Weaknesses

In all of their conditioning studies the authors did not include a control cue. For example, a sound presented the same number of times but unrelated to US (shock or robot) presentation. This does not detract from their behavioral findings. However, it means the authors do not know if the observed behavior is a consequence of pairing. Or is a behavior that would be observed to any cue played in the setting? This is particularly important for the experiments using the robot US.

The authors make claims about the contribution of CGRP neurons to freezing and fleeing behavior, however, all of the optogenetic manipulations are centered on the US presentation period. Presently, the experiments show a role for these neurons in processing aversive outcomes but show little role for these neurons in cue responding or behavior organizing. Claims of contributions to behavior should be substantiated by manipulations targeting the cue period.

Appraisal

The authors achieved their aims and have revealed a much greater role for parabrachial CGRP neurons in threat alarm.

Discussion

Understanding neural circuits for threat requires us (as a field) to examine diverse threat settings and behavioral outcomes. A commendable and rigorous aspect of this manuscript was the authors decision to use a new behavioral paradigm and measure multiple behavioral outcomes. Indeed, this manuscript would not have been nearly as impactful had they not done that. This novel behavior was combined with excellent recording and optogenetic manipulations - a standard the field should aspire to. Studies like this are the only way that we as a field will map complete neural circuits for threat.

Reviewer #2 (Public Review):

-Summary of the Authors' Aims:<br /> The authors aimed to investigate the role of calcitonin gene-related peptide (CGRP) neurons in the parabrachial nucleus (PBN) in modulating defensive behaviors in response to threats. They sought to determine whether these neurons, previously shown to be involved in passive freezing behavior, also play a role in active defensive behaviors, such as fleeing, when faced with imminent threats.

-Major Strengths and Weaknesses of the Methods and Results:<br /> The authors utilized an innovative approach by employing a predator-like robot to create a naturalistic threat scenario. This method allowed for a detailed observation of both passive and active defensive behaviors in mice. The combination of electrophysiology, optogenetics, and behavioral analysis provided a comprehensive examination of CGRP neuron activity and its influence on defensive behaviors. The study's strengths lie in its robust methodology, clear results, and the multi-faceted approach that enhances the validity of the findings.

No notable weakness found.

-Appraisal of Aims and Results:<br /> The authors successfully achieved their aims by demonstrating that CGRP neurons in the PBN modulate both passive and active defensive behaviors. The results clearly show that activation of these neurons enhances fear memory and promotes conditioned fleeing behavior, while inhibition reduces these responses. The study provides strong evidence supporting the hypothesis that CGRP neurons act as a comprehensive alarm system in the brain.

-Impact on the Field and Utility of Methods and Data:<br /> This work has significant implications for the field of neuroscience, particularly in understanding the neural mechanisms underlying adaptive defensive behaviors. The innovative use of a predator-like robot to simulate naturalistic threats adds ecological validity to the findings and may inspire future studies to adopt similar approaches. The comprehensive analysis of CGRP neuron activity and its role in defensive behaviors provides valuable data that could be useful for researchers studying fear conditioning, neural circuitry, and behavior modulation.

-Additional Context:<br /> The study builds on previous research that primarily focused on the role of CGRP neurons in passive defensive responses, such as freezing. By extending this research to include active responses, the authors have provided a more complete picture of the role of these neurons in threat detection and response. The findings highlight the versatility of CGRP neurons in modulating different types of defensive behaviors based on the perceived intensity and immediacy of threats.

Overall, this manuscript makes a significant contribution to our understanding of the neural basis of defensive behaviors and offers valuable methodological insights for future research in the field.

Reviewer #3 (Public Review):

Strengths:<br /> The study used optogenetics together with in vivo electrophysiology to monitor CGRP neuron activity in response to various aversive stimuli including robot chasing to determine whether they encode noxious stimuli differentially. The study used an interesting conditioning paradigm to investigate the role of CGRP neurons in the PBN in both freezing and flight behaviors.

Weakness:<br /> The major weakness of this study is that the chasing robot threat conditioning model elicits weak unconditioned and conditioned flight responses, making it difficult to interpret the robustness of the findings. Furthermore, the conclusion that the CGRP neurons are capable of inducing flight is not substantiated by the data. No manipulations are made to influence the flight behavior of the mouse. Instead, the manipulations are designed to alter the intensity of the unconditioned stimulus.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

We thank reviewers for their comments and constructive criticisms of our study. We have implemented corrections* that were suggested for the manuscript, and we have also clarified any concerns that were raised in our responses below. *

*Reviewer #1 *

Overall technology development is good though as they claim that they are first is not true as it has been used earlier by https://doi.org/10.1128/msphere.00160-22. Hence may be that they have used to decipher the cell cycle.

The cited paper used FUCCI in the host cells and not in the parasites themselves. Our study thus reports the first FUCCI model in a unicellular *eukaryote. *

• *

The manuscript is extremely dense and at times very difficult to read and to be clear if they are focussing on the technology or cell cycle. The technology may be a better part of manuscript but the dissection of cell cycle is not very novel and at times very confusing to follow. Many of these aspects has been dissected out previously from their own group and many group in Toxoplasma and Plasmodium and it is quite known about that the cell cycle in Apicomplexa is very complex.

We adapted FUCCI to the Toxoplasma model to help dissect the organization of its cell cycle, which as the reviewer noted, is highly complex. While overlaps between some phases were anticipated based on prior data, these overlaps had not been measured. We were able to determine the extent of these overlaps in the post-G1 period and describe the organization of the non-conventional cell cycle of T. gondii.

Another aspect that most FUCCI use Geminin and CDT1 factors and since Geminin is not present it would have been better to validate that with CDT1 that is present in Apicomplexa and may be more relevant than PCNA1.

Unfortunately, the Toxoplasma ortholog of CDT1 (TgiRD1) cannot be used as a FUCCI marker for the reasons stated in lines 116-117; the expression of TgiRD1 is not limited to a specific cell cycle phase (Hawkins et al., 2024). PCNA1 can be (and has been) used as a FUCCI marker, but it was not considered an ideal marker in mammalian cells due to its relatively low expression levels. However, Toxoplasma PCNA1 is highly abundant in tachyzoites, and its expression correlates with the period of DNA replication. Furthermore, Plasmodium ortholog of PCNA1 had been used as a DNA replication sensor in the recent studies (35353560). *Altogether, it validates PCNA1 as an appropriate S-phase FUCCI probe. *

The first part of the manuscript only deals with first to identify the function and localisation of PCNA1 and then develop FUCCI technology and then go on to study cell cycle. So the focus of the manuscript is not clear. It seems three different results are just assembled together in one manuscript with out clear focus. In order to get clear focus the authors should clear set out the focus as to why they developed FUCCCI and how they decipher either replication, budding, apical or basal complex, centrosome or cytokinesis as well to be used for drug discovery The way it is organised it is not flowing well and confuses the reader who may not be aware of different compartment of Toxoplasma cell or not a molecular parasitologist.

