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  1. Dec 2022
  2. betasite.razorpay.com betasite.razorpay.com
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  3. www.scienceintheclassroom.org www.scienceintheclassroom.org
    Apoptosis propagates through the cytoplasm as trigger waves
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    1. willagbe 01 Dec 2022
      glutathione S-transferase

      A protein that is popularly used as a tag for the purification of recombinant proteins. It can be fused to either ends of the desired protein, usually the end that does not affect the function of the target protein.

      A recombinant protein is produced by cloning a gene into a system that allows the expression of that gene and the translation of its gene product.

      Glossary
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  4. Nov 2022
  5. hypothes.is hypothes.is
    Hypothesis
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  6. www.biorxiv.org www.biorxiv.org
    Activation-pathway transitions in human voltage-gated proton channels revealed by a non-canonical fluorescent amino acid
    1
    1. biophysics_sciencecolab 29 Nov 2022

      Authors' response (28 November 2022)

      GENERAL ASSESSMENT

      This interesting preprint by Suárez-Delgado et al. explores the mechanism by which activation of the Hv1 voltage-activated proton channel is dependent upon both the voltage and pH difference across the membrane. The authors are the first to incorporate the fluorescent unnatural amino acid, Anap, into the extracellular regions of the S4 helix of human Hv1 to monitor transitions of S4 upon changes in voltage or pH. The authors first checked that Anap is pH insensitive for practical use in Hv1, where changes in local pH are known to occur when the voltage sensor activates and the proton pore opens. Anap was incorporated at positions throughout the S3-S4 linker and the extracellular end of S4 (up to the 202nd residue) of hHv1 and some positions showed clear voltage-dependent changes in fluorescence intensity. The authors also obtained fluorescence spectra at different voltages and observed no spectral shifts, raising the possibility that voltage dependent changes in fluorescence intensity could primarily be due to fluorescence quenching. Upon mutation of F150, the Anap signal at the resting membrane voltage increased, suggesting dequenching upon removal of F150. The authors also discovered that the kinetics of Anap fluorescence upon membrane repolarization have two phases (rapid and slow) under certain pH conditions and that there is a pH-dependent negative shift of the conductance-voltage (G-V) relation compared with the fluorescence-voltage (F-V) relation in some mutants. The biphasic kinetics of the fluorescence decay upon repolarization were explained by modelling a slower transition of return from intermediate resting state to a resting state. The pH-dependent shift of the G-V relation from the F-V relation provides insight into mechanisms of ΔpH-dependent gating of Hv1, a longstanding enigma. Overall, the approaches are rigorous, the figures show important results, and this work paves the way for the use of Anap fluorescence to study Hv1 gating and modulation.

      We thank the reviewers for the careful reading and assessment of our manuscript and for the constructive criticism. We have tried to respond to all the essential revisions, both by rewriting sections and performing some experiments or new analysis. Below we respond one by one to all the points raised. Please also note that we have added an author to the manuscript, who has carried out new experiments included in this revised version of the preprint.

      RECOMMENDATIONS

      Revisions essential for endorsement:

      1) In its current form, the narrative of the preprint has two threads. One on the mechanisms of Anap fluorescence changes (mainly quenching) and another on a previously unappreciated transition of the voltage sensor, as revealed by Anap. Our impression is that the preprint suffers somewhat from this split focus, which could be resolved by explaining why Anap was used to explore voltage sensor activation in Hv1 in the introduction. Perhaps the authors could also explain the advantage of smaller sized fluorophores compared to other maleimide-based fluorophores earlier in the introduction, or the utility of being able to insert Anap into transmembrane segments. The authors should more clearly point out how they exploited the advantages of Anap as a tool in this study. It would furthermore be helpful to discuss previous studies using nongenetic tools for VCF and spell out how they have delineated key aspects of Hv1, which would help to emphasize how several positions studied here (for example, 201 and 202) could not be labelled with cysteine-based fluorophores.

      We think that this is a very useful suggestion and we have expanded the introduction to more pointedly indicate the contributions of previous voltage-clamp fluorometry experiments in Hv1 channels and to clearly explain why we chose to pursue the use of a genetically-encoded small fluorophore such as Anap.

      2) We think the authors should be cautious about understanding the physicochemical nature of Anap using prodan as a model. It would be helpful to discuss the possibility that undetected spectral shifts due to a nonquenching mechanism could be overlooked, even though major signal changes can be explained by fluorescence quenching in their data. Regarding the mechanisms of remaining voltage-dependent fluorescence changes of F150A-A197Anap, it would be helpful for the authors to suggest possible ideas about which residues might account for remaining signals.

      The beautiful spectral data for Anap is impressive. However, the physicochemical basis of the fluorescence change of Anap cannot be understood by simple extension of findings for prodan, which shows structural similarity to Anap. Our understanding is that changes in Anap fluorescence can only reveal a change in the structural relationship between Anap and one of its neighbors because the physicochemical basis of Anap fluorescence is complicated. For example, fluorescence could also be affected by the electrostatic environment, stretch of peptide bond, etc. Previous studies, including those of TRP channels, showed that the kind of environmental changes that Anap faces in ion channels do not necessarily induce large spectral shifts, unlike in cell-free spectral analyses using distinct solvents. Further, only minor shifts in spectra occur upon local structural change, as seen in previous work including Xu et al. Nat. Commun. 2020 11:3790. Such minor shifts could be perhaps overlooked even when Anap is incorporated into S4 and exposed to environmental change. Therefore, it is not easy to decode the physicochemical basis of Anap fluorescence changes. F150A-A197Anap has increased fluorescence and no change in spectral pattern, leading the authors to conclude that F150 quenches Anap fluorescence of A197 position. However, a significant amount of fluorescence change still occurs upon changes in membrane potential after F150 is changed to alanine (Figure 4). It is very likely that quenching is not the only mechanism underlying the observed voltage induced change of Anap fluorescence of Hv1. The authors suggest that remaining voltage-dependent fluorescence change of F150A-A197Anap could be due to interaction with other aromatic residues, but this has not been tested.

      Thank you for pointing out our oversimplified discussion of the mechanisms of Anap fluorescence changes in Hv1 channels. We have taken into account your comments and present a more nuanced and toned-down discussion of the possible mechanisms at play in our experimental system.

      3) The current version of the preprint is missing important control experiments, ideally performed using western blots to measure protein expression or, if that is not possible, proton current and fluorescence measurements, to demonstrate that protein expression or functional channels are not seen for all mutants in the absence of ANAP (but in the presence of the tRNA and Rs construct). A similar control for imaging would be to use ANAP alone without encoding.

      We thank the reviewers for this recommendation. We show that the number of cells showing mCherry fluorescence is greatly diminished in the absence of L-Anap, but in the presence of the tRNA and synthetase. As suggested, we have included results of control experiments in which we attempted to record currents from cells expressing the constructs: F150A-A197tag, Q191tag, A197tag and L201tag co-expressed with the tRNA and synthetase-coding plasmid (pANAP) and in the absence of L-Anap. We struggled to find red fluorescing cells and recorded currents from a relatively small number of these cells, most of which was leak current. We now include these data in Figure 1-Supplement 1B. These control experiments show that there is very little leakage of expression of channels that did not incorporate Anap.

      4) Aromatics in the S4 segment were ruled out as potential quenchers on the assumption that they would move together with Anap during gating. It should be noted, however, that Hv1 is a dimer and therefore a fluorophore attached to S4 in one subunit could be quenched by S4 aromatics in the neighboring subunit if were close to the dimer interface. In Fujiwara et al. J. Gen. Physiol. 2014 143:377-386, for example, W207 does not appear very far from labeled positions in the adjacent S4. This possibility should be mentioned in the discussion.

      We appreciate the reviewers' concern regarding the role of other aromatic residues near Anap incorporation sites, especially the ones close to the subunit interface given that Hv1 is a dimer. We now mention the possibility that other residues could be quenching groups, especially given the fact that some quenching remains in the double mutant F150A-A197Anap (line 272 in results and line 432 in discussion). We have also included a new analysis of the ratio of Anap/mCherry fluorescence (at resting membrane potential) for all insertion sites. This shows a decreased ratio as Anap gets inserted in residues closer to the c-terminus of S4, which is evidence of a quenching group located near the center of the transmembrane domains (Figure 4-Supplement 1).

      5) It is not clear whether the Anap spectra purely represent Hv1 incorporated into the plasma membrane or perhaps include signals from the cytoplasm or channels in internal membranes (whether assembled or incompletely assembled). It would be helpful to provide a more complete presentation of the data obtained and to provide more information in the Methods Section. In the Methods section, it is stated "The spectra of both fluorophores (Anap and mCherry) were recorded by measuring line scans of the spectral image of the cell membrane, and the background fluorescence from a region of the image without cells was subtracted". How are signals from cell membranes specified in this method being discriminated from those associated with the cytoplasm and intracellular membranes? If spectral data include signals from free Anap in the cytoplasm or Hv1 in intracellular membranes, spectral shifts upon membrane potential changes will be difficult to detect, even when Anap is incorporated into Hv1 and senses environmental change by voltage-induced conformational change. In Figure 3E, wavelength spectra were shown as standardized signals for different voltages. Amplitude change would be demonstrated (spectrum at different voltages without standardization should be shown).

      We appreciate the concern related to the origin of the fluorescence signals and we have improved both the presentation and the associated figures. Since this is also a concern for the experiments that determined the pH-dependence of Anap incorporated at position Q191, we have included a figure supplement 1 to Figure 2 in which we explain how the membrane was visualized. We use mCherry fluoresce as an indication of plasma membrane-associated channels, since its red fluorescence is easier to detect in the membrane than Anap fluorescence (even though cytoplasm dialysis in whole-cell should diminish the amount of free Anap, it is difficult to distinguish Anap fluorescence in the membrane by itself). Once the membrane associated mCherry fluorescence is detected, the measurement of the spectrum from a very small membrane area is insured because the spectrograph slit delimits light collection to a very small vertical area and the horizontal line scan further limits light measurement. These procedures are now made explicit in methods section and supplementary figure mentioned before. Moreover, we explain that they were also followed in experiments where the cell was under voltage-clamp. The spectral data in Figure 3E is now presented without normalization to show the voltage-dependent change in amplitude without changes in peak emission wavelength.

      In Figure 4, spectra were compared between A197Anap and F150A-A197Anap, showing increases of fluorescence in F150A-A197Anap. Was this signal measured at resting membrane potential? How does the spectrum change when the membrane potential is changed?

      As in the experiments of figure 1E, the spectra were obtained in non-patched cells. Thus, the signal was measured at the HEK cell resting potential (~ -30 mV) and a ΔpH ≈ 0.2. We have now incorporated that information in the methods section and the figure description. On the other hand, we did not perform experiments measuring the double mutant spectra at different voltage steps, so we cannot respond to the second question.

      Rationales for the confirmation of signals originating from the cell surface for Hv1 Anap might include the observations that: a) some mutants showed slightly different spectral patterns (in particular, Q191Anap showed a small hump at longer wavelengths, which is proposed to represent FRET between mCherry and Anap) and b) signal intensity was voltage dependent (if signals originate from endomembranes, they should not be voltage dependent). Mentioning these two points earlier in the text might help to alleviate concerns about the location of the protein that contributes to the measured signals.

      These are great suggestions and we have incorporated them to the text (lines 156, 190 and 216 Results section), along with a better explanation of procedures followed to measure mostly membrane-associated fluorescence (see new Figure 2-Supplement 1).

      6) In Fig 5, the fluorescence kinetics do not really match the current activation kinetics for panels A, B, and C. Is there an explanation for this mismatch? It would be helpful to have the fitted data in the figure. A more thorough comparison of the kinetics of currents and fluorescence would be helpful throughout the study.

      We believe that the kinetics of fluorescence and current does not match because the current activation rate is overestimated due to a small amount of proton depletion present in recordings from large currents. This is an unavoidable problem in proton current recordings, even with the high concentration of proton buffer used in our experiments and the long time-intervals between each voltage pulse. For this reason, we did not undertake a systematic exploration of kinetics. Nonetheless, the current and fluorescence rates are very close and have the same voltage dependence, indicating a close correlation between voltage-sensor movement and current activation. We now explain this limitation in the manuscript text (line 223 and 327, results section).

      7) Which construct of hHv1 was used to obtain the data in Figure 6? Unless we missed it, this information is not provided in the text or figure legend. Is it for L201Anap? This figure also shows an intriguing finding that the G-V relationship is negatively shifted from the F-V relationship at pHo7-pHi7 but not at pHo5.5-pHi5.5. A shifted G-V relation with the same ΔpH contrasts with what has been reported in other papers. However, the authors did not really discuss this surprising finding in the light of previous references. Could the shift of the G-V relation between two pH conditions with the same ΔpH be due to any position-specific effect of Anap? If Figure 6 represents L201Anap mutant, the presence of Anap at L201 probably makes such shift of G-V curve in Figure 6C? The authors should openly discuss this finding in relation to what has been reported in the literature.

      Yes, construct L201Anap was used in Figure 6. This is stated now in the figure legend and in the corresponding main text. We agree that the leftward shift of the GV with respect to the FV in pHi7-pHo7 is an intriguing finding, suggesting that coupling between S4 movement and proton permeation can be regulated by the absolute value of the pH. We discuss this in the results section. The DeCoursey group has shown evidence in W207 mutants of hHv1 that the absolute value of pH can modulate the voltage dependence of the conductance. Although we had mentioned these results, we now mention them more prominently and also discuss the possibility that this might be a unique feature of introducing Anap at L201.

      8) The authors suggest that the small hump near 600 nm in Figure 1E represents FRET between Anap and mCherry. It is surprising that FRET can take place across the membrane. Can the authors point to another case of FRET taking place across a cell membrane? One possibility might be that misfolded proteins place mCherry and Anap close to each other. It is also curious that only A191Anap did not show such a FRET-like signal. Also, if there is FRET, why wouldn't this also contribute to the voltage-dependent changes in fluorescence?

      We thank the reviewers for bringing up this point. Based on published data, we assumed that mCherry could not be excited by 405 nm radiation, thus our conclusion that the observed emission near 604 nm is FRET between Anap and mCherry. We have now measured the excitation of the Hv1-mCherry construct and observe that the 405 nm laser is capable of exciting mCherry and produced ~2 % emission (as compared to 514 nm excitation), which is almost the same as that observed for the Hv1Anap-mCherry channels. We now conclude that the second hump in the emission spectrum near 600 nm is due to direct excitation of mCherry.

      On the other hand, FRET across the membrane has been demonstrated for the membrane-bound hydrophobic anion dipicrylamine and membrane-anchored GFP (Chanda, et al. A hybrid approach to measuring electrical activity in genetically specified neurons. Nature neuroscience, 2005, vol. 8, no 11, p. 1619-1626.) and dipicrylamine and GFP in the c-terminus of CNG channels (Taraska & Zagotta, Structural dynamics in the gating ring of cyclic nucleotide–gated ion channels. Nature structural & molecular biology, 2007, vol. 14, no 9, p. 854). Finally, single-molecule FRET between dyes placed extracellularly and intracellularly in Hv1 channels has been demonstrated (Han et al. eLife 2022;11:e73093. DOI: https:// doi. org/ 10. 7554/ eLife. 73093).

      A191Anap shows the hump at ~600 nm, but we think it's less evident because Anap at 191 is less quenched (see Figure 4-Supplement 1 and answer to point 4 above).

      9) F150A-A197Anap shows a leftward shift of the F-V relation compared with the G-V relation only when ΔpH=1. Another unusual finding with F150A-A197Anap is the very small shift of the G-V relation between ΔpH=0 and ΔpH=1, when other reports in the literature suggest it should be 40 mV or more. Are these peculiar properties simply due to the absence of Phe at position 150, which might play a critical role in gating as one of the hydrophobic plugs of Hv1? To address this possibility, it would be ideal to compare different ΔpH values with and without F150 when Anap is incorporated at a different position (such as L201Anap). Regardless, it would be helpful to discuss this point.

      We now discuss these changes in the discussion (lines 440-446).

      10) In Figure 1E, I202Anap exhibits a blue shift in its spectrum suggesting the environment of Anap on I202 is more hydrophobic than the other sites. We presume these spectra were obtained at a negative membrane voltage, but the text or legend should clearly state how these were obtained. The authors should also explain whether the whole cell or edge was imaged. If these are at negative membrane voltages, might the Anap spectrum shift to higher wavelengths (i.e. more hydrophilic) when the membrane is depolarized? Did the authors find any spectral shift for I202Anap when doing a similar test as depicted in Figure 3E?

      Yes, the spectrum of I202Anap was obtained at the resting potential (~ -30 mV), as were all spectra in Figure 1E. We now indicate this clearly in the methods section and in the figure legend. Fluorescence was measured from the membrane region as indicated by mCherry fluorescence and as illustrated in Figure 2-Supplement 1. We did not explore this mutant further and we cannot answer the question of whether a depolarizing potential might produce a red shift of the spectrum.

      11) In Figure 3E, spectra are shown as normalized signals for different voltages, but an amplitude change should also be demonstrated by providing raw spectra at different voltages.

      We have changed figure 3E to show non-normalized data that now show the increase in fluorescence intensity and no wavelength shift in the fluorescence spectrum of Anap (see also response to point 5).

      12) In Figure 4, spectra are compared between A197Anap and F150A-A197Anap, showing increase of fluorescence in F150A-A197Anap. Were these obtained at a negative membrane voltage? How do these spectra change when membrane potential is changed?

      See response to point 4 of "Revisions essential for endorsement" section.

      Additional suggestions for the authors to consider:

      1) The authors propose that Anap fluorescence tracks an S4 movement involved in the opening of the channel. They also argue that the existence of more than one open state could explain why the increase in florescence upon depolarization lags the proton current in most cases. While they convincingly show that Anap is not pH sensitive per se, when incorporated into the protein, the fluorescence efficiency of the fluorophore could still be affected by protonation of channel residues in the immediate environment when the channel opens, even after S4 has completed its movement. To address this alternative explanation, the authors could use Hv1 mutants with strongly reduced proton conductance. Channels bearing mutations corresponding to N214R or D112N were used successfully to isolate Hv1 gating currents from the much larger proton currents (De La Rosa & Ramsey, Biophys. J. 2018 114:2844-2854; Carmona et al. PNAS 2018 115:9240-9245; Carmona et al. PNAS 2021 118: e2025556118). Perhaps, they could be used with patch clamp fluorometry as well?

      This is an interesting suggestion that could be explored in a follow up study.

      2) The data showing that Hv1-197Anap is quenched by Phe at position 150 are very nice. Yet, it would be useful to show that the quenching is specific to F150 using a negative control. F149, for instance, is just next to F150 but points in a different direction, so its mutation to alanine should not affect Hv1-197Anap fluorescence.

      This is an interesting suggestion, but, as suggested by reviewers, we think there is a possibility that other aromatic residues could contribute to quenching. Given the absence of a reliable structure for Hv1, prediction of the relative positions of any resides is very difficult and thus we did not attempt the suggested experiment.

      3) A major finding of this work is the identification of a slow kinetic component that is highly sensitive to ΔpH. Earlier studies found that the ability of Hv1 to sense ΔpH is altered by some channel modifications, e.g., in the loop between TMH2 and TMH3 (Cherny et al. J. Gen. Physiol. 2018 150:851-862). Did the authors check whether any of these modifications alter the transition responsible for the slow kinetic component? For instance, a suppression of the transition resulting from a H168X mutation would help tighten the link to ΔpH sensing.

      We did not carry out any of these experiments.

      4) We understand that it is difficult to tightly control intracellular and extracellular pH when Hv1 is heterologously expressed in mammalian cells. The G-V relation is not always reliable because accumulation of protons or depletion of protons upon Hv channel activity will alter gating, as the authors have previously published (De La Rosa et al., J. Gen. Physiol. 2016 147:127-136). Could the kinetic analysis of Anap fluorescence be affected by similar alterations to proton concentration in the vicinity of Hv1? It would be helpful for the authors to comment on this specifically.

      Thanks for this suggestion. Yes, we think that the kinetics, specially of ionic currents can be affected by even small changes in the pH gradient, for this reason we did not attempt a systematic kinetic analysis. We mention this in the text where we compare the voltage dependence of current and fluorescence activation for construct A197Anap (line 223).

      5) Quenching of Anap by Phe could be verified in cell free conditions using a spectrophotometer with different concentrations of Phe, or citing the literature if it has already been reported.

      We attempted this experiment but were unsuccessful in observing Anap quenching by phenylalanine at the concentrations of phenylalanine that can be attained in aqueous solution. We suspect that Phe quenching of Anap could happen by electron transfer or ground-state complex formation, in which case near proximity is necessary and higher concentrations of Phe would be required to detect quenching in solution. However, we measured the absorbance of Anap in the absence and presence of phenylalanine (Phe) (and tyrosine (Tyr)) at the concentrations that can be achieved in aqueous solution (8 mM and 1mM, respectively). Absorbance measurements can detect ground-state complex formation even at relatively low concentrations (J.R. Lakowicz, 1999, Principles of Fluorescence Spectroscopy). We observed that the absorbance of Anap is modified by the presence of Tyr or Phe, indicating that these amino acids indeed interact with Anap, possibly through ground-state complex formation. We include this data for the reviewers to inspect.

      6) The authors did not cite any example of Anap incorporation into S4 helices, but there are several recent papers where Anap was utilized to probe motion of S4 in other channels. Examples include Dai et al., Nat. Commun. 2021 12:2802 and Mizutani et al. PNAS 2022 119:e2200364119.

      Thanks for this observation, we have included these important results in the discussion.

      7) In the Anap-free negative control (with only A197TAG plasmid transfection), the mCherry signal seems positive (Supplementary Figure 1, left row, second from the top). Is this due to unexpected skipping of the TAG codon to make mCherry-containing partial polypeptides? It would seem like an explanation is needed.

      Thanks for bringing this up. We do not know the exact origin of these leak expression of red fluorescence. We think that, as suggested, there is a possibility that skipping of the Amber codon can lead to a methionine at the end of S4 acting as a second translation initiation site, giving rise to truncated channels that would express mCherry but not currents. This is consistent with the fact that we cannot detect currents in the absence of Anap but we see a small number of red cells.

      8) The data of Figure 3E are shown as data with different membrane voltages. But there is no information about membrane voltage for Fig. 1E and Fig. 2A and Fig. 4B. Are these from unpatched cells? Please clarify.

      See response to point 4 of "Revisions essential for endorsement" section.

      9) G-V relations are shown for F150A-A197Anap, but current traces of F150A-A197Anap are missing.

      We have modified the figure to include current and fluorescence traces.

      10) On Page 11, Line 303 says "experimental F-V relationship is positively shifted by 10 mV with respect to the G-V curve". But looking at the data Fig5D, the shift at ΔpH=2 seems the opposite. Perhaps "positively" should be "negatively" in this sentence?

      Thanks for pointing out this mistake. We have found that this misunderstanding was provoked because of a mistake with the image labeling of F-V and G-V curves for the ΔpH=2 data, we have now corrected the figure. The shift of F-V is indeed positive to G-V as stated before.

      (This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

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    biorxiv.org/content/10.1101/2022.06.30.498345v1
  7. brainsteam.co.uk brainsteam.co.uk
    A Week with Hypothes.is
    1
    1. chrisaldrich 27 Nov 2022

      https://brainsteam.co.uk/2022/11/26/one-week-with-hypothesis/

      I too read a lot of niche papers and feel the emptiness, but because I'm most often writing for myself anyway, its alright. There are times, however, when I see a growing community of people who've left their associative trails behind before I've found a particular page.

      I've used the phrase "digital exhaust" before, but I like the more positive framing of "learning exhaust".

      If you've not found it yet, my own experimentations with the platform can largely be found here: https://boffosocko.com/tag/hypothes.is/

      James Ravenscroft Hypothes.is read digital exhaust learning exhaust learning in public reply
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  8. www.thaipbsworld.com www.thaipbsworld.com
    Beauty queen: Your biggest mission might be the simplest one
    1
    1. TappyToppy 25 Nov 2022
      She competed in the beauty contest with her campaign tag line #realsizebeauty.

