5,905 Matching Annotations
  1. Feb 2022
    1. This article is for those who want to keep traveling despite restrictions due to covid. Basically giving tips on how to navigate the multiple governmental restrictions and policies including links to airline or country websites for choosing destinations. Because of this trend in travel advice in covid times, we may see attitudes towards travel shift to travel knowing the risks involved (quarantine, masks requirements, etc.) and hence see tourism rise again. Last minute covid holiday packages. What if the trend for remaining home also stayed the same for next five years and the adventure seekers become the avatars for the folks who want to stay at home.

      The crisis is changing the way how people will enjoy their international holiday, with an extra concern on testing and quarantine expenses and risk taking. That may have an impact on the tourism market, asking the airline companies to provide flexible policies /products and may witness the booming of travel insurance market.

  2. Jan 2022
    1. Dr. Thrasher wrote a book! (2022, January 8). My cousin wanted to get tested. She waited in an auto testing line for 6.5 hours, and stayed in it bc she was traveling to bury her Daddy. How many people give up in such long lines? How many cases upwards of a million are we losing bc Biden et all failed on home tests? Https://t.co/Q7WVy5qD4v [Tweet]. @thrasherxy. https://twitter.com/thrasherxy/status/1479826389142491146

    1. Depuis longtemps, je suis d’avis que la rigueur d’un cours ne se mesure pas à la quantité de connaissances dont l’enseignant fait étalage, mais aux apprentissages que les étudiants font.

      Which can lead to an assessment of pedagogical efficacy. It's funny, to me, that those who complain about "grade inflation" (typically admins) rarely entertain the notion that grades could be higher than usual if the course went well. The situation is quite different in "L&D" (Learning and Development, typically for training and professional development in an organizational context). "Oh, great! We were able to get everyone to reach the standard for this competency! Must mean that we've done something right in our Instructional Design!"

  3. Dec 2021
    1. El púlsar binario PSR J0737 como banco de pruebas de la relatividad general Por Francisco R. Villatoro, el 16 diciembre, 2021. Categoría(s): Astronomía • Ciencia • Física • Noticias • Physics • Relatividad • Science ✎ 3

      l púlsar binario PSR J0737 como banco de pruebas de la relatividad general Por Francisco R. Villatoro, el 16 diciembre, 2021. Categoría(s): Astronomía • Ciencia • Física • Noticias • Physics • Relatividad • Science ✎ 3

      Hulse y Taylor recibieron el Premio Nobel de Física en 1993 por su estudio del púlsar binario PSR B1913+16 (el primero que se descubrió en 1974), que observó de forma indirecta la emisión de ondas gravitacionales. Se publica en Physical Review X un análisis similar del púlsar binario PSR J0737−3039A/B, descubierto en 2003. El púlsar binario PSR J0737 es un banco de pruebas único para el estudio de la relatividad general ya que está situado a solo dos mil años luz de la Tierra, ambas estrellas de neutrones se observan como púlsares y su inclinación orbital es muy próxima a 90 °, luego se puede observar cómo el espaciotiempo curvo del plano orbital modifica los pulsos emitidos. Las observaciones durante 16 años de la precesión del periastro siguen la fórmula de la emisión gravitacional cuadripolar de Einstein con un error menor del 0.013 % (el resultado obtenido tras 2.5 años de observaciones tenía un error del 0.05 % y se publicó en 2006 en Science). Sin lugar a dudas un púlsar binario que habrá que seguir durante las próximas décadas para mejorar estas estimaciones.

      Además de probar la fórmula cuadripolar de Einstein, se ha probado el retraso debido al efecto de Shapiro (en un espaciotiempo curvo las señales de radio viajan durante más tiempo y las observamos retrasadas). También se han realizado otras pruebas de la relatividad que hasta ahora no se habían podido realizar con otros púlsares binarios. Por ejemplo, se ha medido la deformación relativista de la órbita (debido al acoplamiento relativista entre el espín (rotación de las estrellas de neutrones) y el momento angular de su órbita). En estas pruebas los resultados tienen mucha mayor incertidumbre, pero en todos los casos son compatibles con las predicciones de la relatividad general de Einstein. Esta teoría, a la que muchos físicos quieren matar cuanto antes, además de muy bella es muy robusta y promete reinar en la física durante muchas décadas.