We believe the reviewer has described the logic of our study. Our goal was to dissect the cell cycle. Consequently, we adapted a suitable technology, FUCCI. We described the relevant experiments that allowed us to produce a new molecular tool for an apicomplexan model, and illustrated how we used this tool to better understand the complicated processes of its cell division. Therefore, we organized our study accordingly and included our goal, plans, results, and conclusions that support the success of adopted technology and establishment of the cell cycle organization. We hope this brief explanation can provide some clarification for the reviewer.

Some of the conclusion on the that Replication starts at centromere region is not novel and has been studied previously.

We agree that the centromeric start of DNA replication is not a novel feature, which is stated in the text. This result was shown to demonstrate that Toxoplasma replicates its DNA according to the rules* conserved across eukaryotes. *

The manuscript needs revising by writing precisely eliminating too much literature reference in the result section with clear focus. Some of these references can be elaborated in the introduction and discussion to keep the focus.

We examined the results section, and as much as we wanted to comply with this reviewer, we found no references that could be eliminated or transferred to the introduction. We believe that to aid the reader, some foundational knowledge needs to be presented together with obtained results to support those findings.

• *

Some points with respect to figures: Generally with image panels, arrows don't stand out well

We* have adjusted the images.

*

Fig1: no scale bars and the green arrow do not stand out. So may be to make white.

*The scale bar can be found in the bottom right image, which applies to every image in the panel. We changed the color of the arrows. *

Fig 2E: state the time point in the fig without IAA treatment (-IAA)

The requested information was added to the figure legend.

Fig4: no bell shaped curve

We rephrased the description. The” bell-shape” analogy applies to the temporal dynamics of DNA replication, which starts with a single aggregate, expands to numerous replication foci, and is reduced to a few aggregates at the end of replication. We attempted to quantify aggregates, but their irregular shape makes this task impossible. Our statement is supported by steady-state images and real-time microscopy of the DNA replication included in the manuscript.

Fig 5D: it isn't obvious what the numbers on the right hand side of the graph mean. If it is size, there should be a unit given

We provided an explanation in the figure legend*.

*

Figure 6 - how do they determine that the tachyzoites have progressed through 61% of S phase? Make this clearer here.

*We examined only DNA replicating parasites (S-phase) and determined the fraction of BCC0-positive (39%) and BCC0-negative (61%) tachyzoites. Quantifications can be found in Table S4, in the S-C worksheet. *

• *

Fig7: it a strange way of ordering the figure as FigE is after Fig F hence no logical order. Thank you, we have corrected the order of these panels*. *

Fig 8H is not mentioned in the text

*Thank you, we referenced the wrong panel. Fig. 8H is now included in the text. *

Figure 9 is nice and useful but the arrows could be made proportional of time spent in each cell cycle phase. They're a little off in the conventional cell cycle at the minute

• *These schematics are intended to illustrate the dramatic difference in cell cycle organization rather than to directly describe cell cycle organization, the latter of which can be found in Figure 6.

Some comment on the text in the manuscript: Line 137: describing the expression pattern: the following papers first described the expression pattern of PCNA1 and 2 can be cited in the result. https://doi.org/10.1016/j.molbiopara.2005.03.020 We added the reference.

Line 154: Provide schematic for AID HA cloning and confirmation.

The schematics and PCR confirmations* can be found in the supplemental figure S2.

*

Line 157: Fig 2 after 4 h treatment FACS analysis shows more than 1 and less than 2n genomic content. Does this study have any -IAA treated control for 4h and 7h to compare as what should the standard genomic content to be there at this time point of development. At 4 h of development can the authors provide any statistical analysis with their 3 experiments to prove their point that the replication is actually stalled. Downregulation of TgPCNA1 as shown is western blot is still basal protein left to carry the genomic replication in 7 mins. Can authors also state that TgPCNA 2 which is although non-essential but has no redundant role in the replication machinery.

The -IAA control is indicated as 0h and is shown in blue. The statistical analysis of three independent experiments showing the increase of the S-phase population is included in Table S3. The Fig. 2 WB shows over 99% TgPCNA1 degradation, and the residual >1% would be insufficient to carry out full DNA replication. This residual signal is likely due to PCNA1 remaining in complex, which would resist *proteolysis. Unfortunately, we do not feel comfortable to make the final statement suggested. We believe that the lack of TgPCNA2 complementation with yeast PCNA1 (Guerini et al, 2005) is insufficient to draw the conclusion that TgPCNA2 plays a non-redundant role in Toxoplasma replication machinery. *

Line 178 : typing error "that that

Thank you, this has been corrected*.

*

Line 179: states the role of TgPCNA 1 in DNA1 replication, however line 159 and 160 states the TgPCNA1 deficient can fulfil DNA replication. Can author confirm this contrast in the results. Results trying to illustrate the same fact TgFUCCIs or TgPCNA1ng that TgPCNA1 first aggregates at centromeres and then distributed on many replication forks and disappears late during cytokinesis. The part of the result can be merged.

We apologize for the *confusion. We rephrased our statements and supported them by corresponding references. Although it may seem repetitive, but it was our intention to emphasize a consistent spatial-temporal expression of TgPCNA1-HA and TgPCNA1-NG. *

Line 189: Typing error, should say "such as nucleus", currently as is missing

Thank you, this has *been corrected.

*

Line 346-349: basically explaining the same thing twice.

We apologize for the confusion, the first sentence describes compartments where MORN1 is located. The second sentence describes how MORN1 localization changes during cell cycle progression, information which is used later in our quantitative IFA of cell cycle phases*. *

• *

Line 347 - immunfluorescent should be immunofluorescence

Thank you, this has been corrected*.

*

Line 395-399: does this study has any non-inhibited (-IAA control) at 4h and 7 h. for fig 7C & 7G. Can the authors provide any statistical analysis for the significance with their 3 experiments.

The untreated control (7h mock) is shown as 0h treatment (first bar in each panel). The figure also shows the results of the statistical analysis (t-test, numbers above) that can also be found in Table* S7.

*

Line 415: Why the authors have not used the TgFUUCI sc lines which expresses the TgPCNAng and IMCmch both. This could have helped to understand the real time dynamics of DNA replication and budding initiation (cytokenesis), rather then fixing and staining with TgIMC.

*The recent study by Gubbels lab identified the earliest known budding marker BCC0. Unfortunately, BCC0 is a low abundant factor and cannot be used in FUCCI. IMC3 emerges in the midst of budding when the daughter conoid and polar rings are assembled and thus does not signify either the beginning or the end of cytokinesis. We added IMC3 as a supporting budding marker, while our primary focus remains on the DNA replication marker PCNA1. *

Overall good technology development as FUCCI but the rest of the manuscript is extremely dense and the focus of the study is not clear after technology part. The complexity of the cell cycle is known and hence not much novelty here and extremely descriptive and hard read. Science can be simplified.