      She wants to show the world that no matter how your body is, your beauty doesn't depend on it. She created the trend called #realsizebeauty to let people recognize and be confidence to yourself.

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  9. Local file Local file
    Direct phosphorylation of HY5 by SPA kinases to regulate photomorphogenesis in Arabidopsis
    5
    1. KateBethanyM 24 Nov 2022
      trep-tagge

      The Strep-tag system is a method which allows the purification and detection of proteins by affinity chromatography. The Strep-tag II is a synthetic peptide consisting of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin, a specifically engineered streptavidin, and can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. Because the Strep-tag elutes under gentle, physiological conditions, it is especially suited for generation of functional proteins.

    2. KateBethanyM 21 Nov 2022
      nonphosphorylated forms of HY5 (GST-HY5-S36A) have higheraffinity to MBP-COP1. GST-HY5 and all other phosphorylation mutant proteins were pulled down by MBP-COP1 using maltose agarose beads.

      So this blot has affinity for the MBP tag on COP1- it will therefore be attached at all time. The HY5 and HY5 mutants are being washed over this background (each row has 1x HY5 added). The MBP-only column is a negative control to show that the HY5 do not interact with the MBP tag on its own.

    4. KateBethanyM 20 Nov 2022
      (b)

      TAP-SPA1--- is this a control? Paik et al. 2019 paper uses both-- need to check if used for different things? Is this to show that the LUC tag is not having an effect in the complementation mutants? By showing that the phenotype is unaffected by the addition of the TAP-SPA1? BUT shouldn't they have done a LUC-SPA1 only expressing plant (with no spaQ mutant background) as well, to show this? That TAP-SPA1 and LUC-SPA1 produce the same phenotype?

    5. KateBethanyM 20 Nov 2022
      20 lM Phos-tag (Fig. S2C). However, in spaQ mutant, nomobility shift was observed under these conditions and HY5-GFP showed a faster migrating band than that in WT (Fig. 2c),suggesting a complete absence of phosphorylation of HY5in vivo.

      So the Phospho-tagged SDS-PAGE gel will enhance the difference in mobility of phosphorylated---- or not. The lack of band shift is shown between the B and CIP conditions in fig.2c. This is the same result as shown for the HY5-S26A, so the phosphorylation is at this residue by the SPA kinase

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  10. www.goutpal.com www.goutpal.com
    Gout Skin Rash and Itching Forum Archives - Your Gout Friend
    1
    1. KeithTaylor 23 Nov 2022
      Gout Skin Rash and Itching Forum

      See Is Gout Itchy and associated new pages.

      GoutPal Links Documentation

      Notes like this are part of my GoutPal Links service. Which is currently in the pre-launch phase. I will add documentation for this feature as I answer requests from readers. So if you need help using this feature, the best way is to subscribe to my free GoutPal Links Newsletter. Where you can email and message me directly. Or you can use the feedback options near the end of every page.

      Gout Change
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  11. www.biorxiv.org www.biorxiv.org
    New submission 23/11/2022, 10:55:23
    1
    1. Public_Reviews 23 Nov 2022

      Joint Public Review:

      In this work Malis et al introduce a novel spin-labeling MRI sequence to measure cerebrospinal fluid (CSF) outflow. The glymphatic system is of growing interest in a range of diseases, but few studies have been conducted in humans due to the requirement for and invasiveness of contrast injections. By labeling one hemisphere of the brain the authors attempt to assess outflow through the superior sagittal sinus (SSS), one of the major drainage pathways for CSF, signal changes across time were assessed to extract commonly used metrics. Additionally, correlations with age are explored in their cohort of healthy volunteers. The authors report the movement of labeled CSF from the subarachnoid space to the dura mater, parasagittal dura, and ultimately SSS, evidence of leakage from the subarachnoid space to the SSS, and decreases in CSF outflow metrics with older age.

      1. I don't think that the description of Parasagittal dura in figure 1 is correct. There is no anatomical structure at the top of SSS that is known as PSD. The location of the lymphatic structures is also incorrect. Please review "Anatomic details of intradural channels in the parasagittal dura: a possible pathway for flow of cerebrospinal fluid" Neurosurgery 1996 Fox at al. There is usually no obvious tissue between the upper wall of the SSS and the calvarium, which can also be seen in the authors' fig 2A and 2B. All of the tissues located lateral to the SSS are known as PSD. Also, the SSS wall is not as thick as the authors stated and is known as PSD in this region. For this reason, the authors need to revise Fig 1 and it should be changed to PSD in the areas referred to as the SSS wall in the article.

      2. The authors described tagged CSF in two pathways: from dura mater to PSD and SAS into the SSS and directly from SAS to SSS. Flow from dura mater to PSD and SAS in the main and supplement cannot be seen. Only a flow from PSD to SSS can be seen. Also, regular dura cannot carry flow-collagen-rich fibrous tissue, except parasagittal dura. There is no flow from dura to the CSF in the figures.

      3. The authors have conducted many tests to prevent venous contamination. However, measurements were made based on SSS flow rates in all tests. Small parenchymal venous structures, and small cortical-SAS veins might be tagged due to different flow patterns and T2- Relaxation times.

      4. The rate of CSF formation in humans is 0.3 - 0.4 ml min-1. ( Brinker et al 2014. Fluids Barriers CNS). We can assume that the absorption rate is also similar to the CSF formation for the entire system brain and Spine. Therefore, the absorption rate of this very small amount of CSF by SSS is very low in seconds. It is hard to detect by MR and especially CSF flow from the PSD to SSS. The authors concluded that using this technique the rate averaged less than a couple of seconds, rather than on the order of hours or days as previously reported with the use of intrathecal administration of GBCA (Ringstad et al., 2020).

      5. Overall, I think that the CSF flow from the PSD to the CNS described by the authors - the CSF flow, might be the venous flow that drains into the SSS slowly, predominantly in the rich venous channels, venous lacunae, and previously described channels in the PSD. Additional explanations are needed.

      6. The study is generally well described and to the best of my knowledge an innovative approach. The findings are broadly consistent with what might be expected from the literature and the authors make a good argument in support of their findings. However, the lack of validation is a major limitation of the presented work. In introducing a novel technique a comparison with an existing approach, such as Gd enhanced contrast techniques, or phase contrast would have been expected. Several considerations could have been mentioned/addressed in more detail e.g. what effect labeling efficiency, tortuosity of vessels, lack of gating, the effectiveness of the intensity thresholding to remove the signal from blood, etc may have on the quantification, etc. Without a more thorough validation, it is difficult to evaluate the findings. While scans were conducted on two volunteers to assess reproducibility this is a very small sample and it is notable that scans were conducted consecutively, which might be expected to reduce variance relative to scans further apart e.g. on different dates, scanned by a different operator and no information is provided on how the two scans were positioned (i.e. separately vs copied from the first to the second scan), some metrics showed large percentage differences, which were more pronounced in one subject than the other. Without further data, it is difficult to interpret the reproducibility results. No assessment of the effect of physiological parameters e.g. breathing, cardiac pulsations, or factors affecting glymphatic clearance e.g. amount of sleep the evening before was given.

      7. Given these limitations it is hard to adequately assess the likely impact or utility. In recent years several groups have published work e.g. doi.org/10.1038/s41467-020-16002-4 , doi.org/10.1016/j.neuroimage.2021.118755 assessing the blood-CSF barrier. However, previous work has generally focused on larger structures, and by labeling in the oblique-sagittal plane it is unclear how drainage and blood flow rates may affect the presented values here.

      8. Some validation data would greatly increase the value of the reported work. I would therefore encourage the authors to consider acquiring some additional datasets to compare measures of CSF draining against another method e.g. 2-D or 4-D phase contrast, or Gd-based contrast-enhanced techniques. Some additional points to consider are noted below.

      8. Abstract

      CSF outflow may also be imaged with phase contrast MRI (albeit in a limited way).<br /> Demographics would fit better in Results, breakdown could be given for the young and old groups i.e. n, ages, sex.<br /> Conclusion - unless further validation can be provided I think some of the claims should be toned down.

      9. Introduction

      The authors emphasise the role of Nedergaard, however, there was some relevant earlier work (e.g. Rennels et al, PMID: 2396537).

      10. Methods

      It would be more conventional to summarise the volunteer characteristics in the Results.<br /> Given the age difference between the two groups, and the fact that for conventional ASL we know of differences in labelling efficiency and the need for a different post-labelling duration in more elderly patients how did the authors account for this?<br /> More broadly what would the effect of differences in labeling efficiency be, given the labeling plane is unlikely to be perpendicular to the draining vessels?<br /> While the authors mention circadian effects there is no mention of controlling for other factors before the scan e.g. caffeine consumption, smoking, etc.<br /> Various mechanisms have been hypothesised to drive glymphatic pulsations. Assessing how physiological signals correlated with the flow may have been a useful proof of concept. Why was it not considered necessary to use a gated acquisition? Did the authors consider the potential impact of respiratory and cardiac pulsations on their measurements?<br /> ROI segmentation - manually selected by two raters, was this done independently and blinded? How were consensus ROIs agreed?<br /> Intensity values outwith MEAN +/- 2 SD were excluded from further analyses. This is justified as removing pulsatile blood. However, was this done independently for tag-on and tag-off? Does this mean slight differences were present in the number of voxels between the two?<br /> The starting points and parameter ranges are given in Eq'n 3, how were the ranges defined? Was there a reason for constraining the fit to positive values only, is there a risk of bias from this?<br /> While the main results appear to have a reasonable sample size n=2 for the reproducibility analysis is very limited. Additional datasets would be useful in properly interpreting the results.

      11. Results<br /> While the authors have taken some measures to reduce potential contamination from blood I would be concerned about the risk of surface vessels affecting the signal, and there does not seem to be an evaluation of how effective their measures are.<br /> The labeling pulse is applied in the oblique sagittal orientation, but in tandem with differing rates of blood flow and CSF drainage from the labeling plane does that not risk circulating flow from other slices potentially affecting the values?<br /> Figure 4. The authors focus on the parasagittal dura, but in both the subtraction image and panel C showing different slices at TI=1250 ms some movement appears visible in the opposing hemisphere. Similarly in S2 If the signal does represent CSF movement then this seems counterintuitive and should be explained.<br /> In Figures 4 and 5 the angulation of the TIME-SLIP tag pulse seems quite different. What procedure was used to standardise this, and what effect may this have on the results?

      12. Discussion<br /> Phrasing error 'which will be assessed in future studies'.<br /> I would suggest that some of the claims of novelty be moderated e.g. 'may facilitate establishment of normative values for CSF outflow' seems a stretch given multiple pathways exist and this is only considered one.<br /> More consideration should be given to some of the points mentioned in the results. The lack of validation should be properly discussed.