      El artículo es M. Kramer, I. H. Stairs, …, G. Theureau, «Strong-field Gravity Tests with the Double Pulsar,» Physical Review X 11: 04150 (13 Dec 2021), doi: https://doi.org/10.1103/PhysRevX.11.041050, arXiv:2112.06795[astro-ph.HE] (13 Dec 2021); más información divulgativa en Lijing Shao, «General Relativity Withstands Double Pulsar’s Scrutiny,» Physics 14: 173 (13 Dec 2021) [web].

      Una manera de destacar la excepcionalidad del púlsar binario PSR J0737 es compararlo con el famoso PSR B1913, que ha sido estudiado durante 35 años. Esta figura muestra la precesión del periastro de la órbita; la diferencia en la densidad de puntos entre 0 y −20 es notable. Así se explica que el nuevo resultado para PSR J0737 tras 16 años tenga un error menor del 0.013 %, cuando para PSR B1913 solo se alcanzó el 0.2 %; por cierto, para las fusiones de agujeros negros observadas por LIGO-Virgo el error típico ronda el 20 %. No le he dicho, pero supongo que sabrás que el periastro de una órbita elíptica es el punto donde la distancia entre ambos cuerpos es mínima; se llama perihelio cuando uno de los cuerpos es el Sol y perigeo cuando es la Tierra. El fenómeno que mide esta figura es análogo a la precesión del perihelio de la órbita de Mercurio, que Einstein usó como guía hacia la formulación correcta de su teoría de la gravitación.

      ASTROFÍSICACIENCIAEXPERIMENTOFÍSICANOTICIASPÚLSARTEORÍA DE LA RELATIVIDAD GENERAL

      3 Comentarios Mario dice: 17 diciembre, 2021 a las 5:10 pm Francis Hay una frase que no entiendo, favor revisar: «…,luego sus señales se observa cómo el espaciotiempo curvo del plano orbital modificada la señal que observamos». Atte Mario

      RESPONDER Francisco R. Villatoro dice: 17 diciembre, 2021 a las 9:15 pm Gracias, Mario.

      RESPONDER Mario dice: 19 diciembre, 2021 a las 10:07 pm Francis, entiendo que por el efecto shapiro las señales de radio se ven retrasadas; pero para notar tal retraso tiene que haber una referencia. Cuál es esa referencia?

      RESPONDER Deja un comentario

    1. For the evaluation of thermal stability, each separator was positioned between slide glasses and subjected to certain tensile shear stress. Then, the temperature was raised to 150℃ and 180℃ at 5℃·m−1, and both separators were kept for 12 h at each temperature in order to compare their contraction percentages

      how to test heat shrinkage

  4. Nov 2021
    1. e Netherlands and analyzingtest scores on externally standardized tests, the studyreports a learning loss of 3.16 percentiles on a com-posite index of math, reading, and spelling, an effectthat varies in size by socioeconomic background andschool composition

      the Netherlands and analyzingtest scores on externally standardized tests, the studyreports a learning loss of 3.16 percentiles on a com-posite index of math, reading, and spelling, an effectthat varies in size by socioeconomic background andschool composition

    1. evernote之类的拥有自己的很强大的clipper剪藏工具,我觉得logseq作为笔记学习工具也应该有一个,在logseq的API开放后大佬们可以考虑开发一个.

      一个简单的测试

    1. verb (used with object), set, set·ting.to put (something or someone) in a particular place: to set a vase on a table.