The reviewer agrees the apicomplexan cell cycle is highly complex, and the field has worked diligently to piece together what we can about it, which contributes to the density of the manuscript. We hope that the targeted audience will find it thoughtful, and we strove to provide sufficient information for those outside our field. We also respectfully disagree that our study offers little novelty; while it is known how complex the apicomplexan cell cycle is, there is still much to uncover. While overlapping cell cycle phases exist in other eukaryotes, there were no such studies that showed the degree of these overlaps across the entire T. gondii cell cycle. We believe there are valuable insights to be gained from the identification of the composite cell cycle phase, which in turn could help draw attention to other understudied features of the cell cycle in non-conventional eukaryotes*. *

*Reviewer #2 *

1. It is not always clear where apical and basal ends of the parasite are. E.g. in Fig 3F are the two parasites on the right facing down with their apical end? In Fig 4 it is even harder to see. Might be helpful to turn these images with their apical end up to make comparative interpretation of figures easier. In the text it mentions that PCNA1 concludes at the 'proximal' end of the nucleus (or with the nucleus proximal, which is not clear either??). Please define clearly where the proximal site is, as it is not clear in the figures or in the movie (the 'last focus' marker in Fig 4D??). Thank you for the suggestion. We rotated images in Fig. 3 and marked the parasite ends in Fig. 4. We also indicated parasites’ polarity in the movies.

Synchrony of replication cycle. Tight synchronization depends on the retention of the cytoplasmic bridge, as mentioned by the authors. In larger vacuoles, it is very conceivable not all parasites are connected with each other (notably in large cysts with bradyzoites), which could lead to loss of tight synchrony. The results section states "One plausible explanation is that the rosette split shortens the communication path between tachyzoites". This is somewhat cryptic language: does a 'rosette split' imply the rupture of the cytoplasmic bridge? This statement should be clarified. Another factor could be centrosome maturation, with the mother centrosome ready sooner than the daughter, which is a model proposed in schizogony, where the nuclear cycles in a shared cytoplasm are even more asynchronous/independent.

Yes, by ‘rosette split’, we refer to the break of the connection, or a cytoplasmic bridge. The model based on centrosome maturation is interesting, however, it does not explain the synchronization of a vacuole of 16, unless centrosome age resets at that point*. *

Centrosome duplication. This has been documented to occur at the basal side of the nucleus (the whole nucleus rotates for centrosome duplication). The images as depicted in Fig 6 do not seem to indicate this event, possibly because it is not easy to track apical and basal end of the cell (#1 above). Please comment, as this could be an additional spatial cue to the specific stage of the cycle.

This is a very interesting suggestion, thank you. Indeed, the centrosome often duplicates away from the apical end (disconnects from the Golgi), sometimes on the side or the basal end, but quickly rotates back to the apical position to reconnect with co-segregating organelles. Centrosome traveling is an interesting feature, and it is possible that this reorientation back to the apical end signifies budding initiation. We will explore this hypothesis in future studies.

• Specific experimental issues that are easily addressable.

• The term "Apicomplexan" should be spelled with a lower case "apicomplexan", which is not consistently applied throughout the manuscript. Thank you, we have corrected the spelling*. *

* 2. Line 567 the term used in 2008 was "tightly knit" not "closely woven". We wanted to avoid the exact citation and rephrased the title of the review.

*

*Reviewer #3 *

-The authors choose to describe PCNA1 and IMC3 as FUCCI markers. The efficiency of this system in mammalian cells is based on the proof that the markers are regulated through a rapid proteolysis process. However, the data available for these markers point toward a transcriptional regulation of these markers (Toxodb and (1)). In contrast, the authors do not provide any data indicating that these proteins are true FUCCI markers. Consequently, they should not use the term FUCCI throughout the paper unless they prove that the cell cycle expression depends on proteolysis. For example, the authors could express these genes with a promoter that is not cell cycle regulated.

PCNA1 was one of the original FUCCI markers for mammalian cells, later replaced by the more abundant geminin. PCNA1 ubiquitination is well supported across all eukaryotes, and we believe there is much data to support this same turnover mechanism acts to regulate PCNA1 in Toxoplasma. Transcriptional profiles show that TgPCNA1 mRNA is constantly present in cells, never dropping below 80%, making this mRNA is among the most abundant in the cell. It also indicates that proteolysis, rather than halted transcription, controls TgPCNA1 protein levels, since TgPCNA1 protein expression drops to nearly undetectable levels in early G1 and budding (Fig. 1). In addition, TgPCNA1 is highly conserved in structure (Fig. S1) and in function (TgPCNA1 interactome, Fig. 1). The TgPCNA1 Ub sites were detected in global ubiquitome analyses (ToxoDB), supporting the fact that TgPCNA1 protein abundance is regulated by ubiquitin-dependent degradation. Furthermore, PCNA1 as a FUCCI marker in model eukaryotes was not tested for proteolysis because it was unquestionable that PCNA1 is regulated by proteolysis. In addition, Plasmodium ortholog of PCNA1 had been used as a DNA replication sensor in the recent studies (35353560), which validates PCNA1 as an appropriate S-phase FUCCI probe. The modern FUCCI probes are fragments of CDT1 and Geminin mimicking the spatiotemporal expression of the corresponding cell cycle regulators. The transcriptional profile of TgIMC3 is also largely unchanged across the cell cycle, which heavily implies that proteolysis control*s its dynamic protein expression. Therefore, we believe that the term FUCCI applies to TgPCNA1 and TgIMC3. *

-The authors show that the localization of PCNA1 change during the cell cycle and indicate that the PCNA1 aggregates observed are replication forks. They do not provide data supporting this. They should co-localize these aggregates with other markers such as ORC, MCM proteins or DNA polymerase to better characterize these aggregates. There are number of techniques that could be used to localize the origin(s) of replication. Similarly, ExM should be used to characterize the colocalization between PCNA1 aggregates and the centromeres. As such, the images provided are of poor quality and do not support the author conclusions. The few PCNA1 aggregates toward the end of the S phase are also not characterized. Are they telomeres?

Although this is an important point, such detailed analyses of the DNA replication machinery is out of the scope of the current study and will be examined in a follow-up study. Data that suggest the aggregates correspond to replication forks include proteomics analyses of chromatin-bound PCNA1 that identified replisome components such as the MCM, high conservation of TgPCNA1 sequence and structure (Fig. S1), and its conserved interactions (Fig. 1). Recent studies used Plasmodium ortholog of PCNA1 to trace DNA replication dynamics during schizogony (35353560), *Therefore, we doubt that TgPCNA1 would perform functions outside of its role as a DNA replication factor, which has been extensively studied in other eukaryotes. *

• The authors characterized the proteins associated with PCNA1. All the proteins found to potentially interact are chromatin-bound and are not naturally found in other localization (2). It is unclear why the authors insist on the fact that there are two PCNA1 complexes (one chromatin-bound and one non-chromatin bound). More concerning is the lack of verification of this dataset through reciprocal IP for example.