      Review 1
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    Partners Group, CVC Team Up to Rival Celanese for CeramTec
    1
    1. Dvorak 22 Nov 2022
      Partners Group, CVC Team Up to Rival Celanese for CeramTecBC said to seek over $4.7 billion for technical-ceramics makerNext-round bids due around July 19 as buyout activity surgesByDinesh Nair, Jan-Henrik Foerster, and Kiel Porter+FollowJuly 14, 2021 at 12:51 PM EDTUpdated onJuly 15, 2021 at 3:49 AM EDTShare this articleCopiedFollow the authors@DNair5+ Get alerts forDinesh Nair@JanFoe+ Get alerts forJan-Henrik Foerster@kielporter+ Get alerts forKiel PorterBuyout firms Partners Group Holding AG and CVC Capital Partners have teamed up against chemicals company Celanese Corp. in the bidding for German technical-ceramics maker CeramTec GmbH, according to people familiar with the matter.Owner BC Partners has called for next-round bids around July 19 and is seeking a valuation of at least 4 billion euros ($4.7 billion), the people said, asking not to be identified because discussions are private.LIVE ON BLOOMBERGWatch Live TVListen to Live RadioVideo Player is loading.Play VideoPlayUnmuteCurrent Time 0:00/Duration 0:00Loaded: 0%Progress: 0%Stream Type LIVERemaining Time -0:00 Playback Rate1xChaptersChaptersCaptionscaptions settings, opens captions settings dialogcaptions off, selectedFullscreenThis is a modal window.An error has occurred. 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surges"],"adCode":"bloomberg\/markets\/deals\/article","adTargeting":{"suid":"QW88EUT1UM0W01","page":"articlejav","currentResource":"Story|QW88EUT1UM0W01","ni":["ASSETMGMT","AUTOMOTIVE","BON","BUSINESS","EQUITYKEY","FAMOFFNEWS","FIALL","FIASST","FIN","INI","MARKETS","PE"],"tagr":[],"kwl":["biz_cartier","biz_googlelisttwo","biz_schwab","biz_generic","biz_lexus2","biz_singlecountry2","biz_United","biz_BMW","biz_boeing","biz_att6","biz_facebook1","biz_facebook2","biz_mulberry","biz_Fidelity_investopedia","biz_hsbcpb","biz_jpmorgan","biz_morg","biz_morgan1","biz_mobkoivca","biz_mobkoirichemont19","biz_kpmg","biz_socgenoctnov19","biz_wellspop","biz_porsche","biz_porsche2019","biz_burberryhk","biz_porsche2020","biz_kpmgpg","biz_mobkoifacebookpolicyaffairs","biz_mstouts2020","biz_signet","biz_cartier3","biz_signet2","biz_mobkoiintel","biz_socgen2020","biz_vancleef1","biz_cigna","biz_vca","biz_mobkoiintel2","biz_mobkoicastrol","biz_msci","biz_facebookpgemea21","biz_vacheron_2021","biz_iwc_2021","biz_panerai","biz_vancleef_2022","biz_cartier2022","biz_vcawatches2022","biz_Chanel","biz_chanelbleu","biz_vac2022"],"sites":["markets","deals"],"tickers":[],"language":"en","gs_cat":["hnwi_aiq_custom","csuite3_aiq_custom","sme2_aiq_custom","gs_economy_markets","travel_aiq_custom","gs_economy","pos_ibm","gs_science_misc","gs_business_sme","private_equity_custom","gs_science","gs_auto_misc","gv_safe"]},"archived":true,"assistance":["Aaron Kirchfeld"],"attributor":"bn","authoredRegion":"Global","authors":[{"id":"18657817","name":"Dinesh Nair","slug":"ARyyGQR8v_w\/dinesh-nair","title":null,"bio":null,"columnist":false,"contributor":false,"editorialBoard":false,"headshot":{"id":"386512166","baseUrl":"https:\/\/assets.bwbx.io\/images\/users\/iqjWHBFdfxIU\/iAHQo9umsWls\/v1\/200x200.jpg","origWidth":2000,"origHeight":2000,"caption":null,"type":"image","themes":null},"facebookHandle":null,"facebookUrl":null,"twitterHandle":"DNair5","twitterUrl":"https:\/\/www.twitter.com\/DNair5"},{"id":"17673321","name":"Jan-Henrik Foerster","slug":"AQ2saY71wbs\/janhenrik-foerster","title":null,"bio":null,"columnist":false,"contributor":false,"editorialBoard":false,"headshot":{"id":"386352019","baseUrl":"https:\/\/assets.bwbx.io\/images\/users\/iqjWHBFdfxIU\/ifa_pJeO2tgQ\/v2\/200x200.jpg","origWidth":2000,"origHeight":2000,"caption":null,"type":"image","themes":null},"facebookHandle":null,"facebookUrl":null,"twitterHandle":"JanFoe","twitterUrl":"https:\/\/www.twitter.com\/JanFoe"},{"id":"18043877","name":"Kiel Porter","slug":"ARNT5T6VBW0\/kiel-porter","title":null,"bio":null,"columnist":false,"contributor":false,"editorialBoard":false,"headshot":{"id":"110348767","baseUrl":"https:\/\/assets.bwbx.io\/images\/users\/iqjWHBFdfxIU\/iodhiLzBpXTw\/v1\/200x200.jpg","origWidth":160,"origHeight":211,"caption":null,"type":"image","themes":null},"facebookHandle":null,"facebookUrl":null,"twitterHandle":"kielporter","twitterUrl":"https:\/\/www.twitter.com\/kielporter"}],"blensQuoteIds":[{"id":"7473980Z:FP"},{"id":"CE:US"},{"id":"3711Z:GR"},{"id":"PGHN:SW"},{"id":"1872421D:LN"}],"body":"<div class=\"inline-newsletter-top\"><\/div><p>Buyout firms <a href=\"\/quote\/PGHN:SW\" title=\"Company Overview\"><meta content=\"PGHN SW Equity\"><meta content=\"SecurityLink\">Partners Group Holding AG<\/a> and <a href=\"\/quote\/2270Z:LN\" title=\"Company Overview\"><meta content=\"2270Z LN Equity\"><meta content=\"SecurityLink\">CVC Capital Partners<\/a> have teamed up against chemicals company <a href=\"\/quote\/CE:US\" title=\"Company Overview\"><meta content=\"CE US Equity\"><meta content=\"SecurityLink\">Celanese Corp.<\/a> in the bidding for German technical-ceramics maker <a href=\"\/quote\/3711Z:GR\" title=\"Company Overview\"><meta content=\"3711Z GR Equity\"><meta content=\"SecurityLink\">CeramTec GmbH<\/a>, according to people familiar with the matter.<\/p><p>Owner <a href=\"\/quote\/7473980Z:FP\" title=\"Company Overview\"><meta content=\"7473980Z FP Equity\"><meta content=\"SecurityLink\">BC Partners<\/a> has called for next-round bids around July 19 and is seeking a valuation of at least 4 billion euros ($4.7 billion), the people said, asking not to be identified because discussions are private.<\/p>\n <div id=\"outstream-video-1-QW88EUT1UM0W01\" class=\"outstream-ad outstream-ad--default paywall\" data-position=\"outstream-video\" data-ad-placeholder=\"Advertisement\">\n \n <script type=\"application\/javascript\">window.__bloomberg__.ads.enqueue(\"outstream-video-1-QW88EUT1UM0W01\");<\/script>\n <script class=\"params\" type=\"application\/json\">{\"contentId\":\"QW88EUT1UM0W01\",\"position\":\"outstream\",\"dimensions\":{\"large_desktop\":[[300,250],[1,8],[3,3]],\"small_desktop\":[[300,250],[1,8],[3,3]],\"tablet\":[[300,250],[1,8],[3,3]]},\"strategy\":\"viewable\",\"type\":\"Outstream Video Native Ad\",\"targeting\":{\"position\":\"outstream\",\"url\":\"\/news\/articles\/2021-07-14\/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding\"},\"containerId\":\"outstream-video-1-QW88EUT1UM0W01\"}<\/script>\n \n <\/div>\n \n <div id=\"outstream-video-2-QW88EUT1UM0W01\" class=\"outstream-ad outstream-ad--mobile paywall\" data-position=\"outstream-video\" data-ad-placeholder=\"Advertisement\">\n \n <script type=\"application\/javascript\">window.__bloomberg__.ads.enqueue(\"outstream-video-2-QW88EUT1UM0W01\");<\/script>\n <script class=\"params\" type=\"application\/json\">{\"contentId\":\"QW88EUT1UM0W01\",\"position\":\"outstream\",\"dimensions\":{\"mobile\":[[300,250],[1,8],[3,3]]},\"strategy\":\"viewable\",\"type\":\"Outstream Video Native Ad\",\"targeting\":{\"position\":\"outstream\",\"url\":\"\/news\/articles\/2021-07-14\/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding\"},\"containerId\":\"outstream-video-2-QW88EUT1UM0W01\"}<\/script>\n \n <\/div>\n <p class=\"paywall\">Other investment firms and companies have also looked at the asset, the people said. Bloomberg News <a href=\"https:\/\/www.bloomberg.com\/news\/articles\/2021-07-01\/bc-partners-said-to-explore-options-for-4-billion-ceramics-firm\" title=\"BC Partners Said to Mull Options for $4 Billion Ceramic Firm (1)\" target=\"_blank\"><meta content=\"QVLX12T1UM0W\"><meta content=\"StoryLink\">reported<\/a> earlier this month that BC Partners started exploring options, including a sale or initial public offering, in a deal that could value the business at 3.5 billion euros or more.<\/p><aside class=\"left-rail-newsletter paywall\"><\/aside><p class=\"paywall\">CeramTec produces industrial and technical ceramics for the medical, automotive, electronics and chemicals industries, making everything from hip joints to car parts. The company, which traces its <a href=\"https:\/\/www.ceramtec-group.com\/en\/about-us\/history\" title=\"History\" target=\"_blank\" rel=\"noopener\"><meta content=\"WebLink\">roots<\/a> back to a porcelain factory from 1903, employs more than 3,400 globally and had over 550 million euros in 2020 sales, according to its <a href=\"https:\/\/www.ceramtec-group.com\/en\/about-us\" title=\"related website\" target=\"_blank\" rel=\"noopener\"><meta content=\"WebLink\">website<\/a>.<\/p><p class=\"paywall\">Private equity firms&#x2019; divestments in Europe have risen more than 150% to $70 billion this year, according to data compiled by Bloomberg. <a href=\"\/quote\/277924Z:LN\" title=\"Company Overview\"><meta content=\"277924Z LN Equity\"><meta content=\"SecurityLink\">TDR Capital<\/a> agreed <a href=\"https:\/\/www.bloomberg.com\/news\/articles\/2021-06-27\/brookfield-unit-said-to-near-deal-for-tdr-backed-modulaire-group\" title=\"Brookfield Unit to Buy TDR-Backed Modulaire Group for $5 Billion\" target=\"_blank\"><meta content=\"QVEGAXT1UM0Z\"><meta content=\"StoryLink\">last month<\/a> to sell Modulaire Group, a designer of modular work spaces, to <a href=\"\/quote\/BBU-U:CN\" title=\"Company Overview\"><meta content=\"BBU-U CN Equity\"><meta content=\"SecurityLink\">Brookfield Business Partners LP<\/a> for about $5 billion.<\/p>\n <div id=\"box-jc6JU4A\" class=\"mobile-box page-ad paywall\" data-position=\"mobile-box\" data-ad-placeholder=\"Advertisement\">\n \n <script type=\"application\/javascript\">window.__bloomberg__.ads.enqueue(\"box-jc6JU4A\");<\/script>\n <script class=\"params\" type=\"application\/json\">{\"contentId\":\"QW88EUT1UM0W01\",\"position\":\"box\",\"dimensions\":{\"mobile\":[[300,250],[3,3],[1,1],\"fluid\"]},\"type\":\"Mobile Body Box Ad\",\"positionIncrement\":1,\"targeting\":{\"position\":\"box1\",\"positionIncrement\":1,\"url\":\"\/news\/articles\/2021-07-14\/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding\"},\"containerId\":\"box-jc6JU4A\"}<\/script>\n \n <\/div>\n <div class=\"for-you__wrapper paywall\"><\/div><p class=\"paywall\">No final decisions have been made, and there&#x2019;s no certainty talks will lead to a transaction, the people said. Representatives for BC Partners, Celanese, CVC and Partners Group declined to comment.<\/p>\n <div id=\"desktop-in-article-1-QW88EUT1UM0W01\" class=\"desktop-in-article page-ad paywall\" data-position=\"desktop-in-article\" data-ad-placeholder=\"Advertisement\">\n \n <script type=\"application\/javascript\">window.__bloomberg__.ads.enqueue(\"desktop-in-article-1-QW88EUT1UM0W01\");<\/script>\n <script class=\"params\" type=\"application\/json\">{\"contentId\":\"QW88EUT1UM0W01\",\"position\":\"desktop-in-article1\",\"dimensions\":{\"large_desktop\":[[300,250],[5,4],[3,3]],\"small_desktop\":[[300,250],[5,4],[3,3]]},\"type\":\"Desktop in article Native Ad\",\"targeting\":{\"position\":\"desktop-in-article1\",\"url\":\"\/news\/articles\/2021-07-14\/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding\"},\"containerId\":\"desktop-in-article-1-QW88EUT1UM0W01\"}<\/script>\n \n <\/div>\n <p class=\"paywall\">A consortium led by BC Partners <a href=\"https:\/\/www.bcpartners.com\/news\/bc-partners-led-consortium-including-psp-investments-and-ontario-teachers-acquires-ceramtec-a-leading-international-manufacturer-and-supplier-of-technical-ceramic\" title=\"Link\" target=\"_blank\" rel=\"noopener\"><meta content=\"WebLink\">agreed<\/a> to acquire CeramTec from private equity firm <a href=\"\/quote\/9990648Z:LN\" title=\"Company Overview\"><meta content=\"9990648Z LN Equity\"><meta content=\"SecurityLink\">Cinven<\/a> in 2017. Canada&#x2019;s Public Sector Pension Investment Board and Ontario Teachers&#x2019; Pension Plan also joined the deal. That acquisition valued CeramTec at about 2.6 billion euros including debt, Bloomberg News <a href=\"\/news\/terminal\/OXM9ER6KLVRX\" title=\"Cinven Is Said Near $3 Billion CeramTec Sale to BC Partners (1)\" class=\"terminal-news-story\" target=\"_blank\"><meta content=\"OXM9ER6KLVRX\"><meta content=\"StoryLink\">reported<\/a> at the time.<\/p><p class=\"paywall\">Elsewhere in Germany, BC Partners this month agreed to take a <a href=\"\/news\/terminal\/QVZJPOT0AFBE\" title=\"BC Partners Reaches Deal for German Laboratories Group Tentamus\" class=\"terminal-news-story\" target=\"_blank\"><meta content=\"QVZJPOT0AFBE\"><meta content=\"StoryLink\">stake<\/a> in Tentamus Group GmbH amid strong private equity demand for laboratory assets in Europe. The deal values the food and pharmaceutical-testing company at about 1 billion euros, people familiar with the matter said.<\/p>\n <div id=\"in-article-1-QW88EUT1UM0W01\" class=\"in-article page-ad hide_on_small_desktop hide_on_large_desktop paywall\" data-position=\"in-article\" data-ad-placeholder=\"Advertisement\">\n \n <script type=\"application\/javascript\">window.__bloomberg__.ads.enqueue(\"in-article-1-QW88EUT1UM0W01\");<\/script>\n <script class=\"params\" type=\"application\/json\">{\"contentId\":\"QW88EUT1UM0W01\",\"position\":\"in-article1\",\"dimensions\":{\"mobile\":[[5,19],[300,250],[3,3],[1,1],\"fluid\"],\"tablet\":[[5,11],[728,90],[1,1]]},\"type\":\"In Article Flex Native Ad\",\"positionIncrement\":1,\"targeting\":{\"position\":\"in-article1\",\"positionIncrement\":1,\"url\":\"\/news\/articles\/2021-07-14\/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding\"},\"containerId\":\"in-article-1-QW88EUT1UM0W01\"}<\/script>\n \n <\/div>\n <p class=\"paywall\"><em>&#x2014; With assistance by Aaron Kirchfeld<\/em><\/p><div class=\"trashline paywall\">(<span>Adds BC Partners Germany deal in final paragraph.<\/span>)<\/div><ol class=\"noscript-footnotes paywall\"><\/ol><div class=\"inline-newsletter-bottom paywall\"><\/div>","brand":"markets","canonical":"https:\/\/www.bloomberg.com\/news\/articles\/2021-07-14\/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding","byline":"Dinesh Nair, Jan-Henrik Förster and Kiel Porter","categories":["markets"],"charts":[],"checksum":"6638bc1c8e358f7bda223c21d7a55eba","columnists":[],"corrected":false,"dek":null,"disableAds":false,"disclaimer":"","embeds":[],"facebookStatus":"Buyout firms Partners Group Holding AG and CVC Capital Partners have teamed up against chemicals company Celanese Corp. in the bidding for German technical-ceramics maker CeramTec GmbH, according to people familiar with the matter.","featureVersion":null,"footer":"<meta itemprop=\"NewsFooterAttributionType\" content=\"http:\/\/bloomberg.com\/StoryFormat\/NewsIndividualAttribution\"><p class=\"news-rsf-assists\">--With assistance from <span itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Person\"><link itemprop=\"additionalType\" href=\"http:\/\/bloomberg.com\/StoryFormat\/ContactInfo\"><meta itemprop=\"url\" content=\"bbg:\/\/people\/profile\/15014888\"><meta itemprop=\"pepl\" content=\"15014888\"><meta itemprop=\"uuid\" content=\"3925253\"><meta itemprop=\"email\" content=\"akirchfeld@bloomberg.net\"><meta itemprop=\"telephone\" content=\"+44-20-35258830\"><span itemprop=\"workLocation\" itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Place\"><meta itemprop=\"name\" content=\"London\"><\/span><meta itemprop=\"role\" content=\"assist\"><span itemprop=\"attribution\" itemscope=\"itemscope\"><meta itemprop=\"indicator\" content=\"assist\"><meta itemprop=\"ordinal\" content=\"4\"><\/span><span itemprop=\"name\">Aaron Kirchfeld<\/span><\/span>.<\/p><p class=\"news-rsf-contact-reporter\">To contact the reporters on this story:<br><span itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Person\"><link itemprop=\"additionalType\" href=\"http:\/\/bloomberg.com\/StoryFormat\/ContactInfo\"><meta itemprop=\"url\" content=\"bbg:\/\/people\/profile\/18657817\"><meta itemprop=\"pepl\" content=\"18657817\"><meta itemprop=\"uuid\" content=\"11900697\"><meta itemprop=\"telephone\" content=\"+44-20-35253212\"><meta itemprop=\"role\" content=\"by\"><meta itemprop=\"role\" content=\"reporter\"><span itemprop=\"attribution\" itemscope=\"itemscope\"><meta itemprop=\"indicator\" content=\"by\"><meta itemprop=\"indicator\" content=\"reporter\"><meta itemprop=\"ordinal\" content=\"1\"><\/span><span itemprop=\"name\">Dinesh Nair<\/span> in <span itemprop=\"workLocation\" itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Place\"><span itemprop=\"name\">London<\/span><\/span> at <span itemprop=\"email\">dnair5@bloomberg.net<\/span><\/span>;<br><span itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Person\"><link itemprop=\"additionalType\" href=\"http:\/\/bloomberg.com\/StoryFormat\/ContactInfo\"><meta itemprop=\"url\" content=\"bbg:\/\/people\/profile\/17673321\"><meta itemprop=\"pepl\" content=\"17673321\"><meta itemprop=\"uuid\" content=\"11757890\"><meta itemprop=\"telephone\" content=\"+44-20-35254287\"><meta itemprop=\"role\" content=\"by\"><meta itemprop=\"role\" content=\"reporter\"><span itemprop=\"attribution\" itemscope=\"itemscope\"><meta itemprop=\"indicator\" content=\"by\"><meta itemprop=\"indicator\" content=\"reporter\"><meta itemprop=\"ordinal\" content=\"2\"><\/span><span itemprop=\"name\">Jan-Henrik F&#xF6;rster<\/span> in <span itemprop=\"workLocation\" itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Place\"><span itemprop=\"name\">London<\/span><\/span> at <span itemprop=\"email\">jforster20@bloomberg.net<\/span><\/span>;<br><span itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Person\"><link itemprop=\"additionalType\" href=\"http:\/\/bloomberg.com\/StoryFormat\/ContactInfo\"><meta itemprop=\"url\" content=\"bbg:\/\/people\/profile\/18043877\"><meta itemprop=\"pepl\" content=\"18043877\"><meta itemprop=\"uuid\" content=\"10594416\"><meta itemprop=\"telephone\" content=\"+1-312-443-5967\"><meta itemprop=\"role\" content=\"by\"><meta itemprop=\"role\" content=\"reporter\"><span itemprop=\"attribution\" itemscope=\"itemscope\"><meta itemprop=\"indicator\" content=\"by\"><meta itemprop=\"indicator\" content=\"reporter\"><meta itemprop=\"ordinal\" content=\"3\"><\/span><span itemprop=\"name\">Kiel Porter<\/span> in <span itemprop=\"workLocation\" itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Place\"><span itemprop=\"name\">Chicago<\/span><\/span> at <span itemprop=\"email\">kporter17@bloomberg.net<\/span><\/span><\/p><p class=\"news-rsf-contact-editor\">To contact the editors responsible for this story:<br><span itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Person\"><link itemprop=\"additionalType\" href=\"http:\/\/bloomberg.com\/StoryFormat\/ContactInfo\"><meta itemprop=\"url\" content=\"bbg:\/\/people\/profile\/6720026\"><meta itemprop=\"pepl\" content=\"6720026\"><meta itemprop=\"uuid\" content=\"2920049\"><meta itemprop=\"jobTitle\" content=\"Executive Editor:Deals &amp; Corporate Finance\"><meta itemprop=\"telephone\" content=\"+1-212-617-1697\"><span itemprop=\"workLocation\" itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Place\"><meta itemprop=\"name\" content=\"New York\"><\/span><meta itemprop=\"role\" content=\"editor\"><meta itemprop=\"role\" content=\"responsible\"><span itemprop=\"attribution\" itemscope=\"itemscope\"><meta itemprop=\"indicator\" content=\"editor\"><meta itemprop=\"indicator\" content=\"responsible\"><meta itemprop=\"ordinal\" content=\"6\"><\/span><span itemprop=\"name\">Daniel Hauck<\/span> at <span itemprop=\"email\">dhauck1@bloomberg.net<\/span><\/span><br><span class=\"news-rsf-editor-byline\"><span itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Person\"><link itemprop=\"additionalType\" href=\"http:\/\/bloomberg.com\/StoryFormat\/ContactInfo\"><meta itemprop=\"url\" content=\"bbg:\/\/people\/profile\/21714985\"><meta itemprop=\"pepl\" content=\"21714985\"><meta itemprop=\"uuid\" content=\"29472435\"><meta itemprop=\"email\" content=\"fsahloul@bloomberg.net\"><meta itemprop=\"telephone\" content=\"+44-20-35253357\"><span itemprop=\"workLocation\" itemscope=\"itemscope\" itemtype=\"http:\/\/schema.org\/Place\"><meta itemprop=\"name\" content=\"London\"><\/span><meta itemprop=\"role\" content=\"editor\"><meta itemprop=\"role\" content=\"primary\"><span itemprop=\"attribution\" itemscope=\"itemscope\"><meta itemprop=\"indicator\" content=\"editor\"><meta itemprop=\"indicator\" content=\"primary\"><meta itemprop=\"ordinal\" content=\"5\"><\/span><span itemprop=\"name\">Fareed Sahloul<\/span><\/span><\/span><\/p>","footnotes":{},"franchise":"deals","headline":"Partners Group, CVC Team Up to Rival Celanese for CeramTec","headlineText":"Partners Group, CVC Team Up to Rival Celanese for CeramTec","hedAndDekPosition":"above","id":"QW88EUT1UM0W01","isPressRelease":false,"isTrending":false,"imageAttachments":{"373530619":{"id":"373530619","baseUrl":"https:\/\/assets.bwbx.io\/images\/users\/iqjWHBFdfxIU\/i6CB2JgDX_TI\/v0\/-1x-1.jpg","origWidth":811,"origHeight":608,"caption":null,"type":"image","alt":"ceramtec","themes":null},"373530732":{"id":"373530732","baseUrl":"https:\/\/assets.bwbx.io\/images\/users\/iqjWHBFdfxIU\/i2B9vOvxzOdU\/v0\/-1x-1.jpg","origWidth":1215,"origHeight":608,"caption":null,"type":"image","alt":"ceramtec SOCIAL","themes":null},"373752001":{"id":"373752001","baseUrl":"https:\/\/assets.bwbx.io\/images\/users\/iqjWHBFdfxIU\/ikeljb901_vY\/v0\/-1x-1.jpg","origWidth":1215,"origHeight":608,"caption":null,"type":"image","alt":"Partners Group, CVC Team Up to Rival Celanese for CeramTec","themes":null}},"label":null,"language":"en","ledeAttachment":null,"ledeCaption":null,"ledeCredit":null,"ledeDescription":null,"ledeImageUrl":null,"ledeKind":"not_quite_full_width","ledeMediaKind":"","ledeSize":"","locale":"en","magazine":null,"magazineMetadata":null,"marketcards":[],"metadata":{"hiddenInlineAttachments":[],"magazine":false,"suppressComments":false,"excludeFromPaywall":false,"theme":null,"background":null,"isMetered":false,"newsletterSlug":null,"newsletterToutLabel":null,"cobrand":null,"terminalBlogId":null},"mostRelevantTags":["Capital Partners","Private Equity","Valuation","IPOs","Automotive","Europe"],"moved":false,"pillar":null,"premium":false,"primaryCategory":"markets","primarySite":"markets","publishedAt":"2021-07-14T16:51:30.758Z","readings":{"url":"https:\/\/assets.bwbx.io\/s3\/readings\/QW9WWDT0AFB41626336911620.mp3","durationMs":135262},"relatedStories":[],"resourceType":"Story","revision":"QW9WWDT0AFB4","secondaryBrands":["markets","business"],"seoHeadline":"Partners Group, CVC Team Up to Rival Celanese for CeramTec","slug":"2021-07-14\/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding","socialDescription":"Buyout firms Partners Group Holding AG and CVC Capital Partners have teamed up against chemicals company Celanese Corp. in the bidding for German technical-ceramics maker CeramTec GmbH, according to people familiar with the matter.","socialHeadline":"Partners Group, CVC Team Up to Rival Celanese for CeramTec","socialImageUrl":"https:\/\/assets.bwbx.io\/images\/users\/iqjWHBFdfxIU\/i2B9vOvxzOdU\/v0\/1200x600.jpg","storythreads":[],"summary":"Buyout firms Partners Group Holding AG and CVC Capital Partners have teamed up against chemicals company Celanese Corp. in the bidding for German technical-ceramics maker CeramTec GmbH, according to people familiar with the matter.","summaryText":"","suppressComments":false,"textHeadline":"Partners Group, CVC Team Up to Rival Celanese for CeramTec","theme":"markets","timeline":{},"topic":"Capital Partners","trashline":"(<span itemprop=\"description\">Adds BC Partners Germany deal in final paragraph.<\/span>)","twitterDescription":"Buyout firms Partners Group Holding AG and CVC Capital Partners have teamed up against chemicals company Celanese Corp. in the bidding for German technical-ceramics maker CeramTec GmbH, according to people familiar with the matter.","twitterHandle":"markets","twitterText":"Partners Group and CVC have teamed up against chemicals company Celanese in the bidding for German technical-ceramics maker CeramTec, sources say","twitterTitle":"Partners Group, CVC Team Up to Rival Celanese for CeramTec","type":"archived","updatedAt":"2021-07-15T07:49:08.909Z","url":"\/news\/articles\/2021-07-14\/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding","videoAttachments":{},"webOriginal":false,"wssTags":[{"id":"Europe","type":"Region","directScore":0.5449411764705883,"derivedScore":8.858547871735478},{"id":"DE","type":"Country","directScore":0.1251764705882353,"derivedScore":5.632072944712481},{"id":"CA","type":"Country","directScore":0.23294117647058823,"derivedScore":0.4639285714285714},{"id":"1125977D:GR","type":"Company","directScore":0.09835294117647059,"derivedScore":0.09835294117647059},{"id":"BBU-U:CN","type":"Company","directScore":0.3952941176470588,"derivedScore":0.3952941176470588},{"id":"1872421D:LN","type":"Company","directScore":0.42023529411764704,"derivedScore":0.42023529411764704},{"id":"PGHN:SW","type":"Company","directScore":3.824317135549872,"derivedScore":3.824317135549872},{"id":"3711Z:GR","type":"Company","directScore":5.629854175079643,"derivedScore":5.629854175079643},{"id":"CE:US","type":"Company","directScore":6.306546237717054,"derivedScore":6.306546237717054},{"id":"7473980Z:FP","type":"Company","directScore":7.619764705882353,"derivedScore":7.619764705882353},{"id":"food","type":"Topic","directScore":0.048,"derivedScore":0.048},{"id":"debt","type":"Topic","directScore":0.15058823529411763,"derivedScore":0.15058823529411763},{"id":"pension-plan","type":"Topic","directScore":0.2,"derivedScore":0.2},{"id":"automotive","type":"Topic","directScore":0.6291764705882353,"derivedScore":0.6291764705882353},{"id":"ipos","type":"Topic","directScore":0.7049411764705882,"derivedScore":0.7049411764705882},{"id":"valuation","type":"Topic","directScore":0.8536470588235294,"derivedScore":0.8536470588235294},{"id":"private-equity","type":"Topic","directScore":1.6324705882352941,"derivedScore":1.6324705882352941},{"id":"capital-partners","type":"Topic","directScore":3.8010741687979537,"derivedScore":3.8010741687979537},{"id":"markets","type":"Classification","directScore":0,"derivedScore":6},{"id":"finance","type":"Classification","directScore":0,"derivedScore":8.210859861717834},{"id":"GB","type":"Country","directScore":0,"derivedScore":0.42023529411764704},{"id":"industrials","type":"Topic","directScore":0,"derivedScore":0.3952941176470588},{"id":"FR","type":"Country","directScore":0,"derivedScore":7.619764705882353},{"id":"infrastructure","type":"Topic","directScore":0,"derivedScore":0.6291764705882353},{"id":"US","type":"Region","directScore":0,"derivedScore":6.322191868855642},{"id":"fixed-income","type":"Topic","directScore":0,"derivedScore":0.15058823529411763},{"id":"CH","type":"Country","directScore":0,"derivedScore":3.824317135549872},{"id":"materials","type":"Topic","directScore":0,"derivedScore":6.317207134650508},{"id":"bonds","type":"Topic","directScore":0,"derivedScore":0.15058823529411763},{"id":"US","type":"Country","directScore":0,"derivedScore":6.306546237717054},{"id":"transportation","type":"Topic","directScore":0,"derivedScore":0.6291764705882353},{"id":"technology","type":"Classification","directScore":0,"derivedScore":8.81943180439255},{"id":"UK","type":"Country","directScore":0,"derivedScore":0.42023529411764704}],"validatedAt":"2022-11-22T16:52:27.557Z","teaserBody":"<p>Buyout firms <a href=\"\/quote\/PGHN:SW\" itemprop=\"StoryLink\" itemscope=\"itemscope\" title=\"Company Overview\"><meta itemprop=\"security\" content=\"PGHN SW Equity\"><meta itemprop=\"type\" content=\"SecurityLink\">Partners Group Holding AG<\/a> and <a href=\"\/quote\/2270Z:LN\" itemprop=\"StoryLink\" itemscope=\"itemscope\" title=\"Company Overview\"><meta itemprop=\"security\" content=\"2270Z LN Equity\"><meta itemprop=\"type\" content=\"SecurityLink\">CVC Capital Partners<\/a> have teamed up against chemicals company <a href=\"\/quote\/CE:US\" itemprop=\"StoryLink\" itemscope=\"itemscope\" title=\"Company Overview\"><meta itemprop=\"security\" content=\"CE US Equity\"><meta itemprop=\"type\" content=\"SecurityLink\">Celanese Corp.<\/a> in the bidding for German technical-ceramics maker <a href=\"\/quote\/3711Z:GR\" itemprop=\"StoryLink\" itemscope=\"itemscope\" title=\"Company Overview\"><meta itemprop=\"security\" content=\"3711Z GR Equity\"><meta itemprop=\"type\" content=\"SecurityLink\">CeramTec GmbH<\/a>, according to people familiar with the matter.<\/p><p>Owner <a href=\"\/quote\/7473980Z:FP\" itemprop=\"StoryLink\" itemscope=\"itemscope\" title=\"Company Overview\"><meta itemprop=\"security\" content=\"7473980Z FP Equity\"><meta itemprop=\"type\" content=\"SecurityLink\">BC Partners<\/a> has called for next-round bids around July 19 and is seeking a valuation of at least 4 billion euros ($4.7 billion), the people said, asking not to be identified because discussions are private.<\/p>"},"greenDataSnippet":{"js":"","css":"","html":""},"coronavirusDataSnippet":{"js":"","css":"","html":""},"isNewsletter":false,"mostPopular":[{"brand":"technology","site":"technology","byline":"","headline":"Billionaire Investor Carl Icahn Is Betting Against GameStop 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Already

      CVC looks like Ceramic with the "era" starting with "emblem"

      ** not affiliated with QWERTY either. ECMA

    Visit annotations in context

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    URL

    bloomberg.com/news/articles/2021-07-14/partners-group-cvc-team-up-against-celanese-in-ceramtec-bidding
  13. www.biorxiv.org www.biorxiv.org
    New submission 21/11/2022, 14:27:26
    2
    1. Public_Reviews 22 Nov 2022

      Author Response

      Reviewer #1 (Public Review):

      The authors have generated a set of seven nanobody tools against two of the largest Drosophila proteins, which are related to vertebrate titin and essential for muscle function. The study of such gigantic proteins is a challenge. They show that each of these nanobodies recognizes their epitope with high affinity (as expected from antibodies), fails to generate a signal after immune-fixation of a mutant for the cognate protein, do not cross-react with each other, and generates a signal in the muscle that makes sense with what one would anticipate for fly titin homologs. In addition, they show that these nanobodies have better penetration and labeling efficiency than conventional antibodies in thick tissues after classical paraformaldehyde fixation. Using these nanobodies, they could deduce the organization of the epitopes in different muscle types and propose a model for Sallimus and Projectin arrangement in muscles, including in larvae which are difficult to label with traditional antibodies due to their impermeable chitin skeleton. Finally, they could fuse the gene encoding one of the nanobodies to the open reading frame of NeonGreen and express the corresponding fusion protein in animals to use the probe in FRAP assays.

      The work is very well performed and convincing. However, given its significant redundancy in terms of biological conclusions with the companion study "Nanobodies combined with DNAPAINT super-resolution reveal a staggered titin nano-architecture in flight muscles" by the same authors, and other published papers, I recommend the authors further prove the use of their nanobodies in live assays. In particular, the authors should test whether they can use the nanobodies to induce protein degradation either permanently or conditionally.

      Thanks for this nice summary of our findings. We have now extended the analysis of the Nanobody-NeonGreen fusion expressing larval muscles and provide first proof of principle analysis of new fly strains that we generated that contain Sls-Nano2 or Sls-Nano42 nanobodies fused to a degradation signal. These induce lethality of the animals suggesting that Sls protein is partially non functional. We verified this by providing quantitative stainings of various Sls epitopes in these muscles suggesting that Sls is not fully degraded but rather partially modified in the Sls-Nano-deGrad expressing muscle fibers. These will be interesting tools to study Sls function during sarcomere homeostasis.

      Reviewer #2 (Public Review):

      The data presented in this manuscript are sound but rather descriptive. The contribution - as presented - is mostly of a technical nature. The authors correctly state that anti-GFP nanobodies, while used extensively across many model organisms, have limited utility for in vivo applications when the GFP-tagged protein in question displays abnormal behavior or is non-functional. The creation of nanobodies that are uniquely specific for the protein(s) of interest is therefore a significant improvement, especially since the Sallimus and Projectinspecific reagents reported here react with PFA-fixed material. At least one of these nanobodies, when expressed in vivo, decorates the appropriate target. The source of antigens used for the construction of the nanobody library is Drosophila-derived. The extent of homology of Drosophila Sallimus and Projectin with related proteins in other species is not discussed. Whether the nanobodies reported here would be useful in other (closely related?) species, therefore, remains to be established. For those studying muscle biology in Drosophila, the nanobodies described here will be publicly available as cDNAs. Ease of production implies a readily shared and standardized resource for the field.

      We thank this reviewer for appreciating that our Sallimus and Projectin nanobodies are useful. We now have extended the collection even further, including anti-Obscurin, αActinin and Zasp52 nanobodies, the latter two will also be useful for researcher studying other tissues, in particular Drosophila epithelial tissues. As always in the Drosophila field, all the here generated fly strains and plasmids will be made easily available to the community by placing them in stock centers or shipping them to the laboratories directly. As indicated, also the plasmids will be deposited at Addgene.