      这是我第一个故事

  5. Oct 2021
    1. Coronavirus Pandemic Data Explorer. (n.d.). Our World in Data. Retrieved March 3, 2021, from https://ourworldindata.org/coronavirus-data-explorer

      is:webpage lang:en COVID-19 graph case death Germany Sweden UK Afghanistan Africa Albania Algeria Andorra Angola Anguilla Antigua Barbuda Argentina Armenia Asia Australia Austria Azerbaijan Bahamas Bahrain Bangladesh Barbados Belarus Belgium Belize Benin Bermuda Bhutan Bolivia Bosnia Herzegovina Botswana Brazil Bulgaria Burkina Faso Burundi Cambodia Cameroon Canada Cape Verde Cayman Islands Central African Republic Chad Chile China Colombia Comoros Congo Costa Rica Cote d'ivoire Croatia Cuba Cyprus Czechia Democratic Republic of Congo Denmark Djobouti Dominica Dominician Republic Ecuador Egypt El Salvador Equatorial Guinea Eritrea Estonia Eswatini Ethiopia Europe Europian Union Faeroe Islands Falkland Islands Fiji Finland France Gabon Gambia Georgia Ghana Gibraltar Greece Greenland Grenada Guatemala Guernsey Guinea Guinea-Bissau Guyana Haiti Honduras Hong Kong Hungary Iceland India Indonesia Iran Iraq Ireland Isle of Man Israel Italy Jamaica Japan Jersey Jordan Kazakhstan Kenya Kosovo Kuwait Kyrgyzstan Laos Latvia Lebanon Lesotho Liberia Libya Liechtenstein Lithuania Luxembourg Macao Madagascar Malawi Malaysia Maldives Mali Malta Mashall Islands Mauritania Mauritius Mexico Micronesia Moldova Monaco Mongolia Montenegro Morocco Mozambique Myanmar Namibia Nepal Netherlands New Zealand Nicaragua Niger Nigeria North America North Macedonia Northern Cyprus Norway Oceania Oman Pakistan Palestine Panama Papua New Guinea Paraguay Peru Philipines Poland Portugal Qatar Romania Russia Rwanda Saint Helena Saint Kitts and Nevis Saint Lucia Saint Vincent Grenadines Samoa San Marino Sao Tome and Principe Saudi Arabia Senegal Serbia Seychelles Sierra Leone Singapore Slovakia Slovenia Solomon Islands Somalia South Africa South America South Korea South Sudan Spain Sri Lanka Sudan Suriname Switzerland Syria Taiwan Tajikistan Tanzania Thailand Timor Togo Trinidad Tobago Tunisia Turkey Turks and Caicos Islands Uganda Ukraine United Arab Emirates USA Uruguay Uzbekistan Vanuatu Vatican Venezuela Vietnam World Yemen Zambia Zimbabwe test vaccine chart map table data case fatality rate mortality

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  6. Sep 2021
    1. Just for fun, I did a little experimenting at home to show how some of these different types of cement hold up. I started by cementing a bunch of materials together with a bunch of different types of cement. I waited 24 hours, then cut each one roughly in half, down the middle.
    1. Best demonstration: glue PVC inside an ABS hub or vice-versa. Cut through the two with a mitre saw and make a nice, clean cut. Look at all the voids where the plastics didn't glue together.
    1. Midget retinal ganglion cells (RGCs) are the most common RGC type in the primate retina. Their responses have been proposed to mediate both color and spatial vision, yet the specific links between midget RGc responses and visual perception are unclear.

      同样做标记

    1. 1840年鸦片战争以后,中国逐步成为半殖民地半封建社会,国家蒙辱、人民蒙难、文明蒙尘,中华民族遭受了前所未有的劫难。从那

      这里是可以做标记的

    1. Create an account using the sidebar on the right of the screen

      HI WORD.

    1. Die Bewertung der direkten Gefährdung durch Laserstrahlung erfolgt mithilfe des Vergleichs der Exposition durch die Laserstrahlung von Auge oder Haut mit den jeweiligen Expositionsgrenzwerten, welche eine Grenze für einen Augen- oder Hautschaden darstellen.

      Was kann ich aus dieser Information lernen?

    2. Bewertung der direkten Gefährdung durch Laserstrahlung erfolgt mithilfe des Vergleichs der Exposition durch die Laserstrahlung

      Noch ein wichtiger Kommentar

  7. Aug 2021
  8. Jul 2021
    1. Teaching Metadata IG

      "Metadata...presents educators and trainers with continually evolving pedagogical challenges."

      "...a forum for exchanging experiences and advice on the content of curricula and on classroom methodologies"

      "innovation in metadata design, implementation & best practice"

    1. Media Cafe

      Visitors to this Second Life coffee shop could wear their own objects.