The PCNA IP was used to confirm its conserved function as a DNA replication factor; similarly to what was observed in other eukaryotes, we detected PCNA in both a chromatin-bound and unbound state. PCNA1 is produced in late G1 (diffuse nuclear stain) but is engaged in the replisome only upon DNA replication initiation (aggregated form). Rather than characterize the function of the highly conserved PCNA1, our primary goal was to determine the Toxoplasma cell cycle organization, which explains our choice of the experimental design.

• Quantification of some of the phenotypes is lacking. For example, the DNA content analysis are shown but the change in number are not. Similarly, there is no quantification of the PCNA1 mutant phenotypes observed by ExM. Quantification of the bell shape observed by video-microscopy in figure 4 should also be provided.

The quantifications supporting the main claims of our study are included in the five supplemental Tables S3-S8, including DNA content and microscopy analysis of the phenotype. *The U-ExM microscopy has been solely used to visualize details of the phenotype. *

• The PCNA1 mutant phenotypes are not sufficiently explored by ExM. What happen to the mitotic spindle? What happens to kinetochore (CenH3 is a centromere protein and does not represent kinetochores)? Many markers for these structures have been described, see (3).

The primary goal of our study was to examine and map out the organization of the tachyzoite cell cycle. PCNA1 deficiency was used to demonstrate that Toxoplasma PCNA1 is a conserv*ed DNA replication factor and can be used as an S-phase marker in FUCCI. Thus, we focused on the mutant-induced changes in the dynamics of DNA replication (DNA content) and arrest prior to mitosis (unresolved centrocone). *

• TgPCNA1NG strain has a number of concerns. The localization to the daughter cells conoids seems artificial since not observed in the HA-AID mutant and the expression pattern seems different as well than the previous mutant suggesting the mNG tag is affecting the localization and expression dynamics. Did the authors explore other fluorescent proteins to verify that these discrepancies where not due to this tag ?

The conoidal PCNA1 accumulation was detected only with NeonGreen-tagged PCNA1. We also built and examined tdTomato- and mCherry-tagged versions and detected minor accumulations in the conoid of tdTomato-tagged PCNA1, but not with the mCherry-tagged variant. We believe these aggregations could be attributed to the partially degraded PCNA1-NeonGreen having an affinity to conoidal proteins, thus producing this unexpected signal. Although not included in the manuscript, our quantifications, based on both PCNA1-HA and PCNA1-NeonGreen, showed similar cell cycle organization (G1, S and budding phases) of tachyzoites. The FUCCI probe is an indicator of the cell cycle phase. It does not have to be a functional protein. As we mentioned before, many FUCCI probes are fragments of the factors that mimic the spatiotemporal expression of the corresponding cell cycle regulators.

-Cytokinesis seems to be only defined by the presence of IMC3. The marker appears early during the budding process and it is not normally considered as a cytokinesis marker. The author should the text to reflect this.

We agree with the reviewer that IMC3 is not a true budding marker, which is why we used BCC0 in our quantifications. IMC3 is proven to broadly define the mid-budding stage, making it a convenient supplemental marker. We are currently exploring and testing alternative and additional FUCCI markers. It is not an easy task, since these markers are required to have high expression levels and to be localized into large organelles. For instance, BCC0 was eliminated due to low abundance.

• Throughout the manuscript, the authors seems to ignore an essential characteristic of the tachyzoite cell cycle: the nuclear cycle and the budding cycle are independently regulated. It is therefore normal they overlap as it has been shown by the authors themselves in previous studies. This should be better described and discussed in the paper to understand the peculiarities of the parasite cell cycle.

We apologize for the confusion, but the tachyzoite cell cycle does not contain a nuclear cycle, it consists of a single budding cycle. The nuclear cycle is only a feature in multinuclear cell cycles such as schizogony and endopolygeny. This is the main reason why the overlap between phases is so surprising.

• l196: "The surface of the growing buds": could the authors rephrase?

We rephrased the statement.

-L217: proximal end of the nucleus rather than "parasite ".

*We clarified the statement. It is, in fact, the shift of the nucleus to the proximal end of the parasite.

*

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Referee #3

Evidence, reproducibility and clarity

This is a manuscript from Batra et al. entitled "A FUCCI sensor reveals complex cell cycle organization of Toxoplasma endodyogeny ". It describes the characterization of PCNA1 as cell cycle marker in the parasite Toxoplasma gondii. Tachyzoite endodyogeny is a simplified division process that is crucial for the proliferation of the parasite. Some studies have used fluorescent markers to describe the segregation of organelles and the nuclear division during endodyogeny but the production of more tools to dissect the cell cycle and better characterize mutants is timely. Most of the experiments are based on characterization of PCNA1 mutant and the use of a strain expressing a PCNA1-mNG construct. Unfortunately, there are a number of concerns in this study that need to be addressed.

Major concerns:

• The authors choose to describe PCNA1 and IMC3 as FUCCI markers. The efficiency of this system in mammalian cells is based on the proof that the markers are regulated through a rapid proteolysis process. However, the data available for these markers point toward a transcriptional regulation of these markers (Toxodb and (1)). In contrast, the authors do not provide any data indicating that these proteins are true FUCCI markers. Consequently, they should not use the term FUCCI throughout the paper unless they prove that the cell cycle expression depends on proteolysis. For example, the authors could express these genes with a promoter that is not cell cycle regulated.
• The authors show that the localization of PCNA1 change during the cell cycle and indicate that the PCNA1 aggregates observed are replication forks. They do not provide data supporting this. They should co-localize these aggregates with other markers such as ORC, MCM proteins or DNA polymerase to better characterize these aggregates. There are number of techniques that could be used to localize the origin(s) of replication. Similarly, ExM should be used to characterize the colocalization between PCNA1 aggregates and the centromeres. As such, the images provided are of poor quality and do not support the author conclusions. The few PCNA1 aggregates toward the end of the S phase are also not characterized. Are they telomeres?
• The authors characterized the proteins associated with PCNA1. All the proteins found to potentially interact are chromatin-bound and are not naturally found in other localization (2). It is unclear why the authors insist on the fact that there are two PCNA1 complexes (one chromatin-bound and one non-chromatin bound). More concerning is the lack of verification of this dataset through reciprocal IP for example.
• Quantification of some of the phenotypes is lacking. For example, the DNA content analysis are shown but the change in number are not. Similarly, there is no quantification of the PCNA1 mutant phenotypes observed by ExM. Quantification of the bell shape observed by video-microscopy in figure 4 should also be provided.
• The PCNA1 mutant phenotypes are not sufficiently explored by ExM. What happen to the mitotic spindle? What happens to kinetochore (CenH3 is a centromere protein and does not represent kinetochores)? Many markers for these structures have been described, see (3).
• TgPCNA1NG strain has a number of concerns. The localization to the daughter cells conoids seems artificial since not observed in the HA-AID mutant and the expression pattern seems different as well than the previous mutant suggesting the mNG tag is affecting the localization and expression dynamics. Did the authors explore other fluorescent proteins to verify that these discrepancies where not due to this tag ? -Cytokinesis seems to be only defined by the presence of IMC3. The marker appears early during the budding process and it is not normally considered as a cytokinesis marker. The author should the text to reflect this.
• Throughout the manuscript, the authors seems to ignore an essential characteristic of the tachyzoite cell cycle: the nuclear cycle and the budding cycle are independently regulated. It is therefore normal they overlap as it has been shown by the authors themselves in previous studies. This should be better described and discussed in the paper to understand the peculiarities of the parasite cell cycle.