      Further characterization of these nanobodies by biochemical methods such as immunoblotting would be challenging, given the size of the target proteins. In view of the technical nature of this manuscript, the authors should perhaps critically discuss the distinction between bulky GFP tags versus the much smaller epitope tags and the nanobodies that recognize them, although this was covered in a recent eLife paper from the Perrimon lab. Insertion of small tags, in conjunction with nanobodies that recognize them, would be less perturbing than the much bulkier GFP tag and lend itself to genome-wide applications. Creating nanobodies uniquely specific for each protein encoded in the Drosophila genome is not realistic, and the targeted approach deployed here is obviously valuable.

      We are discussing the drawbacks of solely relying on GFP nanobodies, which requires GFP tagged proteins to be available and being functional. In particular for the sarcomeric proteins this is often not the case. We also cite the Perrimon paper, which was just published as we prepared this manuscript. We would like to point out to this reviewer that even tagging with a small epitope tag is considerable work in Drosophila and that the Perrimon paper, on which this reviewer is an author, does describe only two endogenously tagged genes with a nanotag (histone H2Av and Dilp2) the other genes described were expressed from a UAS source or in cell culture. We show here 22 nanobodies against 11 target epitopes.

      Nanobodies recognise typically folded epitopes and are rather unlikely to work in immunoblotting.

      The authors apply two different approaches to characterize the newly generated Nanobodies: more or less conventional immunohistochemistry with fluorescently labeled nanobodies, and in vivo expression of nanobodies fused to the fluorescent neongreen protein. The superiority of nanobodies in terms of tissue penetration has been shown by others in a direct comparison of intact fluorescently labeled immunoglobulins versus nanobodies. The authors state that in vivo labeling with nanobody fusions "thus far was done only with nanobodies against GFP, mCherry or short epitope tags." There is no fundamental difference between these recognition events and what the authors report for their Sallimus and Projectin-specific reagents. The section that starts at line 304 is thus a little bit of a 'straw man'. There is no reason to assume that a nanobody that recognizes a muscle protein would behave differently than a nanobody that would recognize that same protein (or another) when epitope- or GFP-tagged. What might be interesting is to examine the behavior of these muscle-specific nanobodies in the course of muscle contraction/relaxation: are there conformational alterations that promote dissociation of bound nanobodies? Do different nanobodies display discrete behavior in this regard? The manuscript is silent on how muscles behave in live L3 larvae. The FRAP experiment seems to suggest that not much is happening, but the text refers to the contraction of larval sarcomeres from 8.5 µM to 4.5 µM. Does the in vivo expressed nanobody remain stably bound during this contraction/relaxation cycle? What about the other nanobodies reported in this manuscript? Since the larval motion was reduced by exposure to diethylether, have the authors considered imaging the contractive cycle in the absence of such exposure?

      We appreciate the expert knowledge about nanobodies by this reviewer. However, nanobodies were not extensively applied in Drosophila tissues. Hence, we believe it is important to characterise their penetration in stainings and compare them carefully to antibodies. Such, the Drosophila reader will be aware of their advantages.

      We have now also included more data on the larval muscle morphology in the nanobody expressing muscles. Their morphology is normal. As larvae move around extensively all the time, the binding of the nanobodies to the target must be stable, otherwise it would not be bound when we fix them or anesthetize them. However, we have not attempted to image them at high resolution while crawling freely. From quantifying the crawling speed (about 1.5 mm per second, see Figure 9 S1) we hope this reviewer appreciates that high resolution imaging of sarcomeres in freely crawling larvae is highly non trivial.

      Given that the nanobodies bind well-folded epitopes with low picomolar dissociations constants, it is hard to imagine that conformational changes of the target would dissociate them. The nanobody would stabilise the recognised conformation by a ΔG of ≈60 KJ/ mole, and we would not expect that the chosen domains undergo major conformational changes.

      Reviewer #3 (Public Review):

      Loreau et al. have presented a well-written manuscript reporting clever, original work taking advantage of fairly new biotechnology - the generation and use of single chain antibodies called nanobodies. The authors demonstrate the production of multiple nanobodies to two titin homologs in Drosophila and use these nanobodies to localize these proteins in several fly muscle types and discover interesting aspects of the localization and span of these elongated proteins in the muscle sarcomere. They also demonstrate that one of these single chain antibodies can be expressed in muscle fused to a fluorescent protein to image the localization of a segment of one of these giant proteins called Sallimus in muscle in a live fly. Their project is well-justified given the limitations of the usual approaches for localizing and studying the dynamics of proteins in the muscle of model organisms such as the possibility that GFP tagging of a protein will interfere with its localization or function, and poor penetration of large IgG or IgM antibodies into densly packed structures like the sarcomere after fixation as compared to smaller nanbodies.

      They achieved their goals consistent with the known/expected properties of nanobodies: (1) They demonstrate that at least one of their nanobodies binds with very high affinity. (2) They bind with high specificity. (3) The nanobodies show much better penetration of fixed stage 17 embryos than do conventional antibodies.

      They use their nanobodies mostly generated to the N- and C-terminal ends of Sallimus and Projectin to learn new information about how these elongated proteins span and are oriented in the sarcomere. For example, in examining larval muscles which have long sarcomeres (8.5 microns), using nanobodies to domains located near the N- and C-termini, they show definitively that the predicted 2.1 MDa protein Sallimus spans the entire I-band and extends a bit into the A-band with its N-terminus embedded in the Z-disk and C-terminus in the outer edge of the A-band. Using a similar approach they also show that the 800 kDa Projectin decorates the entire myosin thick filament except for the H-zone and M-line in a polar orientation. Their final experiment is most exciting! They were able to express in fly larval muscles a nanobody directed to near the N-terminus of Sallimus fused to NeonGreen and show that it localizes to Z-disks in living larvae, and by FRAP experiments demonstrate that the binding of this nanobody to Sallimus in vivo is very stable. This opens the door to using a similar approach to study the assembly, dynamics, and even conformational changes of a protein in a complex in a live animal in real time.

      We thank this reviewer for appreciating the quality and impact of our approach and the our obtained results.

      There are only a few minor weaknesses about their conclusions: (1) They should note that in fact their estimate of the span of Sallimus could be an underestimate since their Nano2 nanobody is directed to Ig13/14 so if all of these 12 Ig domains N-terminal of their epitope were unwound it would add 12 X 30 nm = 360 nm of length, and even if unwound would add about 50 nm of length.

      We are discussing the length contribution of the 12 Ig domains now more extensively in the DNA PAINT super-resolution paper, however not in this resource paper as the 50 nm difference was not resolved with the confocal microscopy applied here to the larval muscle sarcomere.

      (2) They discuss how Sallimus and Projectin are the two Drosophila homologs of mammalian titin, however, they ignore the fact that there is more similarity between Sallimus and Projectin to muscle proteins in invertebrates. For example, in C. elegans, TTN-1 is the counterpart of Sallimus, and twitchin is the counterpart of Projectin, both in size and domain organization. The authors present definitive data to support Figure 9, their nice model for a fly larval sarcomere but fail to point out that this model likely pertains to C. elegans and other invertebrates. In Forbes et al. (2010) it was shown that TTN-1, which can be detected by western blot as ~2 MDa protein and using two polyclonal antibodies spans the entire Iband and extends into the outer edge of the A-band, very similar to what the authors here have shown, more elegantly for Sallimus. In addition, several studies have shown that twitchin (Projectin) does not extend into the M-line; the M-line is exclusively occupied by UNC-89, the homolog of Obscurin.

      We thank this reviewer for pointing out the important C. elegans literature that we have now included in this revised manuscript. We apologise for initially omitting them. They are indeed highly relevant.

      Reviewer #4 (Public Review):

      Authors report the generation and characterisation of several nanobodies for giant Drosophila sarcomeric proteins, Sallimus and Projectin the functional orthologs of titin. They describe an efficient pipeline that could potentially help in designing and producing nanobodies for other proteins. There are several advantages to using nanobodies in comparison to conventional antibodies and the authors nicely demonstrate that the generated nanobodies allow to precisely map subcellular localisation and even the protein orientation in the case of Projectin. They also show that small nanobody molecules have superior penetration and labelling efficiencies with respect to classical antibodies. Finally, the authors select one of the nanobodies to test whether it will efficiently detect native proteins in living tissue. They confirm that Sls-Nano2NeoGreen binds Sls in vivo in muscles of temporarily immobilized 3rd instar larva allowing to reveal sarcomeric Sls pattern and to demonstrate by FRAP experiments that Sls does not exchange during a short time period.

      This work is of significant value to a large audience. It provides a clear and precise pipeline for the generation of efficient nanobodies, which are invaluable tools of modern biology.

      We thank this reviewer for expressing strong support for our manuscript and appreciating its value for a large readership.

      AuthorResponse
    2. Public_Reviews 21 Nov 2022

      Reviewer #2 (Public Review):

      The data presented in this manuscript are sound but rather descriptive. The contribution - as presented - is mostly of a technical nature. The authors correctly state that anti-GFP nanobodies, while used extensively across many model organisms, have limited utility for in vivo applications when the GFP-tagged protein in question displays abnormal behavior or is non-functional. The creation of nanobodies that are uniquely specific for the protein(s) of interest is therefore a significant improvement, especially since the Sallimus and Projectin-specific reagents reported here react with PFA-fixed material. At least one of these nanobodies, when expressed in vivo, decorates the appropriate target. The source of antigens used for the construction of the nanobody library is Drosophila-derived. The extent of homology of Drosophila Sallimus and Projectin with related proteins in other species is not discussed. Whether the nanobodies reported here would be useful in other (closely related?) species, therefore, remains to be established. For those studying muscle biology in Drosophila, the nanobodies described here will be publicly available as cDNAs. Ease of production implies a readily shared and standardized resource for the field.

      Further characterization of these nanobodies by biochemical methods such as immunoblotting would be challenging, given the size of the target proteins. In view of the technical nature of this manuscript, the authors should perhaps critically discuss the distinction between bulky GFP tags versus the much smaller epitope tags and the nanobodies that recognize them, although this was covered in a recent eLife paper from the Perrimon lab. Insertion of small tags, in conjunction with nanobodies that recognize them, would be less perturbing than the much bulkier GFP tag and lend itself to genome-wide applications. Creating nanobodies uniquely specific for each protein encoded in the Drosophila genome is not realistic, and the targeted approach deployed here is obviously valuable.

      The authors apply two different approaches to characterize the newly generated Nanobodies: more or less conventional immunohistochemistry with fluorescently labeled nanobodies, and in vivo expression of nanobodies fused to the fluorescent neongreen protein. The superiority of nanobodies in terms of tissue penetration has been shown by others in a direct comparison of intact fluorescently labeled immunoglobulins versus nanobodies. The authors state that in vivo labeling with nanobody fusions "thus far was done only with nanobodies against GFP, mCherry or short epitope tags." There is no fundamental difference between these recognition events and what the authors report for their Sallimus and Projectin-specific reagents. The section that starts at line 304 is thus a little bit of a 'straw man'. There is no reason to assume that a nanobody that recognizes a muscle protein would behave differently than a nanobody that would recognize that same protein (or another) when epitope- or GFP-tagged. What might be interesting is to examine the behavior of these muscle-specific nanobodies in the course of muscle contraction/relaxation: are there conformational alterations that promote dissociation of bound nanobodies? Do different nanobodies display discrete behavior in this regard? The manuscript is silent on how muscles behave in live L3 larvae. The FRAP experiment seems to suggest that not much is happening, but the text refers to the contraction of larval sarcomeres from 8.5 µM to 4.5 µM. Does the in vivo expressed nanobody remain stably bound during this contraction/relaxation cycle? What about the other nanobodies reported in this manuscript? Since the larval motion was reduced by exposure to diethylether, have the authors considered imaging the contractive cycle in the absence of such exposure?

      Review 2
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  14. www.biorxiv.org www.biorxiv.org
    New submission 22/11/2022, 09:21:30
    2
    1. Public_Reviews 22 Nov 2022

      Author Response

      Reviewer #1 (Public Review):

      In a very interesting and technically advanced study, the authors measured the force production of curved protofilaments at depolymerizing mammalian microtubule ends using an optical trap assay that they developed previously for yeast microtubules. They found that the magnesium concentration affects this force production, which they argue based on a theoretical model is due to affecting the length of the protofilament curls, as observed previously by electron microscopy. Comparing with their previous force measurements, they conclude that mammalian microtubules produce smaller force pulses than yeast microtubules due to shorter protofilament curls. This work provides new mechanistic insight into how shrinking microtubules exert forces on cargoes such as for example kinetochores during cell division. The experiments are sophisticated and appear to be of high quality, conclusions are well supported by the data, and language is appropriate when conclusions are drawn from more indirect evidence. Given that the experimental setup differs from the previous optical trap assay (antibody plus tubulin attached to bead versus only antibody attached to bead), a control experiment could be useful with yeast microtubules using the same protocol used in the new variant of the assay, or at least a discussion regarding this issue. One open question may be whether the authors can be sure that measured forces are only due to single depolymerizing protofilaments instead of two or more protofilaments staying laterally attached for a while. How would this affect the interpretation of the data?

      This work will be of interest to cell biologists and biophysicists interested in spindle mechanics or generally in filament mechanics.

      Thank you for your careful reading of our manuscript, your kind remarks, and your favorable review.

      Reviewers #1 and #2 both mentioned a concern about potential differences between our previous setup with yeast microtubules, versus our new setup with predominantly bovine microtubules, and whether such differences might underlie the different pulse amplitudes we measured. We think this concern comes mainly from a misunderstanding of how the beads in both setups were tethered to the sides of the microtubules, and we apologize for not making this aspect clearer in our original submission.

      It is true that our new setup requires one additional step, pre-decoration of the anti-His beads with His6-tagged yeast tubulin. However, in both cases, the anti-His antibodies were kept very sparse on the beads to ensure that most beads, if they became tethered to a microtubule, were attached by a single antibody. (~30 pM beads were mixed with 30 pM of anti-His antibody, for a molar ratio of 1:1.) And even though the anti-His beads in our previous work did not undergo a separate incubation step for pre-decoration with tubulin, they undoubtedly were decorated immediately after being mixed into the microtubule growth mix, which in that case included ~1 µM of unpolymerized His6-tagged yeast tubulin dimers. Thus, the arrangement with beads tethered laterally to the sides of microtubules via single antibodies was created in both cases by essentially the same three-step process: First, beads decorated very sparsely with anti-His antibodies were bound to unpolymerized His6-tagged yeast tubulin. Second, a bead-tethered His6-tagged yeast tubulin was incorporated into the growing tip of a microtubule (which could be assembling from either yeast or bovine tubulin, depending on the experiment). Third, the tip grew past the bead to create a large extension. Because the beads in both scenarios were tethered by a single antibody to the same C-terminal tail of yeast β-tubulin, the differences in pulse amplitude cannot be explained by differences in the tethering. In our revised manuscript, we now mention explicitly in Results that the beads were tethered by single antibodies (lines 95 to 100). In Methods we significantly expanded the section about preparation of beads and how they became tethered (lines 365 to 393). [We refer here, and below, to line numbers when the document is viewed with “All Markup” shown.]

      You also raise an interesting, open question: Do protofilaments curl outward entirely independently of their lateral neighbors? Or under some conditions might they tend to stay laterally associated during the curling process, perhaps curling outward in pairs rather than as individual protofilaments? We cannot formally rule out the possibility that such lateral associations sometimes persist during protofilament curling. However, changes in lateral association seem unlikely to explain the magnesium- and species-dependent differences we measured in pulse amplitude, for several reasons: First, there is good evidence for lengthening of protofilament curls at disassembling tips (e.g., Mandelkow 1991, Tran & Salmon 1997), but we are not aware of convincing evidence for magnesium or species-dependent increases in the propensity of curling protofilaments to remain laterally associated. Second, an increase in lateral association should increase the effective flexural rigidity of the curls, but under all the conditions we examined, pulse enlargement was associated with a steepening of the amplitude-vs-force relation – i.e., with softening, not stiffening. Our model indicates that this softening can be fully explained by an increase in protofilament contour length, without any change in the intrinsic flexural rigidity of the protofilament curls.

      Reviewer #2 (Public Review):

      Microtubules are regarded as dynamic tracks for kinesin and dynein motors that generate force for moving cargoes through cells, but microtubules also act as motors themselves by generating force from outward splaying protofilaments at depolymerizing ends. Force from depolymerization has been demonstrated in vitro and is thought to contribute to chromosome movement and other contexts in cells. Although this model has been in the field for many years, key questions have remained unanswered, including the mechanism of force generation, how force generated might be regulated in cells, and how this system might be tuned across cellular contexts or organisms. The barrier is that we lack an understanding of experimental conditions that can be used to control protofilament shape and energetics. This study by Murray and colleagues makes an important advance towards overcoming that barrier.

      This study builds on previous work from the authors where they developed a system to directly measure forces generated by outward curling protofilaments at depolymerizing microtubule ends. That study showed for the first time that protofilaments act like elastic springs and related the generated force to the estimated energy contained in the microtubule lattice. Furthermore, they showed that slowing polymerization rate did not diminish force generation. That study used recombinant yeast tubulin, including a 6x histidine tag on beta tubulin that created attachment points for the bead on the microtubule lattice. The current study extends that system to show that work output is related to the length of protofilament curls.

      We are grateful for your very thoughtful and thorough review, which has helped us improve our manuscript.

      Murray and colleagues show this by manipulating curls in two ways - using bovine brain tubulin instead of yeast tubulin and altering magnesium concentration. Previous EM studies indicated that protofilaments on depolymerizing bovine microtubules have similar curvature but are shorter. The authors here use a blend of bovine brain tubulin and bead-linked recombinant yeast tubulin with the 6x histidine tag in their in vitro system and find smaller deflections of the laser-trapped bead than previously observed with pure yeast tubulin. A concern with comparing this heterogeneous bovine/yeast system to the previous work with homogeneous yeast tubulin is that density of 6x histidine-tagged tubulin subunits is likely to be different between the two systems. Also, the rate of incorporation of 6x histidine yeast tubulin into bovine microtubules in the current study may be different from the rate of incorporation into yeast microtubules in the previous study. These differences could lead to changes in the strength of bead attachment to the microtubule lattice and alter the compliance of the bead to deflection by curling protofilaments. These possibilities and lattice attachment strength are not explored in this study, raising concerns about comparing the two systems.

      Reviewers #1 and #2 both mentioned a concern about potential differences between our previous setup with yeast microtubules, versus our new setup with predominantly bovine microtubules, and whether such differences might underlie the different pulse amplitudes we measured. As detailed in our response to Reviewer #1 above, we think this concern comes mainly from a misunderstanding of how the beads in both setups were tethered to the sides of the microtubules, and we apologize for not making this aspect clearer in our original submission. For both our yeast and bovine microtubule experiments, the anti-His antibodies were kept very sparse on the beads to ensure that most beads, if they became tethered to a microtubule, were attached by a single antibody. Because the beads in both scenarios were tethered by a single antibody to the same C-terminal tail of yeast β-tubulin, the differences in pulse amplitude cannot be explained by differences in the tethering. In our revised manuscript, we now mention explicitly in Results that the beads were tethered by single antibodies (lines 95 to 100). In Methods we significantly expanded the section about preparation of beads and how they became tethered (lines 365 to 393).

      The authors go on to show that magnesium increases bead deflection and work output from the system. The use of magnesium was motivated by earlier studies which showed that increasing magnesium speeds up depolymerization and increases the lengths of protofilament curls. The use of magnesium here provides the first evidence that work output can be tuned biochemically. This is an important finding. The authors then go on to show that the effect of magnesium on bead deflection can be separated from its effect on depolymerization speed. They do this by proteolytically removing the beta tubulin tail domain, which previous studies had shown to be necessary to mediate the magnesium effect on depolymerization rate. The authors arrive at a conclusion that magnesium must promote protofilament work output by increasing their lengths. How magnesium might do this remains unanswered. The mechanistic insight from the magnesium experiments ends there, but the authors discuss possible roles for magnesium in strengthening longitudinal interactions within protofilaments or perhaps complexing with the GDP nucleotide at the exchangeable site, although that seems less likely at the concentrations in these experiments.

      The major conclusion of the study is the finding that work output from curling protofilaments is a tunable system. The examples here demonstrate tuning by tubulin composition and by divalent cations. Whether these examples relate to tuning in biological systems will be an important next question and could expand our appreciation for the versatility of depolymerizing microtubules as a motor.

      We fully agree that two very important next questions are whether work output from curling protofilaments is truly harnessed in vivo, and whether protofilament properties in vivo might be actively regulated for this purpose. Based on your recommendations, and as detailed below (under Major point 2), we have expanded our discussion of these possibilities in our revised manuscript.

      Reviewer #3 (Public Review):

      The authors used a previously established optical tweezers-based assay to measure the regulation of the working stroke of curled protofilaments of bovine microtubules by magnesium. To do so, the authors improved the assay by attaching bovine microtubules to trapping beads through an incorporated tagged yeast tubulin.

      The assay is state-of-the-art and provides a direct measurement of the stroke size of protofilaments and its dependence on magnesium.

      The authors have achieved all their goals and the manuscript is well written.

      The reported findings will be of high interest for the cell biology community.

      Thank you for reading and evaluating our manuscript. We are grateful for your positive comments.

      AuthorResponse
    2. Public_Reviews 22 Nov 2022

      Reviewer #2 (Public Review):

      Microtubules are regarded as dynamic tracks for kinesin and dynein motors that generate force for moving cargoes through cells, but microtubules also act as motors themselves by generating force from outward splaying protofilaments at depolymerizing ends. Force from depolymerization has been demonstrated in vitro and is thought to contribute to chromosome movement and other contexts in cells. Although this model has been in the field for many years, key questions have remained unanswered, including the mechanism of force generation, how force generated might be regulated in cells, and how this system might be tuned across cellular contexts or organisms. The barrier is that we lack an understanding of experimental conditions that can be used to control protofilament shape and energetics. This study by Murray and colleagues makes an important advance towards overcoming that barrier.

      This study builds on previous work from the authors where they developed a system to directly measure forces generated by outward curling protofilaments at depolymerizing microtubule ends. That study showed for the first time that protofilaments act like elastic springs and related the generated force to the estimated energy contained in the microtubule lattice. Furthermore, they showed that slowing polymerization rate did not diminish force generation. That study used recombinant yeast tubulin, including a 6x histidine tag on beta tubulin that created attachment points for the bead on the microtubule lattice. The current study extends that system to show that work output is related to the length of protofilament curls.

      Murray and colleagues show this by manipulating curls in two ways - using bovine brain tubulin instead of yeast tubulin and altering magnesium concentration. Previous EM studies indicated that protofilaments on depolymerizing bovine microtubules have similar curvature but are shorter. The authors here use a blend of bovine brain tubulin and bead-linked recombinant yeast tubulin with the 6x histidine tag in their in vitro system and find smaller deflections of the laser-trapped bead than previously observed with pure yeast tubulin. A concern with comparing this heterogeneous bovine/yeast system to the previous work with homogeneous yeast tubulin is that density of 6x histidine-tagged tubulin subunits is likely to be different between the two systems. Also, the rate of incorporation of 6x histidine yeast tubulin into bovine microtubules in the current study may be different from the rate of incorporation into yeast microtubules in the previous study. These differences could lead to changes in the strength of bead attachment to the microtubule lattice and alter the compliance of the bead to deflection by curling protofilaments. These possibilities and lattice attachment strength are not explored in this study, raising concerns about comparing the two systems.