      • like the Matrix-styled sunglasses attached to my avatar's nose with its own website. .
    1. M.I.T. OpenCourseWare

      Here, Hypothes.is annotation layer provides a collaborative space for communication. Next, I will post a comment to this old, old blog post, given readers a link to this public annotation.

    1. Shouldn’t Tech Developers (such as yourself) be reaching out to Schools/ Teachers/ Districts FIRST to create and design tech rather than the other way around?”

      Edit

      Reaching out again with new project MentorsOnline.NET

      Notes: 1st edit of annotation for update of info

      • 2nd use of tag: LTI-test-team
    1. Google Meet training and help

      Scenario: Team building exercise, group tag(s) sorting downstream readers/ viewers (audience).

      Note: First use of tag: LTI-test-team

    1. The list of all Alt Codes for special characters and symbols. Learn How to use Alt Key Codes? for special characters and symbols.

      Drag-n-drop learning.

    1. Da Vinci Theme for Art

      My own use of "widget" began with notes speculating on the use of one particular object.

    1. Sign up for a user account on hypothes.is, our annotation platform.

      This is a test annotation! How cool is this?

    1. If you are interested in developing an integration

      Evernote's UI/UX is top shelf; slick, elegant; but, is it extensible? Does it bring the X in XMPP?

      For example: An empty button could be added to the pop-up. A module that can be customized to inject approved widgets...such as chat/IM, or just a Home button keyed to the school's website.

      I once bought a game add-on ($2-3) that came with an extensible button by default. That was a million dollar seller, if memory serves. (See mystical cookie).

  9. Jun 2021
    1. precedents and landmark cas

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    1. Christophe Fraser 💙 on Twitter: “Reading Cummings accounts of early creation of Test & Trace, a question I have is when and how it was morphed from aiming to find ~30 contacts per index case, needed to contain spread, into a service that contacts 2-4 contacts per index case, mostly within household.” / Twitter. (n.d.). Retrieved June 28, 2021, from https://twitter.com/ChristoPhraser/status/1408454903249477632

    1. Thus, by adding system tests, we increase the maintenance costs for development and CI environments and introduce potential points of failures or instability: due to the complex setup, flakiness is the most common problem with end-to-end testing. And most of this flakiness comes from communication with a browser.
    1. Global mocks introduce magic and technically can reduce test coverage.
    2. Manual mocks are used to mock modules across the entire Jest environment. This is a very powerful testing tool that helps simplify unit testing by mocking out modules which cannot be easily consumed in our test environment.
    3. Another common gotcha is that the specs end up verifying the mock is working. If you are using mocks, the mock should support the test, but not be the target of the test.
    4. Don’t test the library
    5. Testing the hasMetricTypes computed prop would seem like a given here. But to test if the computed property is returning the length of metricTypes, is testing the Vue library itself. There is no value in this, besides it adding to the test suite.
  10. May 2021
    1. Smashing Through the Language Barrier to Global Market Entry

      This is a commentkueqwgkqewkqgewh

    1. Jonathan Rothberg 🦋. (2021, March 2). Testing works. I test daily. Insist on HOME testing. @michaelmina_lab @JoeBiden Research suggests B.1.526 needs to be closely watched “for its ability to evade both monoclonal antibody and, to a certain extent, the vaccine-induced antibody,” said Fauci [Tweet]. @JMRothberg. https://twitter.com/JMRothberg/status/1366755339912306688

  11. Apr 2021
    1. Ich kann irgendwie keinen Text auswählen aber die PageNotes klappen :D

  12. Mar 2021
    1. Create a note by selecting some text and clicking the button

      example

    1. 命令总结 指令 描述 /start Just a greeting /uptime returns Lupin Uptime /ver returns Lupin running Version /help help command (WIP) /anno URL Import hypothesis annotations from URL /importFC Imports your Flashcards into Lupin /srs import alias of /importFC /srs x starts a round of SRS for x flashcards /getMM pageTitle Generates a dynamic MindMap for pageTitle /pullNow Pulls all pages from your Git for fast access /themes calls the theme changer /encryptAll Encrypts all your pages with AGE keys /decryptAll Decrypts all your pages back to clear text