Minor

• l196: "The surface of the growing buds": could the authors rephrase?

• L217: proximal end of the nucleus rather than "parasite ".

• Behnke,M.S., Wootton,J.C., Lehmann,M.M., Radke,J.B., Lucas,O., Nawas,J., Sibley,L.D. and White,M.W. (2010) Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PloS One, 5, e12354.

• Barylyuk,K., Koreny,L., Ke,H., Butterworth,S., Crook,O.M., Lassadi,I., Gupta,V., Tromer,E., Mourier,T., Stevens,T.J., et al. (2020) A Comprehensive Subcellular Atlas of the Toxoplasma Proteome via hyperLOPIT Provides Spatial Context for Protein Functions. Cell Host Microbe, 28, 752-766.e9.
• L,B., N,D.S.P., Ec,T., D,S.-F. and M,B. (2022) Composition and organization of kinetochores show plasticity in apicomplexan chromosome segregation. J. Cell Biol., 221.

Significance

This study provides the characterization of a new cell cycle marker to decipher the tachyzoite cell cycle of the apicomplexan parasite Toxoplasma gondii. A better understanding of the cell cycle is needed to prevent the proliferation of this parasite. This study builds on previous works characterizing organellar segregation in T. gondii. It provides data about the overlap of each cell cycle phase and the synchronicity of the cell cycle in a single vacuole. However, it is limited by the use of a single marker and more data are needed to support the conclusions of this study. This study can be of interest to a broad audience.

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Referee #2

Evidence, reproducibility and clarity

• Are the key conclusions convincing?

The data support the new model put forward in the final figure: a composite cell cycle phase

There are couple of points that need attention:

1. It is not always clear where apical and basal ends of the parasite are. E.g. in Fig 3F are the two parasites on the right facing down with their apical end? In Fig 4 it is even harder to see. Might be helpful to turn these images with their apical end up to make comparative interpretation of figures easier. In the text it mentions that PCNA1 concludes at the 'proximal' end of the nucleus (or with the nucleus proximal, which is not clear either??). Please define clearly where the proximal site is, as it is not clear in the figures or in the movie (the 'last focus' marker in Fig 4D??).
2. Synchrony of replication cycle. Tight synchronization depends on the retention of the cytoplasmic bridge, as mentioned by the authors. In larger vacuoles, it is very conceivable not all parasites are connected with each other (notably in large cysts with bradyzoites), which could lead to loss of tight synchrony. The results section states "One plausible explanation is that the rosette split shortens the communication path between tachyzoites". This is somewhat cryptic language: does a 'rosette split' imply the rupture of the cytoplasmic bridge? This statement should be clarified. Another factor could be centrosome maturation, with the mother centrosome ready sooner than the daughter, which is a model proposed in schizogony, where the nuclear cycles in a shared cytoplasm are even more asynchronous/independent.
3. Centrosome duplication. This has been documented to occur at the basal side of the nucleus (the whole nucleus rotates for centrosome duplication). The images as depicted in Fig 6 do not seem to indicate this event, possibly because it is not easy to track apical and basal end of the cell (#1 above). Please comment, as this could be an additional spatial cue to the specific stage of the cycle.
4. Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

The authors are on the conservative end of interpretations and clearly outline the limitations of their approaches and observations, while discussing alternative interpretations. - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

No, the presented experiments and data are very complete - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

n/a - Are the data and the methods presented in such a way that they can be reproduced?

yes - Are the experiments adequately replicated and statistical analysis adequate?

yes

Minor comments:

• Specific experimental issues that are easily addressable.
• The term "Apicomplexan" should be spelled with a lower case "apicomplexan", which is not consistently applied throughout the manuscript.
• Line 567 the term used in 2008 was "tightly knit" not "closely woven".
• Are prior studies referenced appropriately?

Yes - Are the text and figures clear and accurate?

Yes, exceptional - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

See major point #1 above.

Referees cross-commenting

Comment to Rev 1: https://doi.org/10.1128/msphere.00160-22. reports on use of FUCCI in the host cell, not in the parasite itself. This comment therefore does not apply.

Comment to Rev 3: the technicality on FUCCI acting on the protein level. That is a legit concern that needs attention, and could be fixed by avoiding the term FUCCI, or putting the term in the exact context.

Looks like a shared general concern is that it is not always clear where apical and basal ends are in the presented data. This should be addressed in revision.

Significance

• Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

The presented manuscript reports on a technical innovation in Apicomplexa: establishing a FUCCI system. However they did not stop there and added additional markers to unravel the timing and nature of S/M/G2/C overlaps that illuminated previously underappreciated or unknow details. The tools assembled here will be of great value for understanding not only T. gondii endodyogeny checkpoints and sequence of events, but also paves the way for similar studies in more complex apicomplexan cell division modes, like schizogony and endopolygeny. - Place the work in the context of the existing literature (provide references, where appropriate).

The authors very appropriately provide the wider context and completely cover where the field stands. E.g. this protein microscopy-based work fills in the fine grain details where recent advances in transcriptional profiles by single cell experiments cannot provide resolution. The authors do also an outstanding job in providing the background on the general understanding of molecular players, structures and process controls across eukaryotes that pinpoint where the Apicomplexa are different. - State what audience might be interested in and influenced by the reported findings.

The audience comprises a wide array of people with interests in cell cycle regulation, cell cycle checkpoints, DNA replication, nuclear organization across biological systems, and Apicomplexa in particular - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Toxoplasma gondii cell biology - sufficient expertise across the board

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Referee #1

Evidence, reproducibility and clarity

The manuscript by Batra et al have tried to dissect out two aspect to understand the complex cell cycle of Toxoplasma endodyogeny. One is to development of Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) technology for Toxoplasma gondii and then to use that for understanding the complex cell cycle. The authors have created ToxoFUCCIs and ToxoFUCCIsc probes using TgPCNA1 tagged with NeonGreen and TgIMC3 tagged with mCherry and used to dissect the different phases of cell cycle like S, G2, G1 and cytokinesis. Overall technology development is good though as they claim that they are first is not true as it has been used earlier by https://doi.org/10.1128/msphere.00160-22. Hence may be that they have used to decipher the cell cycle.