      The authors go on to show that magnesium increases bead deflection and work output from the system. The use of magnesium was motivated by earlier studies which showed that increasing magnesium speeds up depolymerization and increases the lengths of protofilament curls. The use of magnesium here provides the first evidence that work output can be tuned biochemically. This is an important finding. The authors then go on to show that the effect of magnesium on bead deflection can be separated from its effect on depolymerization speed. They do this by proteolytically removing the beta tubulin tail domain, which previous studies had shown to be necessary to mediate the magnesium effect on depolymerization rate. The authors arrive at a conclusion that magnesium must promote protofilament work output by increasing their lengths. How magnesium might do this remains unanswered. The mechanistic insight from the magnesium experiments ends there, but the authors discuss possible roles for magnesium in strengthening longitudinal interactions within protofilaments or perhaps complexing with the GDP nucleotide at the exchangeable site, although that seems less likely at the concentrations in these experiments.

      The major conclusion of the study is the finding that work output from curling protofilaments is a tunable system. The examples here demonstrate tuning by tubulin composition and by divalent cations. Whether these examples relate to tuning in biological systems will be an important next question and could expand our appreciation for the versatility of depolymerizing microtubules as a motor.

      Review 2
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  15. canvas.xavier.edu canvas.xavier.edu
    Topic: Do the Math: Which One Doesn't Belong?
    1
    1. RyanMurray 21 Nov 2022

      As I wrote in my feedback on the M4 Discussion in IOCD, "I agree that this prompt has a 'low floor' and 'high ceiling.' The prompt emphasizes observation, creative thinking, and multiple perspectives. You even state, 'There are no wrong answers.' I like how you require students to explain their reasoning when they answer the ... question about which tag doesn't belong."

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  16. engineering.appfolio.com engineering.appfolio.com
    Ruby's Main Object Does What? — Engineering Blog
    1
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    engineering.appfolio.com/appfolio-engineering/2018/8/9/rubys-main-object-does-what
  17. www.embopress.org www.embopress.org
    Environmental signal integration by a modular AND gate
    1
    1. pbk1 19 Nov 2022
      green fluorescent protein containing a degradation tag (GFPmut3_LAA)

      BLAST matches the GFPmut3 sequence in rGFP and fGFP to this - so the Voigt lab authors cloned this with the partial degron tag without the LAA amino acids for this paper :

      Yang, Lei, et al. "Permanent genetic memory with> 1-byte capacity." Nature methods 11.12 (2014): 1261-1266.

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  18. www.biorxiv.org www.biorxiv.org
    New submission 17/11/2022, 16:38:24
    2
    1. Public_Reviews 17 Nov 2022

      Author Response

      Reviewer #2 (Public Review):

      “To describe LLPS or to distinguish between polymer-polymer phase separation and LLPS, recent studies have used single particle tracking, a technique allowing to follow the dynamics of individual proteins in living cells (https://doi.org/10.7554/eLife.60577; https://doi.org/10.7554/eLife.69181; https://doi.org/10.7554/eLife.47098). The authors should mention that such an approach can be a good alternative to avoid the artefact of fixation. Using techniques such as single particle tracking or FCS, it is possible to estimate the effective diffusion coefficient of protein-living cells. When a liquid phase separation is formed, it is also possible to estimate the diffusion coefficient of the protein of interest (POI) inside versus outside of the LLPS.”

      We thank the reviewer for their insight and fully agree that live-cell techniques like SPT and FCS are valuable for investigating LLPS while avoiding fixation artifacts. We have added discussion emphasizing this fact and incorporated the citations recommended by the reviewer in Paragraph 1 on Page 15: “Live imaging techniques that allow estimation of protein diffusion coefficients within specific cellular compartments, e.g., SPT (Hansen et al., 2018 and Heckert et al., 2022) and fluorescence correlation spectroscopy (Lanzanò et al., 2017), can be useful alternative approaches for diagnosing LLPS in vivo without the potential artifact of fixation, as diffusion dynamics are recently shown to be affected by LLPS (Heltberg et al., 2021; McSwiggen et al., 2019a; Miné-Hattab et al., 2021; Chong et al., 2022; and Ladouceur et al., 2020).”

      “The authors say that less dynamic interactions are better captured by PFA fixation. In the simulation part, would it be possible to predict from the diffusion coefficients of the POI inside a condensate the effect of the PFA fixation? […] In the simulation part, they could try to incorporate the diffusion coefficient of the protein of interest and see if it is possible to predict the effect of fixation as a function of the diffusion coefficient.”

      We thank the reviewer for pointing out the absence of this critical piece that connects our experimental observations to our kinetic model. Our model considers association/dissociation rates rather than diffusion coefficients to describe interaction dynamics, but the reviewers’ point is still very insightful and important. As described in Response 2, we compared two proteins: Halo-TAF15(IDR), which is poorly preserved by fixation, and TAF15(IDR)-Halo-FTH1, which is well preserved by fixation. We used SPT to measure the dissociation rates of Halo-TAF15(IDR) and TAF15(IDR)-Halo-FTH1 and showed that the dissociation rate of Halo-TAF15(IDR) from its puncta is much faster than that of TAF15(IDR)-Halo-FTH1, demonstrating more stable homotypic interactions of the latter than the former. The observation that TAF15(IDR)-Halo-FTH1 has less dynamic interactions and is better preserved by fixation compared to Halo-TAF15(IDR) agrees with our model’s prediction that less dynamic interactions are better captured by fixation. Please see Response 2 for more details. Our new data and discussion have been added to the revised manuscript in Paragraph 3 on Page 13 and in Figure 3B, Figure 3E, Figure 6, and Video 2.

      “Finally, the authors propose that in the future, it will be important to design novel fixatives with significantly faster cross-linking rates than biomolecular interactions to eliminate fixation artifacts in the cell. It would be even more interesting if the authors could propose some ideas of potential novel fixatives. Did they test several concentrations of PFA, for example? Did they test different times of PFA incubation? Did they test cryofixation and do they know what would be their effect on LLPS? Do they have novel fixatives in mind? […] To strengthen the manuscript, the authors should try more protocols of fixation.”

      We thank the reviewer for these good questions. As described in Response 1, we have done additional quantification of the change of LLPS appearance in cells upon treatment of 0% PFA (only PBS buffer), 1% PFA, 2% PFA, and 8% PFA as well as 4% PFA supplemented by 0.2% GA. We saw statistically significant changes in the LLPS-describing parameters upon all the PFA and PFA/GA treatments except the 0% PFA control. To examine how fixation artifacts depend on the time of PFA incubation, we acquired a time-lapse movie of a cell overexpressing EGFP-FUS(IDR) immediately after 4% PFA treatment and quantified the number of puncta over time (Video 1). We showed that fixation is complete (the number of puncta becomes constant) by roughly 100 seconds (Figure 1 – figure supplement 2). Our new data also justified our choice of a 10-minute PFA incubation time for analyzing fixation-induced change of LLPS appearance in the rest of the paper. Please see Response 1 for more details. Our new data and discussion have been added to the revised manuscript in Paragraph 3 on Page 3 and in Figure 1 - figure supplement 2 (time dependence of fixation artifacts), Figure 1 - figure supplement 3 (fixation artifact at various PFA concentrations), and Figure 1 - figure supplement 4 (fixation artifact upon treatment of 4% PFA supplemented with 0.2% GA).

      We agree that testing more cell fixation protocols such as cryofixation on LLPS appearance would be interesting. However, given the complexity of novel fixation protocols like cryofixation and highly specialized equipment and reagents they require, testing widely how different fixation methods might change LLPS appearance would be a tremendous amount of work that is enough to fill a separate paper. These experiments would be much more appropriate for a separate study in the future.

      Reviewer #3 (Public Review):

      “Understanding whether/how fixation methods affect the detection of biomolecular condensates is of broad interest given the importance of LLPS in regulating different aspects of cell biology. However, in this manuscript, the authors use only paraformaldehyde as a fixation method and study only fluorescently-labelled IDR proteins. The work would benefit from a comparison between living cells and cells fixed with other fixation methods.”

      We appreciate the reviewer for this suggestion and agree that more fixation protocols should be investigated. As described in Response 1 and Response 18, besides examining PFA fixation, we have quantified how fixation using 4% PFA supplemented by 0.2% GA changes LLPS appearance in cells. We saw statistically significant changes in all the LLPS-describing parameters upon PFA/GA treatments. Please see Response 1 and Response 18 for details. Our new data and discussion have been added to the revised manuscript in Paragraph 3 on Page 3 and in Figure 1 - figure supplement 4.

      “In addition, it would be useful to test the impact of these fixation methods on the detection of endogenous proteins or IDR proteins without fluorescent tag.”

      We appreciate the reviewer for this suggestion and have now investigated an endogenous IDR-containing protein in the revised manuscript. Specifically, we quantified the effect of 4% PFA fixation on endogenously expressed EWS::FLI1 in an Ewing sarcoma cell line A673, which is an oncogenic fusion transcription factor that causes Ewing sarcoma (Grünewald et al., 2018) and known to form local, high-concentration hubs at target genes associated with GGAA microsatellites (Chong et al., 2018). We previously Halo-tagged endogenous EWS::FLI1 in A673 cells using CRISPR/Cas9-mediated genome editing (Chong et al., 2018). Here, we quantified the effect of PFA fixation on endogenous EWS::FLI1 puncta in this knock-in cell line and found no significant difference in the distribution of EWS::FLI1 upon fixation. This result suggests that PFA fixation does not change the intracellular distribution of all proteins. Our new data and discussion have been added to the revised manuscript in Paragraph 1 on Page 8 and in Figure 3C.

      Unfortunately, testing fixation artifacts of IDR-containing proteins without a fluorescent tag has been infeasible as we rely on fluorescence from a tag on the protein of interest to quantitatively compare LLPS appearance in live and fixed cells. Although we have considered using non-fluorescent methods, e.g., phase contrast microscopy, to visualize putative LLPS in cells, its lack of specificity in imaging proteins or cellular structures makes the type of quantification we do for fixation artifact characterization inaccessible.

      AuthorResponse
    2. Public_Reviews 17 Nov 2022

      Reviewer #3 (Public Review):

      The authors compare the detection of biomolecular condensates in living cells overexpressing fluorescently tagged IDR proteins and upon fixation with paraformaldehyde (PFA). Given that they observe differences in the number and size of the condensates in the fixed versus living cells the authors conclude that the fixation method can introduce an artifact in the visualization of these condensates. Next, through kinetic modeling simulations, the authors propose a model in which the extent of the artifact introduced by PFA fixation correlates with the strength of the protein-protein interaction: artifacts are lower when the protein‐protein interactions are stable and less dynamic compared with the overall fixation rate. Based on their comparative analysis of PFA fixation and the kinetic modeling the authors strongly recommend caution in the interpretation of data obtained in PFA-fixed cells and suggest that parallel studies with living cells should be performed.

      Understanding whether/how fixation methods affect the detection of biomolecular condensates is of broad interest given the importance of LLPS in regulating different aspects of cell biology. However, in this manuscript, the authors use only paraformaldehyde as a fixation method and study only fluorescently-labelled IDR proteins. The work would benefit from a comparison between living cells and cells fixed with other fixation methods; in addition, it would be useful to test the impact of these fixation methods on the detection of endogenous proteins or IDR proteins without fluorescent tag.

      Review 3
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  19. github.com github.com
    cognitect-labs/transcriptor: Convert REPL interactions into example-based tests.
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  20. Local file Local file
    Supplementary figures
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  21. www.biorxiv.org www.biorxiv.org
    New submission 15/11/2022, 08:49:43
    2
    1. Public_Reviews 15 Nov 2022

      Author Response

      Reviewer #3 (Public Review):

      The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.

      Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their collaborators, as well as in many other laboratories:

      (i) Different organelles have different transport behaviors regarding speed, the ratio of anterograde to retrograde moving organelles, etc.

      (ii) Organelles move in different ways when they pass specific anatomical landmarks in the axons, such as presynaptic terminals.

      (iii) Activity of a neuron (here measured by calcium imaging) can impact the measured transport parameters, albeit in a subtle and mechanistically not well-defined manner. The chosen experimental design precludes a more detailed analysis, for example of the precise movement behavior (such as defining the exact pausing/movement behavior of organelles, which would require higher imaging speeds) or of a correlation of different organellar behavior at synaptic sites or during activity (which would require three-channel simultaneous imaging of two organelle classes plus a synaptic or activity marker).

      In summary, this publication uses sophisticated in vivo labeling and imaging methods to corroborate and complement previous observations on how different axonal organelles move, and what influences their trafficking.

      We thank the reviewer for the time dedicated to our manuscript. We are thankful for the critical and specific comments, which allowed us to further improve our manuscript. We agree that it would have been beneficial to have higher frame rates and there instead of two imaging channels. However, this would have further added technical complexity to an already complex experimental setup resolving fluorescent puncta with sizes below the resolution limit. And we are convinced that all our main conclusions are justified based on the imaging settings in the current data sets.

      AuthorResponse
    2. Public_Reviews 15 Nov 2022

      Reviewer #3 (Public Review):

      The authors use two-photon imaging to visualize various axonal organelle populations that they have virally labeled with fluorescent proteins, including DCVs and late endosomes/ lysosomes. The latter topic is a bit contentious, as the authors use two labels that tag potentially overlapping and not highly specific markers so that the nature of the tagged organelle populations remains unclear. Notably, the authors also have previously published a detailed account of how DCVs traffic in vivo, so the novelty is mostly in comparing the behavior of different organelles and the potential influence of activity.

      Overall, the reported results mostly corroborate the expectations from previous in vitro and in vivo work on these organelles and other cargoes, performed by the authors and their collaborators, as well as in many other laboratories:<br /> (i) Different organelles have different transport behaviors regarding speed, the ratio of anterograde to retrograde moving organelles, etc.<br /> (ii) Organelles move in different ways when they pass specific anatomical landmarks in the axons, such as presynaptic terminals.<br /> (iii) Activity of a neuron (here measured by calcium imaging) can impact the measured transport parameters, albeit in a subtle and mechanistically not well-defined manner. The chosen experimental design precludes a more detailed analysis, for example of the precise movement behavior (such as defining the exact pausing/movement behavior of organelles, which would require higher imaging speeds) or of a correlation of different organellar behavior at synaptic sites or during activity (which would require three-channel simultaneous imaging of two organelle classes plus a synaptic or activity marker).

      In summary, this publication uses sophisticated in vivo labeling and imaging methods to corroborate and complement previous observations on how different axonal organelles move, and what influences their trafficking.

      Review 3
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  22. github.com github.com
    nic-dgl103-f22/assignment-f-cvs1-Sri01729: assignment-f-cvs1-Sri01729 created by GitHub Classroom
    3
    2. AroraAran 08 Nov 2022
      <ul> <li><h3>Latest News</h3></li> <li>Octtober 1:<br/>Lorem ipsum dolor sit</li><br /> <li>September 15th:<br/>Lorem ipsum dolor sit</li><br /> <li>September 10th:<br/>Lorem ipsum dolor sit</li> </ul>

      i believe there should be more space between the heading tag and the content use after it. and the text size need to bigger for the content like month names, as i see in reference image.

    3. AroraAran 08 Nov 2022
      <li>Octtober 1:<br/>Lorem ipsum dolor sit</li><br />

      its not look same as image reference its better if you use heading tag to make month name as heading.

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  23. view.connect.americanpublicmedia.org view.connect.americanpublicmedia.org
    Untitled document
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    1. chrisaldrich 12 Nov 2022
      Most of the tourist and sporting infrastructure had to be built at enormous expense — estimates range anywhere from$200 billion to $300 billion. Yet the return on investment for huge events like this is rarely positive. The Olympics are infamously pricey  to put on, and the economic benefits for residents of the host city are questionable.  So, with the big price tag and not much to show in return, why do countries like Qatar, Russia and Brazil offer up billions of dollars to host global sporting events? According to Victor Matheson, a professor of economics at the College of the Holy Cross and a former Major League Soccer referee, they may be seeking to burnish their reputations through international media coverage.   “If you’re putting any sort of significant money into infrastructure like Qatar obviously is doing, there’s just no way you can make that back on ticket sales, on media rights, [or] on the amount of money you make from tourists coming to visit your country,” Matheson said in an interview with Marketplace’s David Brancaccio. “So obviously, you’re hoping for some sort of long-run benefits, some sort of legacy, and often that is an improvement in your reputation, either as a tourist destination or as a world player in some ways.” 

      Alternate thesis for why countries and cities vie to host money-losing events like the World Cup and the Olympics: grift.

      With the necessary need for building infrastructure, there's easy and ample opportunity for cooking the books and pushing cash flow into the pockets of contractors and political figures as well as into the pockets of the governing bodies and their officials.

      Cross reference FIFA bribery

      Some of the money may go into the local economy and workers which is good, but who's really benefitting here? Where is the money going? Who is footing the loss? It can't all be written off to goodwill.

      FIFA World Cup economics grift goodwill Olympics Russia Brazil Qatar
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  24. github.com github.com
    assignment-e-cvs1-yonishi1/index.html at main · nic-dgl103-f22/assignment-e-cvs1-yonishi1
    1
    1. Shyam___ 12 Nov 2022
      <div class="evaluation"> <a id="communicate-2"></a> <h3><em>Problem: </em>These webpages don’t have appropriate headings. For example, there is no h1 in “Home” webpage.</h3> <figure> <img src="images/Home.PNG" alt="Home page of Ascent Physiotherapy"> <figcaption>There is no h1 tag. </figcaption> </figure> <h3><em>Solution: </em>Put appropriate headings on each page.</h3> </div> <div class="evaluation"> <a id="communicate-3"></a> <h3><em>Problem: </em><br> There are some difficult words and long sentences in some webpages.</h3> <p>In “Products & Services” page, although there are links to explain the difficult words, but not all words have links.</p> <p>In “Our team” page, some long sentences make users give up reading.</p> <figure> <img src="images/Products&Services.PNG" alt="Difficult_words in Ascent Physiotherapy"> <figcaption>For example, TMJ is an abbreviation. </figcaption> </figure> <figure> <img src="images/Our_team.PNG" alt="Long sentences in Our team page"> <figcaption>For example, TMJ is an abbreviation. </figcaption> </figure>

      looks and nice and you have mentioned nicely

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  25. github.com github.com
    nic-dgl103-f22/assignment-e-cvs1-Jan-elle-Chan: assignment-e-cvs1-Jan-elle-Chan created by GitHub Classroom
    11
    1. Shyam___ 12 Nov 2022
      <article class="card">

      most of the time I use the "Div" tag instead of "article". I feel it gets easy to work with "Divs". "article " is not the issue still

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  26. github.com github.com
    nic-dgl103-f22/assignment-e-cvs1-love0201: assignment-e-cvs1-love0201 created by GitHub Classroom
    2
    1. Sri01729 12 Nov 2022
      <article class="box-1"> <div> <h1>Write to Communicate</h1> <p>While analyzing this site the first thing I observed was how messy the content was. The content is not grouped in the blocks. It is hard to find headings as the color does not have enough contrast. The language used is not descriptive and accurate enough. For example, in the main headline it just says “Trending???” which doesn’t make any sense. It should be like “Looking for Trending fits?” The size should be a bit large and there should be a button for call-to-action. </p> </div> <img src="images/Screenshot (73).png"> </article>

      you can use the "main" tag here for the web content. It'll be good to have both header and footer too.

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  27. hypothes.is hypothes.is
    Hypothesis
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  28. hypothes.is hypothes.is
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  29. www.biorxiv.org www.biorxiv.org
    New submission 11/11/2022, 11:00:59
    2
    1. Public_Reviews 11 Nov 2022

      Author Response

      Reviewer #2 (Public Review):

      Grasses develop morphologically unique stomata for efficient gas exchange. A key feature of stomata is the subsidiary cell (SC), which laterally flanks the guard cell (GC). Although it has been shown that the lateral SC contributes to rapid stomatal opening and closing, little is known about how the SC is generated from the subsidiary mother cell (SMC) and how the SMC acquires its intracellular polarity. The authors identified BdPOLAR as a polarity factor that forms a polarity domain in the SMC in a BdPAN1-dependent manner. They concluded that BdPAN1 and BdPOLAR exhibit mutually exclusive localization patterns within SMCs and that formative SC division requires both. Further mutant analysis showed that BdPAN1 and BdPOLAR act in SMC nuclear migration and the proper placement of the cortical division site marker BdTANGLED1, respectively. This study reveals a unique developmental process of grass stomata, where two opposing polarity factors form domains in the SMC and ensure asymmetric cell division and SC generation.

      The findings of this study, if further validated, are novel and interesting. However, I feel that the data presented in the current manuscript do not fully support some crucial conclusions. The lack of dual-color images is the weakest point of this study. If it is technically impossible to add them, alternative analyses are needed to validate the main conclusions.

      1) Is BdPOLAR-mVenus functional? Although the authors interpret that weak BdPOLAR-mVenus expression partially rescued the bdpolar mutant phenotype in Fig. S4D, the localization pattern visualized by BdPOLAR-mVenus may not be completely reliable with this partial rescue activity.

      This is indeed a valid point. The partial complementation of weakly expressing translational reporters (Figure 3–figure supplement 1D) and the weak effect of BdPOLAR-mVenus overexpression lines (Figure 3–figure supplement 1J) at least suggest partial functionality which is strongly dependent on dosage. Yet the localization pattern and the temporal dynamics might indeed not fully reflect the spatiotemporal dynamics of the endogenous BdPOLAR. This criticism is, however, true for any transgenic reporter line–even when fully complementing–as the requirement for dosage, stability, and turnover likely varies strongly between different protein classes and functions.

      Nonetheless, we have added a sentence on p. 7, which mentions this potential caveat.

      2) Regardless of the functionality of the tagged protein, the authors need to provide more information on their localization. For example, is there a difference in polarity pattern depending on expression level? Does overexpressed BdPOLAR-mVenus invade the BdPAN1 zone? In such cases, might the loss of BdPOLAR polarity in the bdpan1 mutant be a side effect of overexpression, not PAN1 exclusion? Does BdPOLAR expression (no tag) show a dose-dependent effect, similar to the mVenus-tagged protein?

      The difference in polarity patterns in bdpan1 mutants and wild-type does not depend on expression level. BdPOLAR-mVenus was crossed into bdpan1 and mutant and wild-type siblings in the F2 generation were analyzed. This means that the data presented in Fig. 3E and F show exactly the same transgene insertion line in wt and bdpan1 and were imaged with the same setting for comparability. Therefore, the difference in localization is not due to different expression levels but indeed reflects a PAN1-dependent effect.

      To address if BdPOLAR without a tag is also sensitive to dosage, we have generated an untagged complementation line that includes the untagged, genomic locus of BdPOLAR including promoter (-3.1kb) and terminator (+1.1kb). Yet, even though this construct is much better at rescuing the mutant, we still see remaining defects in T0 lines (Figure 3–figure supplement 1K) suggesting that even without a tag we cannot fully recapitulate wild-type functionality. Yet, to actually measure protein levels of untagged BdPOLAR, we would need to raise an antibody against BdPOLAR, which we think is clearly out of the scope of this study.

      3) A major conclusion of this study was that the polarity domains of BdPOLAR and BdPAN1 are mutually exclusive. However, not all the cells in the figures were consistent with this statement. For example, the BdPOLAR signals at the GMC/SMC interphase appear to match BdPAN1 localization (compare 0:03 s in Video 1 and 0:20 s in Video 2 [top cell]). The 3D rendered image in Fig. 2F shows that BdPOLAR is excluded near the GMC on the front side of the SMC, where BdPAN1 is not localized. Some cells did not exhibit polarization (Fig. 3A, bottom left; Fig. 3E, bottom left). The most convincing data are the dual-color images of these two proteins. Otherwise, a sophisticated image analysis is required to support this conclusion.

      We agree that dual-color image analysis would have provided the most convincing data. As mentioned in our answers to the reviewing editor and reviewer 1, we have generated a dual marker line (BdPAN1p:BdPAN1-CFP; BdPOLARp:BdPOLAR-mCitrine), yet the BdPAN1-CFP signal (compared to mCitrine signal) was too weak to visualize the proximal BdPAN1 domain.

      This issue was also raised by reviewer 1 and deemed an essential revision. To determine how BdPOLAR and BdPAN1 relate spatially to each other, we have added data in Figure 2E where we manually traced mature SMC outlines to determine BdPOLAR-mVenus and BdPAN1-mCitrine occupancy along the SMC’s circumference. This confirmed that the polarization is indeed opposite yet not perfectly reciprocal (see details above, Essential Revisions #1).

