5,929 Matching Annotations
  1. Mar 2021
    1. 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.
  2. Feb 2021
    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

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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

  3. Jan 2021
    1. An important aspect of the environmental management literature considers the strength and nature of the relationship between economic development and environmental degradation, more specifically whether the former can feasibly be achieved without the latter. Some research has supported the existence of an Environmental Kuznets Curve (EKC), according to which environmental degradation increases up to a point as economies grow but then declines with further affluence [1–5]; however, a substantial body of research has cast doubts on whether countries can truly “grow” their way out of environmental problems [6–10]. As a result, the conventional economic growth paradigm is being challenged from multiple sources, with critics arguing that not only are we in danger of exceeding planetary boundaries [11,12], but that unrestrained growth is also a root cause of unsustainability and contributes to political disaffection, social inequality,

      Annotation in pdf

  4. Dec 2020
  5. www.investopedia.com www.investopedia.com
    1. Nothing has any purpose. Life is meaningless. Any purposes you imagine you have are illusions, errors, or lies. This is the stance of nihilism. It appears quite logical. It might seem to follow naturally from some scientific facts: everything is made of subatomic particles; they certainly don’t have purposes; and you can’t get purpose by glomming together a bunch of purposeless bits. It is easy to fall into nihilism in moments of despair; but, fortunately, it is difficult to maintain, and hardly anyone holds it for long. Nevertheless, the seemingly compelling logic of nihilism needs an answer. It turns out that it is quite wrong, as a matter again of science and logic. But because that is not obvious, three other stances try (and fail) to find a middle way between eternalism and nihilism.

      Hypothesis is reporting the HTML of this selection contains a number of newlines that don't appear in the HTML on the page.

  6. Nov 2020
    1. If you get a positive PCR test and you want to be sure that what you’re finding is a true positive, then you have to perform a viral culture. What this means is that you take the sample, add it to respiratory cells in a petri dish, and see if you can get those cells to start producing new virus particles. If they do, then you know you have a true positive result. For this reason, viral culture is considered the “gold standard” method for diagnosis of viral infections. However, this method is rarely used in clinical practice, which means that in reality, a diagnosis is often made based entirely on the PCR test.

      [[Z: A positive PCR should be followed by a viral culture test to see if you're dealing with a live infection]]

      After a positive PCR test, you don't know if the virus is alive or not. To find this out you can add it to respiratory cells (in the case of a respiratory virus) and see if they start producing virus particles).

      [[Z: Viral culture tests are rarely used in clinical practice]]

      Positive diagnoses of COVID-19 are done base on PCR only.

  7. Oct 2020
    1. 可惜的是,Hypothesis 官网的检索功能比较简陋,也没有提供批量导出功能。对此,最简单的解决方案是使用前面提到的 Facet 工具

      Q: Facet工具是做什么的

  8. Sep 2020
    1. Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause familialParkinson’s disease, and sequence variations are associated with the sporadic form of the disease.LRRK2 phosphorylates a subset of RAB proteins implicated in secretory and recycling traffickingpathways, including RAB8A and RAB10. Another RAB protein, RAB29, has been reported to recruitLRRK2 to the Golgi,

      this is a test

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    1. DouglasandMichaelsgoontoexplain,“Thenewmomisminvolvesmorethanjustimpossibleidealsaboutwomen’schildrearing;itredefinesallwomen,firstandforemost,throughtheirrelationshipstochildren.
    2. ErikaHorwitz’sstudy-(2003)on empoweredmotheringrevealsthatthepracticeofoutlawmotherhoodmaybecharacterizedbyseventhemes:Theimportanceofmothersmeetingtheirownneeds;BeingaMotherdoesnotfulfillallofawoman’sneeds;Involvingothersintheirchil­dren’supbringing;Activelyquestioningtheexpectationsthatareplacedonmothersbysociety;
  9. Aug 2020
    1. Vogels, C. B. F., Brackney, D., Wang, J., Kalinich, C. C., Ott, I., Kudo, E., Lu, P., Venkataraman, A., Tokuyama, M., Moore, A. J., Muenker, M. C., Casanovas-Massana, A., Fournier, J., Bermejo, S., Campbell, M., Datta, R., Nelson, A., Team, Y. I. R., Cruz, C. D., … Grubaugh, N. (2020). SalivaDirect: Simple and sensitive molecular diagnostic test for SARS-CoV-2 surveillance. MedRxiv, 2020.08.03.20167791. https://doi.org/10.1101/2020.08.03.20167791