The manuscript is extremely dense and at times very difficult to read and to be clear if they are focussing on the technology or cell cycle. The technology may be a better part of manuscript but the dissection of cell cycle is not very novel and at times very confusing to follow. Many of these aspects has been dissected out previously from their own group and many group in Toxoplasma and Plasmodium and it is quite known about that the cell cycle in Apicomplexa is very complex. Another aspect that most FUCCI use Geminin and CDT1 factors and since Geminin is not present it would have been better to validate that with CDT1 that is present in Apicomplexa and may be more relevant than PCNA1. The first part of the manuscript only deals with first to identify the function and localisation of PCNA1 and then develop FUCCI technology and then go on to study cell cycle. So the focus of the manuscript is not clear. It seems three different results are just assembled together in one manuscript with out clear focus. Some of the conclusion on the that Replication starts at centromere region is not novel and has been studied previously.

In order to get clear focus the authors should clear set out the focus as to why they developed FUCCCI and how they decipher either replication, budding, apical or basal complex, centrosome or cytokinesisas well to be used for drug discovery The way it is organised it is not flowing well and confuses the reader who may not be aware of different compartment of Toxoplasma cell or not a molecular parasitologist.<br /> The manuscript needs revising by writing precisely eliminating too much literature reference in the result section with clear focus. Some of these references can be elaborated in the introduction and discussion to keep the focus.

Some points with respect to figures:

Generally with image panels, arrows don't stand out well

Fig1: no scale bars and the green arrow do not stand out. So may be to make white.

Fig 2E: state the time point in the fig without IAA treatment (-IAA)

Fig4: no bell shaped curve

Fig 5D: it isn't obvious what the numbers on the right hand side of the graph mean. If it is size, there should be a unit given

Figure 6 - how do they determine that the tachyzoites have progressed through 61% of S phase? Make this clearer here.

Fig7: it a strange way of ordering the figure as FigE is after Fig F hence no logical order.

Fig 8H is not mentioned in the text

Figure 9 is nice and useful but the arrows could be made proportional of time spent in each cell cycle phase. They're a little off in the conventional cell cycle at the minute

Some comment on the text in the manuscript:

Line 137: describing the expression pattern: the following papers first described the expression pattern of PCNA1 and 2 can be cited in the result. https://doi.org/10.1016/j.molbiopara.2005.03.020

Line 154: Provide schematic for AID HA cloning and confirmation.

Line 157: Fig 2 after 4 h treatment FACS analysis shows more than 1 and less than 2n genomic content. Does this study have any -IAA treated control for 4h and 7h to compare as what should the standard genomic content to be there at this time point of development. At 4 h of development can the authors provide any statistical analysis with their 3 experiments to prove their point that the replication is actually stalled. Downregulation of TgPCNA1 as shown is western blot is still basal protein left to carry the genomic replication in 7 mins. Can authors also state that TgPCNA 2 which is although non-essential but has no redundant role in the replication machinery.

Line 178 : typing error "that that

Line 179: states the role of TgPCNA 1 in DNA1 replication, however line 159 and 160 states the TgPCNA1 deficient can fulfil DNA replication. Can author confirm this contrast in the results. Results trying to illustrate the same fact TgFUCCIs or TgPCNA1ng that TgPCNA1 first aggregates at centromeres and then distributed on many replication forks and disappears late during cytokinesis. The part of the result can be merged.

Line 189: Typing error, should say "such as nucleus", currently as is missing

Line 346-349: basically explaining the same thing twice.

Line 347 - immunfluorescent should be immunofluorescence

Line 395-399: does this study has any non-inhibited (-IAA control) at 4h and 7 h. for fig 7C & 7G. Can the authors provide any statistical analysis for the significance with their 3 experiments.

Line 415: Why the authors have not used the TgFUUCI sc lines which expresses the TgPCNAng and IMCmch both. This could have helped to understand the real time dynamics of DNA replication and budding initiation (cytokenesis), rather then fixing and staining with TgIMC.

Overall good technology development as FUCCI but the rest of the manuscript is extremely dense and the focus of the study is not clear after technology part. The complexity of the cell cycle is known and hence not much novelty here and extremely descriptive and hard read. Science can be simplified.

Significance

The development of FUCCI technology is significant part of the manuscript and to understand cellcycle may be they could have used CDT1 rather than PCNA as there is another PCNA 2 that also exist. The authors have given some convincing result for some aspect of cell cycle of which most are known and only it is quite incremental.at some part. The technology may contribute to the methodology development in Apicomplexa.

Bevoegdheid geven aan minderjarige die eigenlijk voor meerjarigen zijn.

has the same effect bjut one is know

or confound variable

Organize in subheadings. Rewrite so it is less of a "shopping list" of articles and more of a story. Connect articles to each other and to your project.

Write it as if the project has been completed.

Do not use future tense.

shall we show the date of last updates rather than publication ?

Really good idea to have a Resources sections

I would send them to the full list of satellites rather than one specific one. The point is that they choose based on their specific needs

As discussed before, I think .xlsx import/export deserve their own satellites. It's a whole topic in itself, and I feel like we will make this satellites anyways so better to prevent duplication + I would stick to very simple and basics stuff at that stage, let's remember it's probably their first few hours using R. A lot has already been covered until now.

I would show import with .csv only, and them have a satellite that deals with working with .xlsx in R.

I am really not a big fan of the side notes, I find them distracing and actually haven't notice them until now. I would suggest we stick to the three "tasks" callouts, and the three "informational" callout to convey information. Actually such a note, whoch is more of a deepening of a topi/concept should become a tooltip

Can we not simplify and use just here::here() without saving to a new variable ?

Need to fix citations so they appear in proper format.

Break it up into paragraphs and cite.

Break it up into paragraphs to make it easier to read. Cite when stating factual information or defining terms.

Rephrase

Citation?

This needs to include ethical implications of your work.

This should probably be under the previous heading "State of the Art"?

This section needs to provide a summary of the State of the Art

Which comparative framework?

This does not make sense. Rewrite using cohesive sentences that connect to each other and portray the project goals.

Nicely organized!

This should be a subsection within the Goals section.

Needs to be streamlined a bit to make it clear what exactly the goals are. You are stating several purpose/aim/goals and it should be clear how they all connect and align.

A citation?

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si

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de un

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sustituyendo

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El primer paréntesis en el lado izquierdo de la siguiente ecuación no está en la posición correcta.

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Quitar la coma.

Por lo tanto,

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entonces

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¿Quisiste decir i.i.d.?