      Finally, we realized that the 3D image renderings were more confusing than helpful and we removed them from the revised version.

      4) Another central conclusion was that BdPOLAR was excluded at the future SC division site, marked with BdTANGLED1. However, these data are also not very convincing, as such specific exclusion cannot be seen in some figure panels (e.g., Fig. 3A, bottom left; Fig. 3E, all three cells on the left). If dual-color imaging is not feasible, a quantitative image analysis is needed to support this conclusion.

      As for point 3, this was also criticized by reviewer 1 and deemed an essential revision by the reviewing editor.

      To determine whether the absence of BdPOLAR signal and the presence of BdTAN1 signal colocalize, we again manually traced mature SMC outlines to determine BdPOLAR-mVenus and BdTAN1-mCitrine occupancy along the SMC’s circumference. We plotted the relative average fluorescence intensity in Figure 4G-I nicely showing that BdTAN1 indeed resides in the BdPOLAR gaps above and below the GMC (again, details above, Essential Revisions #2).

      5) I could not find detailed imaging conditions and data processing methods. Are Figs. 2B and 2E max-projection or single-plane images? If they are single-plane images, which planes of the SMC are observed? In addition, how were Figs. 2C and 2F rendered? (e.g., number of images, distance intervals, processing procedures). This information is important for data interpretations.

      We agree that we might not have provided sufficient imaging condition details and have added more details regarding image acquisition in the method part (p. 20). We always use a consistent depth and show the midplane of SMCs. As mentioned above, we removed Figs. 2C and 2F and the supplemental movies as these data did not seem to be helpful.

      6) [Minor point] The authors should clearly describe where BdPAN1 is expressed and localized. Is it expressed in the GMC and localized at the GMC/SMC interface? Alternatively, is it expressed and localized in the SMC?

      BdPAN1 is expressed throughout the epidermis but starts to strongly accumulate at the GMC/SMC interface. According to the literature (Cartwright et al 2009 with immunostainings against ZmPAN1 and Sutimantanapi et al. 2014 with PAN1 and PAN2 reporter) and our own observations (Fig. S3), this accumulation occurs in the SMC rather than in the GMC. In Fig. S3A, third panel, second GMC from the top, for example, one can see that the early PAN1 polarity domain expands beyond the GMC/SMC interface suggesting that it is indeed forming in SMCs rather than in GMCs. We have specified this in the text more clearly now (p. 5).

      AuthorResponse
    2. Public_Reviews 11 Nov 2022

      Reviewer #2 (Public Review):

      Grasses develop morphologically unique stomata for efficient gas exchange. A key feature of stomata is the subsidiary cell (SC), which laterally flanks the guard cell (GC). Although it has been shown that the lateral SC contributes to rapid stomatal opening and closing, little is known about how the SC is generated from the subsidiary mother cell (SMC) and how the SMC acquires its intracellular polarity. The authors identified BdPOLAR as a polarity factor that forms a polarity domain in the SMC in a BdPAN1-dependent manner. They concluded that BdPAN1 and BdPOLAR exhibit mutually exclusive localization patterns within SMCs and that formative SC division requires both. Further mutant analysis showed that BdPAN1 and BdPOLAR act in SMC nuclear migration and the proper placement of the cortical division site marker BdTANGLED1, respectively. This study reveals a unique developmental process of grass stomata, where two opposing polarity factors form domains in the SMC and ensure asymmetric cell division and SC generation.

      The findings of this study, if further validated, are novel and interesting. However, I feel that the data presented in the current manuscript do not fully support some crucial conclusions. The lack of dual-color images is the weakest point of this study. If it is technically impossible to add them, alternative analyses are needed to validate the main conclusions.

      1. Is BdPOLAR-mVenus functional? Although the authors interpret that weak BdPOLAR-mVenus expression partially rescued the bdpolar mutant phenotype in Fig. S4D, the localization pattern visualized by BdPOLAR-mVenus may not be completely reliable with this partial rescue activity.<br /> 2. Regardless of the functionality of the tagged protein, the authors need to provide more information on their localization. For example, is there a difference in polarity pattern depending on expression level? Does overexpressed BdPOLAR-mVenus invade the BdPAN1 zone? In such cases, might the loss of BdPOLAR polarity in the bdpan1 mutant be a side effect of overexpression, not PAN1 exclusion? Does BdPOLAR expression (no tag) show a dose-dependent effect, similar to the mVenus-tagged protein?<br /> 3. A major conclusion of this study was that the polarity domains of BdPOLAR and BdPAN1 are mutually exclusive. However, not all the cells in the figures were consistent with this statement. For example, the BdPOLAR signals at the GMC/SMC interphase appear to match BdPAN1 localization (compare 0:03 s in Video 1 and 0:20 s in Video 2 [top cell]). The 3D rendered image in Fig. 2F shows that BdPOLAR is excluded near the GMC on the front side of the SMC, where BdPAN1 is not localized. Some cells did not exhibit polarization (Fig. 3A, bottom left; Fig. 3E, bottom left). The most convincing data are the dual-color images of these two proteins. Otherwise, a sophisticated image analysis is required to support this conclusion.<br /> 4. Another central conclusion was that BdPOLAR was excluded at the future SC division site, marked with BdTANGLED1. However, these data are also not very convincing, as such specific exclusion cannot be seen in some figure panels (e.g., Fig. 3A, bottom left; Fig. 3E, all three cells on the left). If dual-color imaging is not feasible, a quantitative image analysis is needed to support this conclusion.<br /> 5. I could not find detailed imaging conditions and data processing methods. Are Figs. 2B and 2E max-projection or single-plane images? If they are single-plane images, which planes of the SMC are observed? In addition, how were Figs. 2C and 2F rendered? (e.g., number of images, distance intervals, processing procedures). This information is important for data interpretations.<br /> 6. [Minor point] The authors should clearly describe where BdPAN1 is expressed and localized. Is it expressed in the GMC and localized at the GMC/SMC interface? Alternatively, is it expressed and localized in the SMC?

      Review 2
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  30. tagteam.harvard.edu tagteam.harvard.edu
    TagTeam :: Tag Library for oa.wikidata_items
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  31. hypothes.is hypothes.is
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  32. hypothes.is hypothes.is
    Hypothesis
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  33. www.biorxiv.org www.biorxiv.org
    lncRNA H19/Let7b/EZH2 axis regulates somatic cell senescence
    1
    1. Public_Reviews 10 Nov 2022

      Reviewer #2 (Public Review):

      The study aims to characterize the role of lncRNA H19 in senescence and proposes a mechanism involving CTCF and the activation of p53. The authors suggest that H19 loss induces let7b-mediated repression of EZH2, which is a critical component in the regulation of senescence-associated genes. Additionally, the authors state that H19 is required for inhibition of senescence by the mTOR inhibitor rapamycin.

      The experiments appear to be performed to a high standard, and the individual observations, and conclusions about the importance of the individual players in senescence appear solid. For example, the authors convincingly show that H19 decreases in expression in aged cells/tissues and that its knockdown leads to entry into senescence. These results are consistent with recent studies in other systems (e.g., ref 38). Also, the knockdown of CTCF convincingly leads to senescence. However, these observations are largely not very surprising/novel. The premise of the manuscript is a connection between these components into a particular "axis" that regulates entry into senescence. This connection between the different regulators studied (H19, CTCF, EZH2, p53), and in particular, their specificity, which is key to the proposed "axis" remains insufficiently supported, and many of the results, unfortunately, appear to be over-interpreted.

      Major comments

      1. In Figure 1, the authors claim that H19 levels are reduced during aging in vitro and in vivo and that H19 levels are maintained by rapamycin treatment. To state the connection between H19 and rapamycin and its relation to aging, there is a need to show what happens in "young" cells treated with rapamycin.

      Furthermore, the authors state that H19 "is essential for the inhibitory effect of rapamycin on cellular senescence". There doesn't appear to be sufficient evidence to support such a claim; additional data emphasizing the direct connection between H19 and rapamycin is needed - e.g., show that in H19-null cells rapamycin does not affect senescence.

      2. CTCF is a general regulator involved in various cellular processes and supporting progression through the cell cycle; therefore, its perturbation can lead to global effects on cell health that are not necessarily related to H19. The data shown in figure 2 is insufficient to indicate a direct correlation between CTCF and H19. This will require showing that mutating specifically the CTCF binding sites near H19 affects senescence.

      The same applies to the connection between H19 and let-7b shown in Figure 5. It is not very surprising that let-7b, a general antagonist of proliferation, positively regulates senescence. Here as well, the direct connection to H19 is weak. Can the authors rescue the cells that enter senescence following H19 depletion by H19 expression? If so - is this rescue capacity lost when let-7 sites are mutated? Is it possible to rescue by expressing an artificial let-7 sponge instead of H19? Otherwise, let-7b could very well be another factor related to senescence and/or regulated, but not the main mediator of the effects of H19, or part of an axis that includes H19, as proposed in the manuscript.

      3. In figures 2d,3f,5i/j the authors present only representative tracks and regions from CUT&Tag-experiments, and its not clear to what extent these changes are significant when considering genome-wide data, replicates etc., and so these data are uninterpretable. This is important, as these panels are used as evidence for specific connections between members of the axis. The authors should provide a statistical test for all the regions in the genome, based on replicates, and show that these changes are significant to use these data to support their model. Otherwise, the specific connection between CTCF and H19 remains weak, and the specific change in p53 regulation of CTCF in the context of senescence is not convincing. In any case, the number of replicates and the QC of the data should be presented, and the data should be made available to the reviewers.

      4. The authors state in the Discussion that the mechanism that lead to decreased H19 expression as part of the senescence program consists of two phases: an acute response driven by p53 activation and a prolonged response dictated by the loss of CTCF. There doesn't appear to be enough evidence to support this claim, as the individual experiments don't measure any such bi-phasic phenomena.

      Review 2
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  34. betasite.razorpay.com betasite.razorpay.com
    Eligibility Check API
    1
    1. vinita.jagannathan 10 Nov 2022
      { "inquiry": "affordability", // new "amount": 100000, // mandatory "currency": "INR", // mandatory "customer": { "id": "cust_JbRkXMROZUMCVq", "contact": "+919000090000", // mandatory "alternate_contact": "9900099000", // new "imei": "6234672537253752735", // new "ip": "105.106.107.108", // new "referrer": "https://merchansite.com/example/paybill", // new "user_agent": "Mozilla/5.0", // new "addresses": [ // new { "name": "Gaurav Kumar", "line1": "SJR Cyber Laskar", "line2": "Hosur Rd", "landmark": "Adugodi", "zipcode": "560030", "city": "Bangalore", "state": "Karnataka", "contact": "9000090000", "tag": "office", "type": "shipping" }, { "name": "Gaurav Kumar", "line1": "Arena Building", "line2": "Hosur Rd", "landmark": "Adugodi", "zipcode": "560030", "city": "Bangalore", "state": "Karnataka", "contact": "9000090000", "tag": "home", "type": "billing" }, { "name": "Gaurav Kumar", "line1": "SJR Cyber Laskar", "line2": "Hosur Rd", "landmark": "Adugodi", "zipcode": "560030", "city": "Bangalore", "state": "Karnataka", "contact": "9000090000", "tag": "office", "type": "saved" } } }, { "instruments": [ { "method": "emi", "issuers": [ "HDFC" ], "types": [ "debit" ] }, { "method": "cardless_emi", "providers": [ "zestmoney", "walnut369" ] }, { "method": "paylater", "providers": [ "simpl", "lazypay" ] } ] }

      please update the sample code. I have fixed it and am sending on slack

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  35. github.com github.com
    nic-dgl103-f22/assignment-e-dlu-RaviPunia: assignment-e-dlu-RaviPunia created by GitHub Classroom
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  36. meta.stackoverflow.com meta.stackoverflow.com
    Generalize [Dockerfile] to [Containerfile], for now and the future․
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  37. github.com github.com
    assignment-e-dlu-caitgarland/index.html at main · nic-dgl103-f22/assignment-e-dlu-caitgarland
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  38. addons.thunderbird.net addons.thunderbird.net
    Tag Toolbar / Tag Popup
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  39. github.com github.com
    assignment-f-dlu-TessaWarman/index.html at main · nic-dgl103-f22/assignment-f-dlu-TessaWarman
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  40. hypothes.is hypothes.is
    Hypothesis
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    1. chrisaldrich 08 Nov 2022

      https://hypothes.is/search?q=tag%3A%27etc556+etcnau%27

      Randomly ran across a great tag full of education resources...

      Seems to be related to this class:<br /> ETC 556 - Contexts And Methods Of Technology In Adult Education

      Description: This course is designed for adult educators in the various contexts, including: higher education, military, non-profit, health and business settings. Through research, readings and collaborative activities, students will gain an understanding of various adult learning methods that include, but are not limited to, training, professional development, performance improvement, online and mobile learning. Letter grade only.

      https://catalog.nau.edu/Courses/course?courseId=011553&catalogYear=2223

      etc556 etcnau education research bookmark
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  41. github.com github.com
    GitHub - nic-dgl103-f22/assignment-f-cvs1-jcoppel: assignment-f-cvs1-jcoppel created by GitHub Classroom
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  42. github.com github.com
    nic-dgl103-f22/assignment-f-cvs1-Pillairenu: assignment-f-cvs1-Pillairenu created by GitHub Classroom
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  43. github.com github.com
    nic-dgl103-f22/assignment-e-cvs1-Aranarora: assignment-e-cvs1-Aranarora created by GitHub Classroom
    1
    1. Sri01729 07 Nov 2022
      <figure> <img src="images/home.png" alt="" width="900" height="500" /> <figcaption> The Home page of the phiarchitecture website. </figcaption> </figure> <figure> <img src="images/About.png" alt="" width="900" height="500" /> <figcaption> The About page of the phiarchitecture website. </figcaption> </figure> <figure> <img src="images/Project.png" alt="" width="900" height="500" /> <figcaption> The Projrct page of the phiarchitecture website. </figcaption> </figure>

      It's better to have some space between each tag in order to understand the code easy(i.e., visibility must be good).

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  44. github.com github.com
    nic-dgl103-f22/assignment-f-cvs1-Jan-elle-Chan: assignment-f-cvs1-Jan-elle-Chan created by GitHub Classroom
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  45. github.com github.com
    nic-dgl103-f22/assignment-f-cvs1-jcoppel: assignment-f-cvs1-jcoppel created by GitHub Classroom
    1
    1. dhuds1 07 Nov 2022
      line-height: 1.8rem; padding: 10px 0; }

      take off the padding on the li and give it to the a tag so you can hover less over the word to select the link, makes it better for accessibility.

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  46. www.biorxiv.org www.biorxiv.org
    New submission 07/11/2022, 13:36:37
    1
    1. Public_Reviews 07 Nov 2022

      Reviewer #3 (Public Review):

      This manuscript will be of interest primarily to researchers in the field of NADPH oxidases (NOXs) but also to those interested in the wider ferric reductase superfamily, also comprising members of the six-transmembrane epithelial antigen of the prostate enzymes (STEAPs). More limited interest may be expressed by investigators of ferredoxin - NADP reductases, resembling the dehydrogenase region (DH) of NOXs, expressing lesser "visibility" in the structure described in the paper. Considering the fact that NOXs are essentially electron transport machines from NADPH to dioxygen, along a multi-step redox cascade, those interested in hydride and electron transfer, at a more conceptual level, might also want to have a look at the paper. Elucidating structures of NOXs are still rare achievements, with only four published papers, so far (one coming from the group of the present main author) and, thus, any new publication profits from the aura of novelty.

      Introduction<br /> This manuscript offers a detailed and in depth description of the structure of the catalytic core of the human phagocyte NADPH oxidase, NOX2, in heterodimeric association with the protein p22phox. The phagocyte NADPH oxidase is responsible for the production of reactive oxygen species (ROS), the primary molecule of which is the superoxide radical (O2.-), derived by the one-electron reduction of molecular oxygen by NADPH. NOX2 belongs to the NOX family, consisting of 7 members (NOX 1-5, and DUOX1 and DUOX2), sharing common structural characteristics but expressing a wide variety of functions. The principal but not the only function of NOX2 is as a source of ROS for the killing of pathogenic microorganisms (bacteria, fungi, protozoa) engulfed by phagocytes in the course of innate and acquired immunity.

      The structures of C. stagnale NOX5, and that of murine and human DUOX1 were determined by X-ray crystallography (NOX5) and cryo-EM (DUOX1). As sources of potentially dangerous auto-toxic ROS, NOXs are subject to strict functional regulation. Whereas Nox5 and the DUOXs are regulated by Ca2+, NOXs 1, 2, and 3 are regulated by several cytosolic proteins, that associate with the Nox2-p22phox dimer forming the active O2.-generating complex. The paramount model of cytosolic regulation is Nox2 and the "dream" of structure investigators is to elucidate the structure of NOX2 in both resting and activated states.

      Achievements<br /> Note: When this paper was received for review, this reviewer was not aware of any publication dealing with the structure of human Nox2. However, on October 14, 2022 a paper was published on line, dealing with the structure of Nox2 (S. Noreng et al., Structure of the core human NADPH oxidase Nox2, Nature Communications (2022)13:6079). This review will not discuss the present manuscript in relation to the paper by S. Noreng et al.

      This manuscript is successful in describing the structure of the NOX2-p22phox heterodimer using cryo-EM methodology. In order to compensate for the small size of the complex, use was made of the Fab of a monoclonal anti-Nox2 antibody binding an anti-light chain tagged nanobody. In order to mimic as much as possible the milieu of NOX2-p22phox in the phagocyte membrane bilayer, the authors reconstitute the quaternary complex in a nanodisc, using soybean phosphatidylcholine (PC) and a membrane scaffold protein (MSP). To the best of my knowledge, this is the first report of studying a NOX in a nanodisc, for both function and structure. Peptidiscs were used in determining the structure of human DUOX1 by a group led by the main author of this paper, but nanodiscs offer the advantage of adding a phospholipid chosen by the investigator. The purified nanodiscs incorporating the quaternary complex led to successful structure determination of the transmembrane domain (TMD), extracellular and intracellular loops, inner and outer hemes, distances between hemes and FAD to inner heme, and a hydrophilic tunnel connecting the exterior of the cell to the oxygen-reducing center of NOX2. The structure of the dehydrogenase region (DH) was less well defined; the FAD-binding domain (FBD) was more visible than the NADPH-binding domain (NBD). The structure of p22phox and the interface between Nox2 and p22phox are well described.

      The mutations in NOX2 and p22phox causative of the deficient bactericidal function in Chronic Granulomatous Disease are related in detail to the location and role of the mutated residues as revealed by the solved structure.<br /> The authors make it clear that the structure, as presented, is in the resting state. The distances between hemes are suitable for electron transfer but the distance between FAD, in the FBD, and the inner heme is too large for transfer. The poor quality of the obtained structure of the DH (especially, the NBD), even after local refinement focusing, suggests its flexibility (mobility?) relative to the TMD and that, in NOX2, the DH is "displaced" relative to the TMD, when compared to the situation in the activated (by Ca2+) DUOX1. The mobility of NBD in NOX2 also results in weak interaction with FBD, making hydride transfer from NADPH to FAD inefficient

      A major achievement of the work described in this manuscript is what I believe to be the first description of the activation of recombinant NOX2-p22phox in a nanodisc, to generate O2.-, when activated by a trimeric fusion protein (trimera), consisting of the functionally important parts of the three cytosolic components, p47phox, p67phox, and Rac (see Y. Berdichevsky et al., J. Biol. Chem. 282, 22122-22139, 2007). This proves that the resting state structure of NOX2-p22phox has all that is needed to be converted to the activated state. The fact that the nature of the phospholipid in the nanodisc can be varied and that this is known to have a major effect on the affinity of the trimera for NOX2-p22phox, offers additional advantages.

      Weaknesses<br /> A weakness of this, otherwise impressive work, is the difficulty for readers who are not sufficiently "structure educated" to fully understand the "displacement" of the DH of NOX2, shown in the NOX2/DUOX1 overlay (Figure 5). The meaning of "centers of mass" of FBD and FAD, in Figures 5C and 5D, respectively, is not properly explained.

      Yet another weakness is the much too vague wording of the change in NOX2 conformation from the resting to the activated state by cytosolic factors as "the cytosolic factors might likely stabilize the DH of NOX2 in the "docked" conformation which is similar to that observed in the activated DUOX1 in the high-calcium state". First, the evidence from biochemical studies of NOX2 activation indicates clearly distinct targets of individual cytosolic components and not a "block" action. There is also support for the conformational change being the result of the action of a single cytosolic component (p67phox), with the other cytosolic components acting as carriers or activators of one cytosolic component by another, such as Rac-GTP acting as a carrier and inducer of a conformational change in p67phox (see J. El-Benna and P.M-C. Dang, J. Leukoc. Biol. 110, 213-215, 2021, and E. Bechor et al., J. Leukoc. Biol. 110, 219-237, 2021). Also, the concept of "docking of the DH to the TMD" seems like an oversimplification of the many locations and partners of such "docking" and ignores the possible multiple consequence of such docking. Even before the appearance of structural studies of NOXs, revealing precise distances between redox stations (NADPH-FAD; FAD-inner heme; inner heme - outer heme), as first reported for C. stagnale Nox5, by F. Magnani et al., Proc. Natl. Acad. Sci. U.S.A. 114, 6764-6769, 2017, a shortening of the distance between an electron donor and acceptor at specific locations in the redox cascade was proposed. The most popular was the NADPH - FAD hydride transfer, based on structural work by P.A. Karplus on Ferredoxin - NADP reductases, the accepted model for the DH of NOXs.

      An unfair request for an unachieved task<br /> Of course, the dream of those hoping for a structure-based response to solving the molecular mechanism of NOX activation is to see the structure of the activated NOX2 in complex with three cytosolic components. The compelling finding in the present manuscript that a nanodisc-embedded recombinant NOX2-p22phox can be activated to ROS production by the use of a [p47phox-p67phox-Rac] trimera (replacing three cytosolic components) will provoke in all the readers the wish to see the structure of such a complex. The size of the trimera with a GFP tag (108 kDa) might make the use of the anti-Nox2 Fab and anti-light chain nanobody, unnecessary. Prenylation of the trimera at the Rac moiety is bound to markedly enhance its affinity for the phospholipids in the nanodisc and is likely to generate a more stable complex, most suitable for cryo-EM (see A. Mizrahi et al., J. Biol. Chem. 285, 25485-25499, 2010).

      Review 3
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  47. web.hypothes.is web.hypothes.is
    Annotating the law | Hypothes.is
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  48. adriennemareebrown.net adriennemareebrown.net
    esii – adrienne maree brown
    1
    1. indyweb 06 Nov 2022
      This palpable, active, ongoing grief is a non-negotiable part of this period of immense change. Grief is one of the most beautiful and difficult ways we love. As we grieve we feel our humanity and connection to each other. Building the path from this heartbreaking present to a future where we center our collective existence in love and care is where we come in. We are the ones shining light on the lies and inconsistencies in our current reality, and we are the ones dreaming up, remembering and practicing mutual ways of being in community with each other. We are learning how to grieve without disappearing, and we are refusing to normalize this terror. We are scholars of belonging and accountability, releasing ourselves from the reductive protocols of punitive culture. We are protesting injustice wherever we find it, while forging the pathways to a justice we cocreate. We are releasing either/or thinking, and we are outgrowing every construct meant to divide and disempower us. We understand that this is an extinction point, and we are not just interested in survival – we want a just world for future generations and for the earth. Each day, we are the ones creating more possibilities. We at ESII see how this community is showing up to hold each other, to grieve, to care for each other, to practice the future together. We love you, we trust you, we grieve with you, and we change with you.