      tewat

    1. There is obvious connections between the flow paths of a use case and its test cases. Deriving functional test cases from a use case through its scenarios (running instances of a use case) is straightforward.
    2. With content based upon an action or event flow structure, a model of well-written use cases also serves as an excellent groundwork and valuable guidelines for the design of test cases
    1. TRAILBLAZER-STORY will follow as it turned out to be inevitable for setting up application state for tests. Instead of fumbling around with factories and traits in your tests, you “tell a story” about what to create in which order, easily customizable, and all written using activities.
    1. It is absolutely advisable to use factory in combination with let. let(:song) { factory( Song::Create, { title: "Timebomb", band: "Rancid" } ) }
    2. You should always use operations as factories in tests.
    3. There are several helpers to deal with operation tests and operations used as factories.
    4. In production, you will never trigger one specific callback or a particular validation, only. Your application will run all code required to create a Song object, for instance. In Trailblazer, this means running the Song::Create operation, and testing that very operation with all its side-effects.
    5. Unit tests for operations: They test all edge cases in a nice, fast unit test environment without any HTTP involved.
    1. 서울대학교 UX lab에서 하이포테시스를 사용한 첫 사례가 되어보았습니다. 저는 인턴 이윤수입니다.

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    1. Kevin McConway. ‘Media: Worst Ever Week for Test & Trace; They Only Reached 59.9% of Identified Contacts. But the % Reached Went up This Week for Contacts Managed by Local Health Protection Teams AND for Contacts Not Managed by Them (What Used to Be Called “complex” and “Non-Complex” Cases.) How?’ Tweet. @kjm2 (blog), 5 November 2020. https://twitter.com/kjm2/status/1324417367477264386.

    1. Why separate out red tests from green tests? Because my green tests serve a fundamentally different purpose. They are there to act as a living specification, validating that the behaviors work as expected. Regardless of whether they are implemented in a unit testing framework or an acceptance testing framework, they are in essence acceptance tests because they’re based upon validating behaviors or acceptance criteria rather than implementation details.
    2. Conversely, red tests are tests I write after the code is written to lock down some implementation.
    3. Have you ever played the game 20 questions? Most of us have played that game at one point in our lives. One person thinks of something that could be an animal, vegetable, or mineral and then they answer yes/no questions that are asked of them. The point of the game is to ask as few questions as possible in order to accurately guess what the person is thinking.  This is how I think of the unit tests that I write the specified behavior as I’m doing test-first development. I ask what are the fewest tests that I need to write in order to assert the behavior I want to create.
    4. I am a big advocate of having a complete test base and even erring on the side of caution when it comes to quality engineering and software validation but that is not what we’re talking about here. What we’re talking about here are the tests that we write when we’re doing test-first development and I’m proposing that writing those tests from the perspective of specifying the behaviors that we want to create is a highly valuable way of writing tests because it drives us to think at the right level of abstraction for creating behavioral tests and that allow us the freedom to refactor our code without breaking it.
  13. Feb 2021
    1. How do you know if source maps are working correctly? Try adding a syntax error to one of your assets and use the console to debug. Does it show the correct file and source location? Or does it reference the top level application.js file?
    1. Release from cell cycle arrest with Cdk4/6 inhibitors generates highly synchronised cell cycle progression in human cell culture

      [TEST] Reviewer 2: In their manuscript “Release from cell cycle arrest with Cdk4/6 inhibitors generates highly synchronised cell cycle progression in human cell culture” Trotter and Hagan investigated cell synchronization via CDK4 inhibitors as an alternative approach to commonly used thymidine-release regimes. The authors find that CDK4 inhibitor-based synchronization is suitable in 5 out of 25 tested cell lines (they show only data of 4 cell lines!) and provide FACS and Western blot data confirming this notion. Overall, the manuscript has the character of a methods study and does not explore the consequences and potential side-effects of CDK4 inhibitor based synchronization beyond the first two mitoses. Using CDK4 inhibitors to (pre)-synchronize cells in G1 phase is not a novel idea and has been used/published before (e.g. recently by Jackman et al., JCB 2020), however, to my knowledge a methodological study as presented here comparing different cell lines and different CDK4 inhibitors to synchronize cells has not been performed. Precise and as much as possible perturbation-free cell cycle synchronization remains key to cell cycle research, hence I think the presented manuscript has the potential to be a valuable addition to the field.