Aplicando

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for - Youtube - documentary movie trailer - Tukdam - Between Worlds - Deep Humanity - death - clear light meditation - Tukdam - from - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson - https://hyp.is/Cv-qVL38Ee-9WiNDbGvK8w/www.youtube.com/watch?v=JObdEbHqqFA

for - wellbeing - mental illness - drug treatment vs brain changes from mindfulness practices - adjacency - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - adjacency - Tukdam and animal hibernation - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

for - Youtube - Tukdam talk - An Overview Of Center for Healthy Minds at University of Wisconsin-Madison (CHM)’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson - wellbeing - clear light meditation, meditation at time of death - Tukdam

summary - Professor Davidson speaks on the subject of Tukdam, the Tibetan practice of meditation at the time of death practiced by Tantric practitioners - He contextualizes it in the framework that all sentient beings are sacred, and have the capacity for unfolding the intrinsic sacred that each of us is born with - Davidson's team explores the impact of meditation and mindfulness practices on human health and wellbeing and have formulated a wellbeing framework with four pillalrs - Deep Humanity - impacts of meditation - meditation at time of death

to - Youtube - documentary movie trailer - Tukdam: Between Worlds - https://hyp.is/FJg9XL4PEe-M9OfpvdsFQQ/www.youtube.com/watch?v=dDBEl9bSGMQ

for - trivia - measuring tukdam after death - 24 hour period immediately following death is important but to date, no data captured - Youtube - Tukdam talk - An Overview Of CHM’s Work On “Well-Being And Tukdam” - Prof. Richard J. Davidson

opzoeken -> snap het niet helemaal.

Welcome back and in this lesson I want to cover the serverless architecture.

Serverless is a type of architecture which is relatively commonplace within AWS, mainly because AWS includes many products and services which support its use.

The key thing to understand about the serverless architecture, aside from the fact that there are really servers running behind the scenes, is that it's not one single thing.

Serverless is an architecture, but it's more a software architecture than a hardware architecture.

The aim with the serverless architecture and where its name comes from is that as a developer or an architect or an administrator, you're aiming to manage few, if any, servers.

Servers are things which carry overhead, so cost, administration and risk, and the serverless architecture aims to remove as much of that as possible.

In many ways, serverless takes the best bits from a few different architectures, mostly microservices and event-driven architectures.

Within serverless you break an application down into as many tiny pieces as possible, even beyond microservices, collections of small and specialized functions.

These functions start up, do one thing really, really well, and then they stop.

In AWS, logically, because of this, Lambda is used.

But there are other platforms such as Microsoft Azure, which has their own equivalent, namely Azure Functions.

From an architecture perspective, the actual technology which is used is less relevant.

These functions which make up your application, they run in stateless and ephemeral environments.

Why this matters is because if the application is architected to assume a clean and empty environment, then these functions can run anywhere.

Every time they run, they obtain the data that they need, they do something, and then optionally, they store the result persistently somehow, or they deliver that output to something else.

The reason why Lambda is cheap is because it's scalable.

Each environment is easy to provision, and each environment is the same.

So the serverless architecture uses this to its advantage.

Each function that runs does so in an ephemeral and stateless environment.

And another key concept within serverless is that generally everything is event-driven.

This means that nothing is running until it's required.

Any function code that your application uses is only running on hardware when it's processing a system or customer interaction, an event.

Serverless environments should use fast products such as Lambda for any general processing needs.

Lambda as a service is built based on execution duration, and functions only run when some form of execution is happening.

Because serverless is event-driven, it means that while not being used, a serverless architecture should be very close to zero cost until something in that environment generates an event.

So serverless environments generally have no persistent usage of compute within that system.

Now, where you need other systems beyond normal compute, a serverless environment should use where possible managed services.

It shouldn't reinvent the wheel.

Examples are using S3 for any persistent object storage, or DynamoDB, which we haven't covered yet for any persistent data storage, and third-party identity providers such as Google, Twitter, Facebook, or even corporate identities such as Active Directory instead of building your own.

Other services that AWS provides, such as Elastic Transcode, can be used to convert media files or manipulate these files in other ways.

With the serverless architecture, your aim should be to consume as a service whatever you can, code as little as possible, and use function as a service for any general-purpose compute needs, and then use all of those building blocks together to create your application.

Now, let's look at this visually, because I think an architecture diagram might make it easier to understand exactly what a serverless architecture looks like.

So let's step through a simple serverless architecture, and we're going to do so visually.

And I want your default position to be that unless we state otherwise, you're not using any self-managed compute, so no servers and no EC2 instances, unless we discuss otherwise.

So that should be your starting position.

And at each step throughout this architecture, I'll highlight exactly why the parts are serverless and why it matters.

Now, we're going to use a slightly more inclusive example.

This time, we're going to use PetTube.

There was an uproar about PetTube only being for cats, and so it's rebranded to be a little bit more inclusive.

So to start with, we've got Julie using her laptop, and she wants to upload some woofy holiday videos.

And so to do that, she browsers to an S3 bucket that's running as a static website for the PetTube application.

She downloads some HTML, and that HTML has some JavaScript included within it.

Now, one crucial part of the serverless architecture is that modern web browsers are capable of running client-side JavaScript inside the browser.

And this is what actually provides the front end for the PetTube application, JavaScript that's running in the browser of the user that's downloaded from a static website S3 bucket.

So at this point, the application has no self-managed compute that's being used.

We've simply downloaded HTML from an S3 bucket with some included JavaScript that's now running in Julie's web browser.

Now PetTube uses third-party identity providers for its authentication.

Like all good serverless applications, it doesn't use its own store of identity, its own store of users.

It's lower admin overhead, and also remember there's a limit on the number of IAM users that can exist inside one AWS account.

That's 5,000 IAM users per account.

And so if we used IAM users for authentication, then PetTube would be limited to 5,000 users, and each user of the application would need one additional account.

So one additional username and one additional password.

So instead of doing that, we use a third-party identity provider and one that our users are already likely to have an account inside.

So that reduces the number of accounts that our users are required to maintain.

So the JavaScript that's running in Julie's browser communicates with the third-party identity provider, and we're going to assume that we're using Google.

And you'll have seen the screen that's generated if you've ever logged into Gmail or anything that uses Gmail logins, but this could just as easily be Twitter, Facebook, or any other third-party identity provider.

The key thing to understand is that Julie logs into this identity provider.

It's this identity provider that validates that the user claiming to be Julie is in fact Julie, so it checks her username and password.

And if it's happy with the process or if it's happy with the username and password combination that Julie's provided, then it returns to Julie an identity token.

And this token proves that she's authenticated with the Google identity provider.

Now, AWS can't directly use third-party identities, and so the JavaScript that's running in Julie's browser communicates with an AWS service called Cognito.

And Cognito swaps this Google identity token for temporary AWS credentials, and these can be used to access AWS resources.

So the JavaScript in Julie's browser now has available some temporary AWS credentials that it can use to interact with AWS.

And so it uses these temporary credentials to upload a video of Woofy to an S3 bucket.

This is the original bucket of our application, the bucket where the master videos go that our customers upload.

Notice that so far in this process, no self-managed compute, no servers have been used to provision this service.

We've performed all of these activities without using any compute servers or compute instances that we need to manage or design as solutions architects.

It's all delivered by using managed services, so S3, Cognito, and the Google identity provider.

Now, when the Woofy video arrives inside the original's bucket, that bucket is configured to generate an event.

That event contains the details of the object which was uploaded, and it's set to send that event to and invoke a Lambda function to process that video.