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  49. adriennemareebrown.net adriennemareebrown.net
    emergent strategy – adrienne maree brown
    1
    1. indyweb 06 Nov 2022
      adrienne maree brown 'The only way to deal with an unfree world is to become so absolutely free that your very existence is an act of rebellion.' – camus…documenting my liberation

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  50. github.com github.com
    nic-dgl103-f22/assignment-f-cvs2-tatianasuarez23: assignment-f-cvs2-tatianasuarez23 created by GitHub Classroom
    1
    1. TomioM 06 Nov 2022
      <img class="logo-img" src="images/dgl-logo.png" alt="DGL logo" width="80">

      I agree with Sahil. I think creating a link to the homepage by placing the img inside an anchor tag would be ideal .

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  51. github.com github.com
    nic-dgl103-f22/assignment-e-dlu-TessaWarman: assignment-e-dlu-TessaWarman created by GitHub Classroom
    2
    2. r_parker 02 Nov 2022
      <div class="image"> <img src="images/homepageheader.png" alt="homepage"> </div>

      you don't actually need the div class= "image" also I think it's missing a closing tag.

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  52. github.com github.com
    nic-dgl103-f22/assignment-e-dlu-rhiannon114: assignment-e-dlu-rhiannon114 created by GitHub Classroom
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  53. github.com github.com
    assignment-e-cvs1-shyam10121998/index.html at main · nic-dgl103-f22/assignment-e-cvs1-shyam10121998
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  54. stackoverflow.com stackoverflow.com
    .gitattributes: override settings for a single file
    1
    1. TylerRick 04 Nov 2022
      Changing the second line to: foo.txt text !diff would restore the default unset-ness for diff, while: foo.txt text diff will force diff to be set (both will presumably result in a diff, since Git has presumably not previously been detecting foo.txt as binary).

      comments for tag: undefined vs. null: Technically this is undefined (unset, !diff) vs. true (diff), but it's similar enough that don't need a separate tag just for that.

      annotation meta: may need new tag: undefined/unset vs. null/set

      git diff default behavior defaults distinction undefined vs. null annotation meta: may need new tag undefined/unset vs. null/set
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  55. hypothes.is hypothes.is
    Hypothesis
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  56. hub.docker.com hub.docker.com
    Windows Server Core by Microsoft | Docker Hub
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  57. github.com github.com
    nic-dgl103-f22/assignment-f-cvs2-sunnyarora242: assignment-f-cvs2-sunnyarora242 created by GitHub Classroom
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  58. github.com github.com
    assignment-f-cvs2-sahilsrawat27/index.html at main · nic-dgl103-f22/assignment-f-cvs2-sahilsrawat27
    1
    1. TomioM 03 Nov 2022
      <p class="rights1">Copyright © 2022. All rights reserved North Island College DIGITAL Design + Development </p>

      A small tag would give more context to the function of the element

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  59. annotatingdracula.commons.gc.cuny.edu annotatingdracula.commons.gc.cuny.edu
    Annotating Dracula | Tagline TK
    1
    1. BriannaC 02 Nov 2022
      Kept in shorthand.

      Both Jonathan and Mina keep their journals in shorthand, and yet what we read here is in complete sentences. And Dr. Seward's Diary is spoken into a phonograph. We are experiencing this text very differently from how it is created. We combined with the several posts that contain correspondence that was never opened by or delivered to the intended experience (see Unopened or Undelivered tag), there is much of this story that we experience differently from the characters within it.

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  60. github.com github.com
    nic-dgl103-f22/assignment-e-dlu-chelsieadam: assignment-e-dlu-chelsieadam created by GitHub Classroom
    1
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  61. github.com github.com
    assignment-e-dlu-trinitybabichuk/index.html at main · nic-dgl103-f22/assignment-e-dlu-trinitybabichuk
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  62. github.com github.com
    nic-dgl103-f22/assignment-e-dlu-39Pancakes: assignment-e-dlu-39Pancakes created by GitHub Classroom
    1
    1. catbennett 01 Nov 2022
      <article class="homepageimagebox"> <img src="images/homepage.png" width="260" alt="photo of homepage"> </article>

      I would pay attention to the semantic coding of this section. I'm not sure if a single image would make sense as an article, especially since article typically requires a heading. I would personally just tag it as a figure (that way you can also attach a caption in case it doesn't load) and if you're worried about styling apply a div.

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  63. www.biorxiv.org www.biorxiv.org
    New submission 01/11/2022, 17:50:03
    1
    1. Public_Reviews 01 Nov 2022

      Reviewer #2 (Public Review):

      The goal of this study was to understand the molecular mechanism of how transcription factor DUX4, which has a role in cancer, inhibits the induction of genes stimulated by interferon-gamma. The authors achieved this goal, and their results mostly support their conclusions. They found that DUX4, in their experimental model, interacts with STAT1, thereby decreasing STAT1 and Pol-II recruitment to sites of gene transcription.

      The present study has many strengths: The topic is of broad interest, the findings are novel and intriguing, the experiments are well-designed and controlled, the data, with one exception, is carefully interpreted, and the manuscript is very well-written.

      Two major weaknesses were identified. One is that all experiments, except Figure 6, rely on one experimental setup, which is a human skeletal muscle cell line with an integrated doxycycline-inducible transgene. The concern is that both the treatment of cells with the drug doxycycline and the fact that signaling pathways could be disrupted in this (immortalized?) cell line could lead to artifacts that skew results. Indeed, results in Figure 4C indicate that total STAT1 is completely localized in the nucleus even prior to interferon stimulation when it should be in the cytoplasm. The other weakness is the use of the DUX4-C-terminal-domain (DUX4-CTD) mutant for the majority of the mechanistic experiments. The concern here is that although the phenotype of ISG repression is observed in this truncated mutant, important regulatory domains could be missing that modulate the interaction with STAT1 or other proteins. Is the NLS added after the flag tag identical to the endogenous NLS? Related, I disagree with the interpretation of Figure 4C that "this interaction happens within the nuclei of DUX4-CTC expressing cells". The interaction could happen prior to STAT1 shuttling to the nucleus.

      Review 2
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  64. www.biorxiv.org www.biorxiv.org
    Differences in single-motor and multi-motor motility properties across the kinesin-6 family
    1
    1. EMBOpress 01 Nov 2022

      Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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      Referee #1

      Evidence, reproducibility and clarity

      This manuscript reports motility characteristics and load-bearing properties of three human kinesin-6 family proteins that function during late telophase/cytokinesis of mitosis. The authors report single molecule and multiple motor motility assays, and vesicle dispersion assays for the three motors. Because the kinesin motors are important for normal division, their motility characteristics are of interest to workers in the mitosis field. However, data presentation in this manuscript could be greatly improved, along with interpretations of functional differences based on kinesin-6 motility properties.

      Major points are the following:

      1. Quantitation and presentation of the data throughout the manuscript should be improved.

      The criteria used for identifying fluorescent spots as single motors are not given. This is typically based on photobleaching experiments and fluorescence intensity measurements - the authors should show these data to validate that the motility reported is due to single motors.

      A table should be included that shows the single molecule motility parameters that were analyzed and compared for the three motors, rather than just the dwell times for the assays shown in Fig. 1. Other motility characteristics should include run lengths, binding rates, detachment rates, and velocity. The percentage of time that the single motors move directionally, diffuse, or remain stationary should also be given.<br /> The authors refer to imaging rates (1 frame/50ms, p. 5), but do not state the total time of the assays, making the statements uninterpretable, as it is not clear what would be expected without knowledge of the total assay time. The authors also state that a slower imaging rate (1 frame/2 sec) was used to detect slow processive motility, but the logic underlying this statement is not clear, as a longer assay time should reveal the slow processive movement irrespective of the imaging rate. These statements should be clarified.<br /> The authors give the data for the dwell times in single motor assays and velocities in multiple motor assays as the mean + SEM, but the SD rather than SEM should be reported for these assays, given that the data are for individual single motors or individual gliding microtubules. The authors state the number of replicate experiments for the assays, but they should also state the number of data points that were obtained for each replicate. Further, they should evaluate the significance of differences in their data by giving P values obtained using appropriate statistical tests and indicate whether the differences among the motors are significant.<br /> The percentages of processive events (p. 5) are most likely dependent on the amount of inactive or denatured protein in a given preparation, rather than a motility property of the motor protein - this could be determined by analysis of whether the percentages differ from preparation to preparation of each motor and whether the mean+SD of the preparations of a given motor differs from the other motors. The statements by the authors on p. 8 that "the majority of proteins do not undergo unidirectional processive motility as single molecules but rather diffuse along the surface of the microtubule for several seconds" and "It is presently unclear why only a subset of kinesin-6 molecules are capable of directional motility (Figure 1 ..." are not meaningful, as they do not take into account the percentages of the kinesin-6 proteins that are inactivated or denatured during protein preparation.<br /> Again, given that inactive motors are produced during preparation of the proteins, it is not clear what the frequency of processive motility events means. If the authors think that the frequency of processive motility events is informative and a characteristic of each motor, they should present controls showing frequencies of processive motility events for specific well characterized motors. For example, does a control of kinesin-1 show 100% or only 95% processive motility events?<br /> For the multiple motor gliding assays, velocities are shown in Fig. 2 without controls demonstrating the dependence of the velocities on motor concentration in the assays - the gliding assays require dilution experiments to show that the velocities are within the linear range of motor concentration and do not fall within the range of higher concentrations in which motor gliding velocity is inhibited or lower motor concentrations in which the density of motors on the surface is too low to support processive movement. These control experiments of motor concentration vs velocity for the gliding assays should be shown for each of the three motors that was assayed. The authors should state whether the gliding velocities that were determined correspond to the Vmax for each of the motors that was assayed.

      Again, the velocities given on p. 6 should include the SD and evaluation of the significance of the differences among the motors by obtaining P values.

      Proteins for motility assays: Western blots of the purified proteins should be shown as a supplemental figure.

      How are the motility characteristics of the three motors related to their spindle functions? This is the central point of the manuscript but is not clearly stated.

      1. Functional assays should be relevant to motor function.

      Given that the kinesin-6 motors under study are mitotic spindle motors that do not normally transport vesicles, it is not clear why the authors chose to show load dependence using peroxisome and Golgi dispersion assays, rather than assays of spindle function. The authors interpret peroxisomes and Golgi to differ in dispersion load, but this appears to be based on interpretations from assays of highly processive motors, kinesin-1 and myosin V, that function in vesicle trafficking, rather than quantitative data from appropriate controls showing that peroxisomes and Golgi can be dispersed by spindle motors that bear different loads. The problems inherent in the use of these assays for spindle motors are evidenced by the authors' observations on p. 6 that MKLP1- mNG-FRB and KIF20-mNG-FRB in midbodies could not be localized to peroxisomes by rapamycin. There are no data presented showing the dependence of dispersion on protein expression/presence in the cytoplasm, making the dispersion assays difficult to interpret.

      The kinesin-6 motor functional tests would be more relevant if they involved mitotic spindle assays, rather than peroxisome or Golgi dispersion assays. It is not clear how the loads involved in peroxisome or Golgi dispersion are related to kinesin motor function in the spindle. What are the implications of low- vs high-load motors in the spindle? How do the authors envision that motor loads in spindles relate to loads borne by vesicle transport motors?

      Minor points needed for clarity and reproducibility of the data:

      Methods

      Plasmids<br /> "MKLP1(1-711) lacks the insert present in KIF23 isoform 1" - the insert present in KIF23 isoform 1 but missing in MKLP1 (1-711) should be depicted/pointed out in Fig. S1 and information provided as to its predicted or actual structure.

      "KIF20B contained the protein sequence conflict E713K and natural variations N716I and H749L "- the sites of these changes should be indicated in Fig. S1 and information provided as to their effects on predicted or actual structure.<br /> Protein purification: "MKLP1(1-711)-3xFLAG-Avi was cloned by stitching four oligonucleotide primer sequences together into a digested MKLP1(1-711)-Avitag plasmid" - please explain what this means: what do the four oligonucleotide primer sequences correspond to? if they are the 3xFLAG-Avi tags, why were four sequences stitched together instead of three?<br /> The figures showing the kymographs should include labeled X and Y axes, rather than scale bars.

      The significance of the statement that "All motors displayed similar behaviors when tagged with Halo and Flag tags" is not clear, as the Halo and Flag tags were also C-terminal tags, like the 3xmCit tag.

      The figures (Fig. 3-5) that contain grey-scale cell depictions would be more readily interpretable by others if they were labeled with the authors' classification of the dispersion phenotype.

      Significance

      This manuscript reports motility characteristics and load-bearing properties of three human kinesin-6 family proteins that function during late telophase/cytokinesis of mitosis. The authors report single molecule and multiple motor motility assays, and vesicle dispersion assays for the three motors. Because the kinesin motors are important for normal division, their motility characteristics are of interest to workers in the mitosis field. However, data presentation in this manuscript could be greatly improved, along with interpretations of functional differences based on kinesin-6 motility properties.

      My expertise: motors, motor function in division, motility assays, microtubules

      peerReviewed
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  65. Oct 2022
  66. github.com github.com
    assignment-e-cvs1-Manjeet-momi/index.html at main · nic-dgl103-f22/assignment-e-cvs1-Manjeet-momi
    3
    1. jcoppel 31 Oct 2022
      <p>All heading tags are working well but the hierarchy in the webpage is missing which is making the page unstructured. This is somehow missleading the viewers as they will not get the difference between headings and links.So, the mixure of headings and links seems irrelevant here. Instead, the headings could be managed either side of the page and links towards the oposite side. This will be east for the people to scroll the page to get information easily. Most importantly, the align attrribute in h3 tag is a wrong use. </p>

      Because there is no maximum width specified, paragraph elements that are very long like this one show up as very long on the user's browser. This is not very readable.

    2. jcoppel 31 Oct 2022
      <span>Comox Valley Lifeline Society</span> <p>Span tag is not necessary to use here. Instead simple heading tag could work here</p>

      When you use <span> tags here as an example, they do not render on the user's browser. Character entities must be used if you want to display reserved characters like < and >. See this for more information.

    3. jcoppel 31 Oct 2022
      <!--<p align="justify" class="plain">The Comox Valley Lifeline Society offers a variety of medical alert services designed specifically for older adults that provide fast, 24/7 access to expert help in an emergency.&nbsp; These services range from the standard HomeSafe service to the fall detection capability of the HomeSafe with AutoAlert and the freedom of the new GoSafe mobile service. </p>--> <ol> <li>There is again wrong use of align attribute in the paragraph tag</li>

      When you talk about how they use the align attribute wrong, they can't see the code you're talking about.

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  67. github.com github.com
    nic-dgl103-f22/assignment-e-crs1-TentacularScientist: assignment-e-crs1-TentacularScientist created by GitHub Classroom
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  68. store.steampowered.com store.steampowered.com
    AEGYPTUS on Steam
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  69. steamcommunity.com steamcommunity.com
    Key banned :: Mortos Discussions
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  70. github.com github.com
    nic-dgl103-f22/assignment-e-cvs2-elizabethajg: assignment-e-cvs2-elizabethajg created by GitHub Classroom
    1
    1. arjunsadiora 27 Oct 2022

      the css part is done very great and they have right space in between the codes , like we can easily see the each tag and what is used in it. i also saw new thing like we can use h1,h2 tags combine and edit it together

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  71. github.com github.com
    assignment-e-cvs2-tomioM/index.html at main · nic-dgl103-f22/assignment-e-cvs2-tomioM
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  72. github.com github.com
    nic-dgl103-f22/assignment-e-cvs2-elizabethajg: assignment-e-cvs2-elizabethajg created by GitHub Classroom
    2
    2. pmenezes3094 27 Oct 2022
      </li> <li>Use the 60-30-10 rule to balance the three colours.</li>

      You might want to consider consistency in formatting. In the first one the closing tag of list item is placed on a new line and in the next one it is placed right after the content.

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  73. benwerd.substack.com benwerd.substack.com
    The writerly nerd
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    1. chrisaldrich 27 Oct 2022

      Given your talents, if you've not explored some of the experimental fiction side of things (like Mark Bernstein's hypertext fiction http://www.eastgate.com/catalog/Fiction.html, Robin Sloan's fish http://www.robinsloan.com/fish/ or Writing with the Machine https://www.robinsloan.com/notes/writing-with-the-machine/, or a variety of others https://hypothes.is/users/chrisaldrich?q=tag%3A%22experimental+fiction%22), perhaps it may be fun and allow you to use some of your technology based-background at the same time?

      reply experimental fiction technology writing
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  74. boffosocko.com boffosocko.com
    Top Ed-Tech Trends of 2015: Indie Ed-Tech
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    1. paldmytro 27 Oct 2022
      One can’t help but notice the proliferation of specific method names for slightly different practices within the now growing space. These specific names for practices literally give both a name and power to the space and help to make it grow. Some of these names include: Zettelkasten itself as a name for Luhmann’s method; Smart Notes (Sönke Ahrens’ delineation of Luhmann’s method, Linking Your Thinking (aka LYT, Nick Milo’s method); Building a Second Brain (BaSB, Tiago Forte’s method); ANTInet (Scott P. Scheper’s analog branded version of Luhmann’s method); and even Pile of Index Cards (PoIC, Hawk Sugano’s productivity-based method from 2006). The naming tends to expand here as many of these examples have a commercial need to differentiate these practices to make them sellable to a larger audience. Should one really consider it a coincidence that Obsidian is so heavily used by those in Tiago Forte’s Building a Second Brain camp when Obsidian’s tag line on their home page boldly declares “A second brain, for you, forever.”? This naming craze even extends to a proliferation of names for note types within each system including fleeting notes, permanent notes, literature notes, atomic notes, evergreen notes, source notes, point notes, concept notes, claim notes, etc. Of course the power of naming begins to wane here as the over-proliferation of names causes semantic collisions and worries when these systems and their adherents talk about related ideas online in broader overlapping publics. One would presume that over time this list of names will settle down and roughly standardize around a much smaller (dare I say atomic?), possibly mutually exclusive set.

      Another example of marketing serving badly for the concepts being easily studied and used. Positioning and differentiation backfires here. Lack of sources linking is a huge issue in a popular non-fiction.

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  75. cdrh.unl.edu cdrh.unl.edu
    What is XML? | Center for Digital Research in the Humanities | Nebraska
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    1. Safiya899 26 Oct 2022
      XML is not limited to a specific set of tags, because a single tag set would not adapt to all documents or applications that may use XML.

      Unlike HTML XML is more useful and flexible when adapting to other applications while HTML is restricted to only one set of tags

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  76. stackoverflow.com stackoverflow.com
    Using yield inside define_method in Ruby
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    1. TylerRick 26 Oct 2022
      The problem is that the caller may write yield instead of block.call. The code I have given is possible caller's code. Extended method definition in my library can be simplified to my code above. Client provides block passed to define_method (body of a method), so he/she can write there anything. Especially yield. I can write in documentation that yield simply does not work, but I am trying to avoid that, and make my library 100% compatible with Ruby (alow to use any language syntax, not only a subset).

      An understandable concern/desire: compatibility

      Added new tag for this: allowing full syntax to be used, not just subset

      clarification good explanation compatibility allowing full syntax to be used, not just subset
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  77. raku.org raku.org
    Raku Programming Language
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  78. www.biorxiv.org www.biorxiv.org
    Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes
    1
    1. EMBOpress 26 Oct 2022

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

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      Reply to the reviewers

      We would like to thank the Reviewers for their valuable comments and constructive suggestions concerning our manuscript entitled " Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-based transgenes" (RC-2022-01629).

      Please find below our responses to the Reviewers' questions and comments. We have revised the Manuscript following the Reviewers' suggestions. The changes in the Manuscript are indicated in blue.

      Reviewer #1 (Evidence, reproducibility and clarity (Required)): ____ This manuscript by Uhlirova and colleagues identified an unwanted off-target effect in the pVALIUM10 TRiP RNAi lines that are commonly used in the fly community. The pVALIUM10 lines use long double-stranded hairpins and are useful vectors for somatic gene knock-down, hence they are widely used.

      Here the authors find that any pVALIUM10 TRiP RNAi line can create the silencing of any transgenes that were cloned with the commonly used Gateway system. this is caused by targeting attB1 and attB2 sequences, which are also present in other Drosophila stocks including the transgenic flyORF collection. Hence, this is an important and useful information for the fly community that should be published quickly. All experiments are well documented and well controlled. I only have a few minor comments.

      1. I recommend to mention the number of 1800 pVALIUM10 lines in Bloomington in the abstract rather than 11% to make clear that this is an important number of lines. (1800 of 13,698 lines in Bloomiongton are 13 and not 11 per cent?)

      We now include the absolute number of pVALIUM10 lines in the manuscript abstract. The percentages have been corrected. Furthermore, we updated/corrected the total number of RNAi lines available from various stock centers in the Discussion, L153-L156.

      The status on 23.10.2022

      VDRC - 23,411 in total (12,934 GD lines; 9,674 KK lines; 803 shRNA lines)

      Bloomington - 13,410 TRiP lines based on pVALIUM vectors (13,674 in total, including 264 non-pVALIUM, and 48 non-fly genes targeting lines)

      NIG - 12,365 in total (5,676 TRiP lines; 7,923 NIG RNAi lines)

      The authors may consider to call the 'unspecific' silencing effect an 'off-target' effect compared to intended 'on-target'. Such a nomenclature would be more consensus.

      We changed the wording in the manuscript as suggested by the reviewer.

      Ideally, all the imaging results in Figure 2 and 3 would be quantified. The simple 'V10' label in the Figure 3L and 3M is not the most intuitive, at least it took me a while to figure out what the authors compare.

      The labeling in the charts has been changed. We now provide quantifications for the data shown in Figure 2 and 3.

      Does the silencing also affect attR sequences? These are present after cassette exchange in many transgenes, most of the time not in the mRNA though, so it might not be so relevant.

      A 22 nucleotide stretch of the attB2 site indeed shows a 100% match to the attL2 site. See the example alignment below (availbale in word/PDF version of the Letter). While we did not assess this possibility experimentally, attL sites would likely be susceptible to the same undesirable off-target silencing effects if present in the nascent or mature transcript.

      Reviewer #1 (Significance (Required)): This is an important and useful information for the fly community that should be published quickly.

      Reviewer #2 (Evidence, reproducibility and clarity (Required)): ____ Stankovic, Csordas, and Uhlirova show that a specific subset of the TRiP RNAi lines available, namely the pVALIUM10 subset, can cause a knockdown of certain co-expressed transgenes that contain attB1 and attB2 sites. The authors demonstrate that while pVALIUM20 or Vienna KK lines for BuGZ or myc RNAi do not affect RNase H1:GFP expression, pVALIUM10 RNAi lines against BuGZ or myc significantly decrease expression of the RNAseH1:GFP transgene. The authors propose that, due to how these RNAi lines were constructed, the siRNA products could be targeting to attB1 and attB2 sites in transgenes that were made using similar methodology. To support this idea, they ubiquitously express mCherry transgenes encoding mRNAs either containing or lacking attB sites. They find that the knockdown of mCherry seen with several different pVALIUM10 RNAi lines is observed with the reporter mRNA containing attB sites, but is suppressed when the attB sites are removed from mCherry mRNA. They also find that the pVALIUM10 RNAi lines reduce the expression of the FlyORF transgene SmD3:HA.

      The paper is very clearly written and the data presented is convincing.

      Minor suggestions:

      1. Figure 3 L+M The labels for the ubi-mcherry and ubiΔattb-mcherry are switched in these graphs (i.e. ubiΔattb-mcherry should be the one with a higher intensity in the pouch compared to the notum).

      Figure 3M the labels don't match the RNAi lines used in H-K.

      We corrected the labelling in the charts.

      Figure 2 and 3. For the images of the transgenes, it seems as if the BuGZ RNAi line has a more drastic effect on RNaseH1 than mCherry, and vice versa for the myc RNAi lines. Did the authors notice a pattern with the decreased expression. Do some of the RNAi lines have a more consistent/severe impact, or might different transgenes be impacted to different extents?