      Except for the missing information on the number of experimental repeats for most experiments (see below) and a request to also include a tabular overview of all cell lines investigated, I do not have major criticisms precluding publication from my point of view. There are several points, however, the authors should address to make the manuscript stronger and more useful to the community.

      1) Discussion & Introduction

      -In recent years, live imaging studies of asynchronous cells, e.g. by the Meyer, Spencer, Mansfeld, Purvis or Bakal labs, have demonstrated that at least for single cell analyses no synchronization regimes are required anymore because singe cells tracks can be aligned computationally to a common cell cycle starting point after the experiment. Of course, such approaches are not compatible with biochemical experiments, but certainly support diverse functional analyses that previously were only possible by synchronized populations such as monitoring degradation/expression profiles of proteins. Hence, to my opinion at least the introduction would benefit from a short paragraph putting the “old school” bulk synchronization regimes to which also their approach belongs into context with such single cell alternatives.

      -Can the authors provide a reference/results for their statement that RPE-1 cells are “refractory to double thymidine block induction synchronization” and that the levels of synchrony are low after serum starvation (l251ff)?

      -Contact inhibition as an alternative method for synchronization, especially in RPE-1 cells, should be mentioned as well.

      -p21, l516ff and p22, l534ff. Can the authors please indicate the names of the cell lines they refer to and not just the references. Also, does this argumentation then hold true for the altered genotypes we know of the non-responsive cell lines? E.g. MCF10A, which lack p16 and amplify myc signaling? Here, it would make sense to include known restriction-point genotype information in the table of all cell lines tested as suggested below. This would greatly benefit researchers choosing the right synchronization protocol for their individual cell line models.

      -p23, l580ff. The closing statement is weak and does not connect well to the text before. Could be completely omitted.

      -p20, l494. While the authors show based on gH2AX staining that there is not increased DNA damage due to CDK4i synchronization in S phase and mitosis they have no own data showing that chromosome segregation is not affected by CDK4 inhibition. While they reference a personal communication with the Pines lab, they should tone down this claim sufficiently.

      P19, l474. The authors should remove the claim of novelty, because CDK4 inhibitors have been used before to synchronize cells, e.g. by refs 56 and 57 and others, e.g. Spencer et al., 2013 have use MEKK inhibitors, which target CDK4 indirectly via cyclin D.

      P20, l501. This holds also true for contact inhibition!

      2) Figures

      -For data presentations with bar charts showing lower n (<10) it would be useful if the authors over plot the single data points on top of the bar charts to enable the reader estimating the distribution of the data. Also, the precise number of repetitions should be indicated, e.g. Fig 1, 4, 6,7,8, 9. It should be indicated if bars show the mean or median!

      -All Western blot analyses should indicate molecular markers (Figs 3 &5) and in the absence of quantifications, it should be mentioned of how many experiments the data is representative of.

      -Generally, for all experiments/data it should be indicated of how many independent repeats the data is representative/shown. E.g. it is not clear of how many repeats the data without error bars is representative of in basically all figures with such data.

      -Generally, the authors should carefully re-read their figure captions and make sure that the referrals to explanations in other figures are correct and provide sufficient data. As a reader, I find it somewhat cumbersome, if I have read the legends of 3 figures to get all the required information – e.g. legend to figure 8 refers to figure legend 7, which refers to Figure legend …

      -Figure 9, the unit of the concentrations of palbociclib is not given.