That Lambda function takes in the event and it creates jobs within the elastic transcoder service, which is a managed service offered by AWS which can take in media and manipulate that media.

One of the things that it can do is to transcode the media, so generate media of different sizes from one master video file.

Multiple jobs get created, one for each size of video that's required.

The elastic transcoder gets the location of the original video as part of the initiation of the job and it loads in that video at the start of each job processing cycle.

So each job outputs an object to a transcoder bucket, so one object for each different size of the original video.

In addition, details on each of the new videos are added to a database, in this case DynamoDB.

Now again at this stage, notice that we still have no self-managed servers.

The only resources that are consumed are storage space in S3, DynamoDB, and any processing time used for the Lambda function and any elastic transcoder jobs.

With this architecture so far, we've allowed a customer to upload a master video, we've transcoded it into different video sizes, and at no point have we consumed any self-managed compute, no EC2 instances or no other long-running compute services.

It's all managed services or compute that's used in Julie's browser.

Now the last part of the architecture is where Julie, by clicking another part of the client site that's running inside her browser, can interact with another Lambda function, and we'll call this My Media, and this Lambda function will load data from the database, identify which objects in the transcode bucket are Julie's, and return URLs for Julie to access.

And this is how Julie can load up a web page which show all of the videos that she's uploaded to the PetTube application.

Now this is a simplified diagram, in reality it's a little bit more complex.

For example, API Gateway would generally be used between any client-side processing and the Lambda functions, but conceptually this is actually how it works.

We've got no self-managed servers, we've got no self-managed database servers, we've got little, if any, costs that are incurred for base usage.

It's a fully consumption-based model.

It consumes compute only when it's being used, so when events are generated, either from a system-side or a client-side, and it uses third-party services as much as possible.

Now there are many third-party services to choose from, and you can never expect to know them all end-to-end.

The key thing to understand about serverless is the way to do things, and I've covered that in this lesson.

Later in the section you'll experience how to implement a serverless application within the demo lesson called PetCuddleatron.

And this will show you how to implement a serverless application just like the one that's on screen.

It's slightly less complex, but it's one that uses many of the same architectural fundamentals, and it should start to really cement the theory that you're learning right now.

Now before we move on to this demo, there are a few more services that I need to cover, which the PetCuddleatron demo lesson will utilize.

So for now, that's it for this lesson.

Thanks for watching.

Go ahead and complete this video, and then when you're ready, I'll look forward to you joining me in the next.

Welcome back.

In this lesson, I want to cover CloudWatch events.

We've covered CloudWatch earlier in the course, which focused on metrics and monitoring.

We've also covered CloudWatch logs, which focused on the ingestion and management of logging data.

CloudWatch events delivers a near real-time stream of system events.

These events describe changes in AWS products and services.

When an instance is terminated, started or stopped, these generate an event.

When any AWS products and services which are supported by CloudWatch events perform actions, they generate events that the product has visibility of.

Events Bridge is the service which is replacing CloudWatch events.

It can perform all of the same bits of functionality that CloudWatch events can produce.

It's got a superset of its functionality.

In addition, Events Bridge can also handle events from third parties as well as custom applications.

They do both share the same basic underlying architecture, but AWS are now starting to encourage a migration from CloudWatch events over to Events Bridge.

We've got a lot of architecture to cover, so let's jump in and get started.

Both Events Bridge and CloudWatch events perform at a high level the same basic task.

They allow you to implement an architecture which can observe if X happens or if something happens at a certain time, so Y, then do Z.

X is a supported service which generates an event, so it's a producer of an event.

Y can be a certain time or time period, and this is specified using the Unix Cron format, which is a flexible format letting you specify one or more times when something should occur, and Z is a supported target service to deliver the event to.

Events Bridge is basically CloudWatch events version two.

It uses the same underlying APIs, and it has the same basic architecture, but AWS recommend that for any new deployments, you should use Events Bridge because it has a superset of the features offered by CloudWatch events.

Things created in one are visible in the other for now, but this could change in the future.

So as a general best practice, you should start using Events Bridge by default for any of the functions that you can use CloudWatch events for.

Now, both of these services actually operate using a default entity, which is known as an event bus, and both of them actually have a default event bus for a single AWS account.

A bus in this context is a stream of events which occur from any supported service inside that AWS account.

Now, in CloudWatch events, there is only one event bus available, so it's implicit.

It's not really exposed to the UI.

It just exists.

You interact with it, but because there's only one of them, it's not actually exposed as a visible thing.

You just look for events and then send these events to targets when you want something to occur.

So in CloudWatch events, there is only one event bus, and it's not exposed inside the UI.

In Event Bridge, you can create additional buses, either for your applications or third-party products and services, and you can interact with these buses in the same way as the account default event bus.

Now, with CloudWatch events and Event Bridge, you create rules, and these rules pattern match events which occur on the buses, and when they see an event which matches, they deliver that event to a target.

Alternatively, you also have schedule-based rules which are essentially pattern-matching rules but which match a certain date and time or ranges of dates and times.

So if you're familiar with the Unix Cron system, this is similar.

For a schedule rule, you define a Cron expression, and the rule executes whenever this matches and delivers this to a particular target.

So the rule matches an event, and it routes that event to one or more targets which you define on that rule.

And an example of one target is to invoke a specific Lambda function.

Now, architecturally, at the heart of Event Bridge is the default account event bus, which is a stream of events which are generated by supported services within the AWS account.

Now, EC2 is an example of a supported service, and let's say in this case, we've got Bob changing the state of an EC2 instance, and he's changing the state from stopped to running.

When the instance changes state, an event gets generated which runs through the event bus.

Event Bridge, which sits over the top of any event buses that it has exposure to, monitors all of the events which pass through this event bus.

Now, within Event Bridge or CloudWatch events, which I'm going to start calling just Event Bridge from now on because it makes it easier, but within Event Bridge, we have rules.

Now, rules are created, and these are linked to a specific event bus, and the default is the account default event bus.

The two types of rules are pattern matching rules, and these match particular patterns of the events themselves as they pass through the event bus.

We've also got scheduled rules which match particular cron-formatted times or ranges of times, and when this cron-formatted expression matches a particular time, the rule is executed, and in both of these cases, when a rule is executed, the rule delivers the particular event that it's matched through to one or more targets.

And of course, as I just mentioned, examples of these targets could be to invoke a lambda function.

Now, events themselves are just JSON structures, and the data in the event structure can be used by the targets.

So in the example of a state change of an EC2 instance, the lambda function will receive the event JSON data, which includes which instance has changed state, what state it's changed into, as well as other things like the date and time when the change occurred.

So that's a theory of both CloudWatch events and the event bridge, and both of these products are used as a central point for managing events generated inside an AWS account and controlling what to do with those events.

So at this point, that is everything that I wanted to cover.

Go ahead and complete this lesson, and then when you're ready, I look forward to you joining me in the next.

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