  10. Jul 2020

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    1. How to Conduct an Ohm Meter Test You can use an analog or digital multimeter. Set it to its highest ohm (Ω) setting, at least 1 kΩ (1,000 ohms). At this setting, the meter generates a small current when you connect the meter leads to the capacitor terminals. After connecting the leads, hold them there for several seconds. If you're using an analog meter and the capacitor is good, the meter needle will start at a low reading, and as charge builds up in the capacitor, the needle will climb steadily toward infinity. If the capacitor is bad, the needle won't move at all. If you're using a digital multimeter and the capacitor is good, the number on the digital display will increase steadily until the capacitor discharges, then it will return to 0 and begin climbing again. If the capacitor is bad, you'll get a very low resistance reading – possibly 0 – and it won't change. That means the insulating material inside the capacitor has worn out.
  11. Jun 2020
    1. MIT, a visiting professorship at Harvard Law School, and the boards of the John D. and Catherine T. MacArthur Foundation, the John S. and James L. Knight Foundation, and the

      Testing annotations in Outline

    1. It is not customary in Rails to run the full test suite before pushing changes. The railties test suite in particular takes a long time, and takes an especially long time if the source code is mounted in /vagrant as happens in the recommended workflow with the rails-dev-box.As a compromise, test what your code obviously affects, and if the change is not in railties, run the whole test suite of the affected component. If all tests are passing, that's enough to propose your contribution.
  12. May 2020
    1. Regular Expression Functions There are three regular-expression functions that operate on strings: matches() tests if a regular expression matches a string. replace() uses regular expressions to replace portions of a string. tokenize() returns a sequence of strings formed by breaking a supplied input string at any separator that matches a given regular expression. Example:   

      Test question: how many are there regular-expression functions in XSLT?

    2. What’s the difference between xsl:value-of, xsl:copy-of, and xsl:sequence? xsl:value-of always creates a text node. xsl:copy-of always creates a copy. xsl:sequence returns the nodes selected, subject possibly to atomization. Sequences can be extended with xsl:sequence.

      What’s the difference between xsl:value-of, xsl:copy-of, and xsl:sequence?

    3. Types XSLT 2.0 allows you to declare: The type of variables. The return type of templates. The type of sequences (constructed with xsl:sequence) The return type of (user-declared) functions. Both the type and required type of parameters.

      What are the types that one can declare in XSLT 2?

  13. Apr 2020
    1. What is a Function Expression?A JavaScript function can also be defined using an expression.A function expression can be stored in a variable:var x = function (a, b) {return a * b};After a function expression has been stored in a variable, the variable can be used as a function. Functions stored in variables do not need function names. They are always invoked (called) using the variable name.

      Test question: What is function expression?

  14. javascript.info javascript.info
  15. developer.mozilla.org developer.mozilla.org
  16. developer.mozilla.org developer.mozilla.org
    1. Click test (cps tester) is a feature providing clicks per second Tool. Our Tool will fulfill the needs of kohi click test tool in the minecraft server for gamers. Now you can take your Clicking speed test. In this Click test 5 scounds u can click as much as u can and your score will appear in Cps counter. click per second is important for your minecraft skills. we are providing clicks per second tool to check your skills.Long time ago Kohi server removed this From their officle site so we decided to put up cps test (clicks per second) tool for you.

  17. Mar 2020
  18. Dec 2019
    1. No backup regimen would be complete without testing. You should regularly test recovery of random files or entire directory structures to ensure not only that the backups are working, but that the data in the backups can be recovered for use after a disaster. I have seen too many instances where a backup could not be restored for one reason or another and valuable data was lost because the lack of testing prevented discovery of the problem.
  19. Nov 2019
    1. pastrami nostrud culpa tri-tip non id meatloaf consectetur shankle et hamburger. In do aliqua flank, ut kielbasa labore tenderloin nisi swine pastrami. Short ribs elit drumstick nisi ham hock eu. Consequat

      This is a test annotation.

  20. Oct 2019
    1. fter the forage is dry, weigh the bag and forage in grams and record the weight. Empty the forage and weigh the bag to get the net forage weight in grams. Postage scales may be adequate for weighing the forage, but the weights must be converted from ounces to grams (1 ounce = 28.3 g). Forage production in lbs./acre is determined by mul-tiplying the net forage weight (from the 10 hoops) by 10. If the forage from 10 hoop placements (10 x .96 sq. ft. = 9.6 sq. ft.) was 50 grams, total forage production would be 500 lbs. / acre.

      Hello!