      Experiments

      -Western blot analyses in Figures 3 and 5. I am not sure if I understand the data and the corresponding description in the figure legend (l916ff). Eg5 is degraded by the APC/C at the G1 phase (Eguren et al., Cell Rep. 2014) and S10 on histone H3 is phosphorylated at the end of G2 phase and trough-out mitosis. To better define mitosis (and the end of M) the authors should also blot for cyclin B1 and/or cyclin A2. The choice of Eg5 and Wee1 is not ideal as their degradation profile is not as sharp and poorly visible in the data presented. Furthermore, it would benefit their comparison between different cell lines if in both experiments the same markers are analysed. Finally, the pS10 blotting and the FACS profiles in Figure 5 indicate that the cells are still significantly enriched in G2 or M phase as the pS10 peak is still very high by the end of the experiment. This indicates that THP-1 cells have a longer cell cycle than RPE-1 cells or that the release in THP-1 is slower than in RPE-1 cells at a certain cell cycle stage. Can the authors comment on this and better describe their data?

      -The authors should include a table with all 25 cell lines tested indicating, which cell lines are suitable for CDK4 inhibitor based synchronization and which are not. They mention in the text only few non-responsive ones, but this information is important for the reproducibility of their data and will save the time of others applying this approach to non-responsive cells. Also, the identity of 5th cell line that responded well to CDK4i synchronization is not given.

    2. Release from cell cycle arrest with Cdk4/6 inhibitors generates highly synchronised cell cycle progression in human cell culture

      [TEST] Reviewer 1: Release from the cell cycle arrest with Cdk4/6 inhibitors generates highly synchronised cell cycle progression in human cell culture by Trotter and Hagan

      In this very interesting manuscript Trotter and Hagan use Cdk4/6 inhibitors, predominately the drug palbociclib, to arrest hTERT-RPE1 cells close to 100% in G1 phase of the cell cycle. Wash out of the drug then triggers synchronized progression of the cells through the cell cycle. The authors carefully define the conditions that allow high efficient synchronisation with palbociclib and establish that this arrest and release protocol does not cause DNA damage as this is the case for the conventional thymidine block and release scheme. The synchronisation protocol described by Trotter and Hagan will help many researchers to perform highly reproducible and meaningful cell cycle analysis experiments in cell lines that before resisted the conventional protocols. I therefore strongly support publishing of this manuscript in Open Biology. I have only minor points that the authors may want to address before publication.

      Specific points

      1. Line 276: “indicated by black and red line sin the FACS plots”.
      2. Lines 291-295: complicated long sentence. Please simplify.
      3. Line 294; remove “data”.
      4. Line 300: “open black squares”.
      5. Fig. 7d: why do authors use grey and blue colours? I guess that two synchronized cultures were used. The colour code on top should follow the colour code in the bars: light, medium, high. The colour code is not completely clear. I guess EdU means EdU positive (+/- gamma-H2AX) and gamma-H2AX (+/- EdU). Please clarify.
      6. An alternative way of analysing Fig. 7d is counting G2 cells (CENP-F positive) for gamma-H2AX staining with and without synchronization. The synchronized cells would be analysed after 14-16 h. This would address whether the synchronisation scheme leads to cells with DNA damage.
      7. The authors should explain better how the experiment in Fig. 9 was done. I guess that cells were incubated with the given palbociclib concentrations for the stated time. The medium was then changed (no palbociclib + nocodazole) and cells were incubated for 24 h followed by FACS analysis. How do the authors explain the behaviour of A549? Shorter incubation
    1. Cell-density effects are generally thought to be mediatedby external signal molecules that affect gene expressionwhen they reach a certain threshold concentration, aphenomenon called quorum sensing

      Test

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    1. A tramping of sea boots was heard in the entry; the door was flung open, and in rolled a wild set of mariners enough. Enveloped in their shaggy watch coats, and with their heads muffled in woollen comforters, all bedarned and ragged, and their beards stiff with icicles, they seemed an eruption of bears from Labrador. They had just landed from their boat, and this was the first house they entered. No wonder, then, that they made a straight wake for the whale’s mouth—the bar—when the wrinkled little old Jonah, there officiating, soon poured them out brimmers all round. One complained of a bad cold in his head, upon which Jonah mixed him a pitch-like potion of gin and molasses, which he swore was a sovereign cure for all colds and catarrhs whatsoever, never mind of how long standing, or whether caught off the coast of Labrador, or on the weather side of an ice-island.

      Testing the API

  14. Jan 2021