      Throughout the study and multiple experimental trials, we did not observe that the BuGZRNAi and mycRNAi silencing efficiency would depend on whether the monitored reporter was RNase H1::GFP or mCherry. What has been reproducible is the differential impact of the three tested mycRNAi lines on ubi-RNaseH1::GFP transgene. While pVALIUM10-based mycRNAi[TRiP.JF01761] reduces RNaseH1::GFP signal Valium20 mycRNAi[TRiP.HMS01538] enhances it and GD mycRNAi[GD2948] has no effect, although the number of replicates for the latter is lower compared to the other tested lines. Why Valium20 mycRNAi[TRiP.HMS01538] increases RNaseH1::GFP signal remains unclear for now.

      We would like to refrain from directly quantitatively comparing the effects of phenotypically different RNAi lines on differently tagged mRNAs/proteins. As the RNAseH1::GFP fusion protein is nuclear while the mCherry is cytoplasmic, their distinct subcellular localization and/or turnover rate may give a different overall impression on the change in fluorescence intensity (Boisvert et al, 2012; Mathieson et al, 2018). Another confounding factor is the described roles of Drosophila Myc in regulating transcription, translation, and cell growth (Gallant, 2007).

      Line 150 unnecessary comma after Both Line 131 knockdown should be knocked down Line 133 should be "using an additional" Figure legend 1 wing disc should be at least written out when the abbreviation (WD) is first used.

      We thank the reviewer for pointing these out, the relevant corrections were performed.

      Reviewer #2 (Significance (Required)):

      Overall, this manuscript is an informative reminder that RNAi lines can have weaknesses that have not yet been considered, and we appreciate the authors work to inform the fly community about this specific issue. These insights are crucial for fly labs to consider when planning experiments that will use the pVALIUM10 RNAi lines in combination with other transgenesis modalities. The manuscript also provides a cautionary note for the usage of similar resources in other model organisms.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)): Summary: In their manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes", Stankovic et al. describe off-target silencing of transgenes expressed from Gateway systems when expressed in transgenic RNAi drosophila lines from the VALIUM10 collection. Using fluorescence microscopy and immunostaining, the authors show that this unintended silencing is specific to VALIUM20 lines and is not observed with VALIUM20, KK or GD lines that also allow gene-specific RNAi silencing. This pleiotropic silencing effect was observed in 10 different VALIUM20 lines and affected Gateway-based transgene expressed from an ubiquitous promoter (poly-ubiquitin, ubi) or from Gal4/UAS systems. Finally, the authors identify the molecular basis of VALIUM20 pleiotropic silencing on Gateway transgenes as being due to the presence of short sequences used for PhiC31-based recombination in the Gateway and the VALIUM systems, and could lead to the production of siRNAs against PhiC31 recombination sites in VALIUM10 lines. Using Gateway transgenes lacking the recombination sites (attB1 and attB2), the authors could abrogate silencing of the transgene in VALIUM10 lines, confirming the recombination as shared targets between the Gateway and the VALIUM systems.

      Major comments: - The study is well designed and the key conclusions are convincing. - However, the authors provide only fluorescence microscopy data to show decreased transgene expression. To confirm pleiotropic RNAi effect on Gateway transgenes in VALIUM10, the authors should assess silencing with another technique. For instance, expression levels of proteins from Gateway transgenes could be measured by Western blot (e.g.: by assessing protein levels of GFP or other tags present in the Gateway transgenes).

      In the manuscript, we present microscopy data as this is the typical use case for fluorescent reporters. The strength of the microscopy, in contrast to Western Blot or RT-qPCR approach, is that it allows us to directly compare the impact of RNAi silencing on cells that express the dsRNA transgene (cell-autonomous) to surrounding neighbor cells. The fluorescent imaging of WDs where all cells express the reporter construct, but only a subset of cells trigger RNAi-mediated silencing, provides spatial resolution and means for normalization while minimizing artifacts that can arise during tissue processing for WB and RT-qPCR. We provide data on GFP and HA-tagged transgenes, respectively, and untagged mCherry expressed from Gateway vectors under ubiquitin or UAS regulatory sequences with the explicit reason to show that the silencing effect is independent of the type of the protein tag or the expression regulator sequence.

      In addition, the claim on line 141,"These results strongly indicate that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs" , should be modified. The authors only present fluorescence microscopy data to show decreased transgene expression and do not actually provide data on siRNA expression in the pVALUM20 lines. Therefore, with the current data, the authors should only say that their results suggest that the dsRNA hairpin produced from pVALIUM10 RNAi vectors generates attB1- and attB2-siRNAs.

      In order to substantiate their claim about pleiotropic RNAi effects from VALIUM lines on Gateway transgenes due to the production of attB1- and attB2 -siRNAs, the authors should perform an experiment to show attB1- and attB2 -siRNAs production in VALIUM10 lines and not in VALIUM20, KK or GD lines. Deep-sequencing analysis of siRNA (i.e.: miRNA-seq) from tissue expressing the corresponding RNAi transgenes would be an excellent approach to assess siRNA production in multiple samples at once. Alternatively, the authors could search published miRNA-seq datasets from VALIUM10 and other RNAi lines to assess the presence of attB1- and attB2 -siRNAs only in VALIUM10 lines. This would be free and require only a few days of data mining and analysis, if such datasets exist already. Another cheaper and faster approach (if lacking easy access to sequencing platform or bioinformatics capability) would be to perform small RNA northern blots analysis from fly tissues expressing VALIUM10 vs VALIUM20 (or KK or GD lines) and should take only a few days to do as described in doi: 10.1038/nprot.2008.67.

      If such experiments or analyses cannot be performed, then the authors can only conclude that their data suggest that the unintended silencing of Gateway transgenes in VALIUM10 is likely due to the production attB1- and attB2 -siRNAs production.

      We thank the reviewer for the valuable suggestions on experimental approcahes to identify the exact interfering RNAs produced by the VALIUM10-based RNAi constructs, which can be useful for controlling the specificity of knockdown of transgenes in studies using the resources mentioned in this report.

      We believe the fluorescence micrographs and quantifications demonstrate the off-target silencing effects of pVALIUM10-based RNAi lines on transgenic reporters generated using the Gateway LR cloning approach. Furthermore, we provide genetic evidence that removing the attB1 and attB2 sites from the reporter construct, which is otherwise identical to the original transgene (same promoter, same position of insertion, same genetic background), is sufficient to abolish the off-target effect. We would argue that the functional genetic experiments we performed with the original and mutated reporters represent the strongest possible evidence to confirm that silencing is taking effect via the attB sites.

      As we do not attempt to detect siRNA complementary to attB1/attB2 sites directly, we have changed the statements in question as per the recommendation of the reviewer.

      • The current data and methods are adequately detailed and presented, and the statistical analysis adequate.

      Minor comments:

      • The current manuscript does not have specific experimental issues.
      • Prior studies are referenced appropriately
      • Overall the text and figures are clear and accurate except for the following issues with Figure 3 and its legends On lines 396, 397, 399 and 403, the authors refer to "wild-type" ubi-mCherry. This transgene directs the ubiquitous expression of an heterologous reporter gene and thus can not as "wild type". It could instead be referred to as the "original" or "unmodified" transgene.

      We removed "wild-type" from the text.

      Fig.3 L: the x-axis labels are wrong. Decrease in the mCherry intensity ratio is observed with the ubi-mCherry construct and not in the ubi∆attB-mCherry, where the attB sequences thought to be targeted by the pVALIUM10 have been deleted.

      More space should be added between the first row of images (B-G), the second (H-L) and also the third (M-P) to avoid confusion between the labeling of the figures. Finally, to help contextualize their findings and gauging the extent of the risk of using VALIUM10 lines in RNAi screen where a Gateway transgene is involved, the authors could provide information on the overlap between the VALIUM10 collection and VALIUM20, GD and KK collections. Knowing how many genes are uniquely targeted by VALIUM10, could be helpful.

      We corrected the Figure panels according Reviewer 1 and 3’s observation.

      Of the TRiP pVALIUM-based RNAi stocks currently available in BDSC, 686 genes are targeted exclusively by pVALIUM10 RNAi lines. Considering KK, GD and shRNA transgenic lines from VDRC and NIG RNAi collection, 17 genes remain unique targets for pVALIUM10 lines. The graphical overview of the availbale lines is availbale in the word/PDF file of the Response to Reviewers Letter.

      Reviewer #3 (Significance (Required)):

      • The manuscript "Drosophila pVALIUM10 TRiP RNAi lines cause undesired silencing of Gateway-base transgenes" by Stankovic et al. is a technical study that sheds light on potential limitations of using common RNAi drosophila lines, namely the VALIUM10 collection.
      • The study provides information about very specific genetic screens conditions in Drosophila, that are likely to be rare. A rapid Pubmed search with the following terms: "drosophila TRiP screen" returns only 11 citations, while a similar search with "drosophila CRISPR screen" returns 99 citations. This suggests that in vivo RNAi screen in Drosophila using TRiP RNAi collections might not be as common or powerful as CRISPR-based screens.
      • The reported findings might be of interest mostly to a small group of scientists working with Drosophila melanogaster that specifically rely on VALIUM10 lines to perform in vivo RNAi screen in combination with Gateway transgene expression. This very specific combination of parameters is rare, since other RNAi fly stock collections exist (e.g.: VALIUM20, 21, KK, GD...). Furthermore, the advent of CRISPR tools that allows tissue-specific gene knock-out has led to the rapid expansion of CRISPR fly stock collections (https://doi.org/10.7554/eLife.53865). Regardless of the limited scope of the study, this kind information is still valuable, albeit to a very limited audience.
      • My relevant fields of expertise for this study are : insect RNAi, RNAi of RNAi screens and drosophila genetics.

      We would like to raise some points concerning the above comments.

      While TRiP-screen may not be an often-used keyword combination, the use of the TRiP lines is, in fact, ubiquitous in the Drosophila community. The tissue-specific RNA interference is still commonly utilized as a rapid, first-generation screening method that can be performed in a tissue-specific manner, representing one of the key advantages of the Drosophila model. To illustrate, since the submission of our manuscript a new study published by Rylee and co-workers investigated Drosophila pseudopupil formation by screening 3971 TRiP RNAi lines (Rylee et al, 2022). In contrast, genetic screens relying on mutant alleles usually require at least one additional cross, effectively doubling the time of the experiment. In addition, tissue-specific or temporarily restricted knockdown is sometimes required in screens, as full-body loss of function is often lethal or has developmental phenotypes incompatible with assessing gene function later in life.

      The use of tissue-specifically driven Cas9 with integrated gRNA-expressing vectors is indeed becoming more common. However, this technique, much like RNA interference, is not without flaws. First, this produces knockout instead of knockdown, which means it has to be induced early in order for the resulting mutation to take effect. Otherwise, the remaining mRNA/protein may prevent the development of a phenotype. Second, the Cas9 must be titrated as high Cas9 levels have adverse phenotypes (Huynh et al, 2018; Meltzer et al, 2019; Poe et al, 2019; Port et al, 2014). Third, in our personal experience, as well as literature reports (Mehravar et al, 2019; Port & Boutros, 2022), indicate that the resulting phenotype can produce mosaics in the tissue.

      Although the combination of Gateway-based reporters with TRiP-RNAi lines may seem like a fringe case, there are popular reporters that could be screening targets. Potentially the most well-known is the live cell cycle indicator fly-FUCCI system (Zielke et al, 2014), which allows the analysis of the cell cycle in real-time thanks to the expression of two fluorescently tagged degrons. As FUCCI transgenes were constructed with Gateway recombination, they represent targets of the pVALIUM10 TRiP lines. We now include the fly-FUCCI system as an example in addition to 3xHA-tagged FlyORF collection in the Discussion.

      REFERENCES

      Boisvert FM, Ahmad Y, Gierlinski M, Charriere F, Lamont D, Scott M, Barton G, Lamond AI (2012) A quantitative spatial proteomics analysis of proteome turnover in human cells. Mol Cell Proteomics 11: M111 011429

      Gallant P (2007) Control of transcription by Pontin and Reptin. Trends Cell Biol 17: 187-192

      Huynh N, Zeng J, Liu W, King-Jones K (2018) A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues. G3 (Bethesda) 8: 3593-3605

      Mathieson T, Franken H, Kosinski J, Kurzawa N, Zinn N, Sweetman G, Poeckel D, Ratnu VS, Schramm M, Becher I et al (2018) Systematic analysis of protein turnover in primary cells. Nature Communications 9: 689

      Mehravar M, Shirazi A, Nazari M, Banan M (2019) Mosaicism in CRISPR/Cas9-mediated genome editing. Developmental Biology 445: 156-162

      Meltzer H, Marom E, Alyagor I, Mayseless O, Berkun V, Segal-Gilboa N, Unger T, Luginbuhl D, Schuldiner O (2019) Tissue-specific (ts)CRISPR as an efficient strategy for in vivo screening in Drosophila. Nature Communications 10: 2113

      Poe AR, Wang B, Sapar ML, Ji H, Li K, Onabajo T, Fazliyeva R, Gibbs M, Qiu Y, Hu Y et al (2019) Robust CRISPR/Cas9-Mediated Tissue-Specific Mutagenesis Reveals Gene Redundancy and Perdurance in Drosophila. Genetics 211: 459-472

      Port F, Boutros M (2022) Tissue-Specific CRISPR-Cas9 Screening in Drosophila. In: Drosophila: Methods and Protocols, Dahmann C. (ed.) pp. 157-176. Springer US: New York, NY

      Port F, Chen HM, Lee T, Bullock SL (2014) Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A 111: E2967-2976

      Rylee J, Mahato S, Aldrich J, Bergh E, Sizemore B, Feder LE, Grega S, Helms K, Maar M, Britt SG et al (2022) A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation. G3 Genes|Genomes|Genetics: jkac257

      Zielke N, Korzelius J, van Straaten M, Bender K, Schuhknecht GFP, Dutta D, Xiang J, Edgar BA (2014) Fly-FUCCI: A versatile tool for studying cell proliferation in complex tissues. Cell Rep 7: 588-598

      PeerReviewed
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  79. hypothes.is hypothes.is
    Hypothesis
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    hypothes.is/search
  80. roadturtlegames.com roadturtlegames.com
    Impact Materials
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    1. Hassan061 25 Oct 2022
      Impact Materials define what interactions will occur when an object interacts with the tags defined in the Impact Tag Library

      -- the Impact Tag Library is where we define the list of tags for our materials.

      i.e.: Plastic, Glass, Concrete...etc

      Impact Documentation
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  81. roadturtlegames.com roadturtlegames.com
    Impact Tag Library
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  82. mcrcalicante.wordpress.com mcrcalicante.wordpress.com
    Segunda Guerra Mundial – Mónada Republicana
    2
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  83. github.com github.com
    nic-dgl103-f22/assignment-c-cvs1-verdai: assignment-c-cvs1-verdai created by GitHub Classroom
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    1. narendrachn 24 Oct 2022
      href="https://fonts.googleapis.com/css2?family=Roboto&display=swap" rel="stylesheet"> <!-- google font--> <link href="https://fonts.googleapis.com/css2?family=Noto+Sans&display=swap" rel="stylesheet">

      Added 3 different google fonts in the head section by the tag LINK

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  85. www.biorxiv.org www.biorxiv.org
    New submission 23/10/2022, 11:58:11
    1
    1. Public_Reviews 23 Oct 2022

      Reviewer #1 (Public Review):

      This paper has many strengths that support its conclusions. Specifically, the use of natively expressed Piezo1 engineered to carry the HA tag allowed the authors to explore the distribution of the protein from primary cells isolated from a mouse at native expression levels. Thus, over-expression effects could be avoided. The super-resolution imaging is nicely controlled and convicting in its analysis of the distribution of the channel in 3D. The supporting EM data also supports the findings from fluorescence. Likewise, the theory is convincing in proving a mechanistic reason why the channel distributes into this region of the cell. While the data are quite nice and well analyzed, the paper is lacking in an exploration of what function this distribution of the channel would provide to the cell. Likewise, if this distribution was disturbed, would the red blood cell's behavior change? For example, would calcium signals in response to an osmotic challenge or squeezing change if the channel was not concentrated in the dimple? As it stands now, the paper presents a structural view of the distribution of piezo1 in a primary cell plasma membrane but lacks direct experimental evidence for the mechanism of this concentration or mechanistic insight into the effects of this spatial distribution on red blood cell physiology.

      Review 1
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  86. github.com github.com
    nic-dgl103-f22/assignment-c-cvs1-Pillairenu: assignment-c-cvs1-Pillairenu created by GitHub Classroom
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    1. Sri01729 23 Oct 2022
      <p>Located in the heart of the Comox Valley, Hairpins Boutique Salon offers a high-end experience with competitive prices, top of the line products and a warm, welcoming atmosphere.</p>

      The individual element tags can be formatted as

      opening tag

                         content

      closing tag

      for better readability.

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  87. github.com github.com
    assignment-c-cvs1-jcoppel/contact.html at main · nic-dgl103-f22/assignment-c-cvs1-jcoppel
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    nic-dgl103-f22/assignment-c-cvs1-Pillairenu: assignment-c-cvs1-Pillairenu created by GitHub Classroom
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    1. jcoppel 23 Oct 2022
      <address> <b>Hairpins Boutique Salon </b> <br/> #4 - 224 6th Street<br/> Courtenay, BC V9N 1M1<br/> </address>

      Had no idea there was an address tag. Good use of proper semantic labelling.

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    2. JanElleChan 23 Oct 2022
      Live Breaking UK

      Having all of these as H4 will not allow individual styling. Try enclosing each in a span or button tag then you can add background colour to each.

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  90. github.com github.com
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  92. cdrh.unl.edu cdrh.unl.edu
    What is EAD? | Center for Digital Research in the Humanities | Nebraska
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    1. meekalys 21 Oct 2022
      EAD makes use of a tag structure that identifies the components of a document. Each component or part is identified, and noted through the encoding. Because EAD is an application of XML, EAD utilizes the concepts of tags, elements, and attributes for encoding text.

      From my understanding, EAD is used only for archival files, and uses the components mentioned before.

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  93. cdrh.unl.edu cdrh.unl.edu
    Introduction to Text Encoding | Center for Digital Research in the Humanities | Nebraska
    1
    1. meekalys 21 Oct 2022
      What is a Tag?

      Tags must be correctly spelled and in the right position for them to work. A combination of tags cannot be used. This means that beginning tags cannot be used with empty tags or closing tags.

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    assignment-d-cvs1-2001sukh/index.html at main · nic-dgl103-f22/assignment-d-cvs1-2001sukh
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  95. gamefound.com gamefound.com
    Kingdom Come: Deliverance - The Board Game by Boardcubator - The Campaign is Shutting Down, None of You’ll Be Charged
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    1. TylerRick 21 Oct 2022
      First and foremost, we need to acknowledge that even though the funding goal has been met–it does not meet the realistic costs of the project. Bluntly speaking, we did not have the confidence to showcase the real goal of ~1.5 million euros (which would be around 10k backers) in a crowdfunding world where “Funded in XY minutes!” is a regular highlight.

      new tag: pressure to understate the real cost/estimate

      unfortunate annotation meta: may need new tag pressure to understate the true anticipated/estimated cost estimating taking the time to do it right/properly want to do it right
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  96. www.reddit.com www.reddit.com
    r/antinet - Bilingual Antinet?
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    1. chrisaldrich 21 Oct 2022
      Memorization is not about a language, rather about a feeling you have about information. In other words, how deep it resonates with your life. In this sense, I was also exploring the idea that having an Antinet Zettelkasten is almost like having a "diary", not for your personal feelings or emotions, rather for exploring the way in which your entire mind and heart work together over the years in which we discover the world. For me, exploring subjects and studying is an internal discovery.

      in reply to los2pollos<br /> https://www.reddit.com/r/antinet/comments/y5un81/comment/it4jy3c/?utm_source=reddit&utm_medium=web2x&context=3

      You're not the only one to think of a card index as diary. Roland Barthes practiced this as well. His biographer Tiphaine Samoyault came to call it his fichierjournal.

      card index as diary quotes examples reply
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  97. github.com github.com
    assignment-c-cvs1-Jan-elle-Chan/index.html at main · nic-dgl103-f22/assignment-c-cvs1-Jan-elle-Chan
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    1. brar33302 20 Oct 2022
      <br>Contact us today to book an appointment!<br> <a href="contact.html">Contact Us</a> </main> <footer> Content taken from <a style="color: green" href="https://www.hairpins.ca/" >https://www.hairpins.ca</a >. Used for educational purposes only. </footer> <!--an inline style rule in anchor tag above--> </body>

      nice work

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  98. minnstate.pressbooks.pub minnstate.pressbooks.pub
    Working with Ideas
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    1. stephenhay 20 Oct 2022
      In the second case, checking “Connection” in my Index would lead me to this card. I might then compare this thought to others that use the same keyword, to see how it supports or modifies the idea of connection.

      The reliance upon tag-like keywords in physical note-taking of this type seems to be a limitation compared to digital systems that allow full text search. That said, the benefits of full text search might be somewhat overblown, as found search terms say nothing of the context and would need either tags or a quick read of the text to provide that context.

      note-taking writing
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  99. github.com github.com
    nic-dgl103-f22/assignment-c-dlu-RaviPunia: assignment-c-dlu-RaviPunia created by GitHub Classroom
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    1. caitlandia 20 Oct 2022
      Skip to content In this repository All GitHub ↵ Jump to ↵ No suggested jump to results In this repository All GitHub ↵ Jump to ↵ In this organization All GitHub ↵ Jump to ↵ In this repository All GitHub ↵ Jump to ↵ Dashboard Pull requests Issues Codespaces Marketplace Explore Sponsors Settings caitgarland Sign out New repository Import repository New gist New organization Sorry, something went wrong. /  ...  /   nic-dgl103-f22  /   assignment-c-dlu-...  /   Clear Command Palette Tip: Type # to search pull requests Type ? for help and tips Tip: Type # to search issues Type ? for help and tips Tip: Type # to search discussions Type ? for help and tips Tip: Type ! to search projects Type ? for help and tips Tip: Type @ to search teams Type ? for help and tips Tip: Type @ to search people and organizations Type ? for help and tips Tip: Type > to activate command mode Type ? for help and tips Tip: Go to your accessibility settings to change your keyboard shortcuts Type ? for help and tips Tip: Type author:@me to search your content Type ? for help and tips Tip: Type is:pr to filter to pull requests Type ? for help and tips Tip: Type is:issue to filter to issues Type ? for help and tips Tip: Type is:project to filter to projects Type ? for help and tips Tip: Type is:open to filter to open content Type ? for help and tips We’ve encountered an error and some results aren't available at this time. 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Issues Pull requests Releases Discussions Discussions are not enabled for this repository Security alerts Apply Cancel Fork 0 Starred 0 Star 0 Code Issues 0 Pull requests 0 Actions Projects 0 Security Insights More Code Issues Pull requests Actions Projects Security Insights Open in github.dev Open in a new github.dev tab Permalink main Switch branches/tags Branches Tags View all branches View all tags Name already in use A tag already exists with the provided branch name. Many Git commands accept both tag and branch names, so creating this branch may cause unexpected behavior. Are you sure you want to create this branch? Cancel Create assignment-c-dlu-RaviPunia/index.html Go to file Go to file T Go to line L Copy path Copy permalink This commit does not belong to any branch on this repository, and may belong to a fork outside of the repository. RaviPunia Final code Latest commit 3fef99d 4 days ago History 2 contributors Users who have contributed to this file 101 lines (92 sloc) 3.43 KB Raw Blame Edit this file E Open in github.dev . Open in GitHub Desktop Open with Desktop View raw Copy raw contents Copy raw contents Copy raw contents Copy raw contents View blame <!DOCTYPE html> <html lang="en"> <head> <!-- DGL 103 DLU1 - Ravi Punia - Assignment C --> <meta charset="UTF-8"> <meta http-equiv="X-UA-Compatible" content="IE=edge"> <meta name="viewport" content="width=device-width, initial-scale=1.0"> <link rel="stylesheet" href="./style.css"> <link rel="icon" type="image/x-icon" href="images/favicon.ico"> <title>Hairpins Boutique Salon</title> <link rel="preconnect" href="https://fonts.googleapis.com"> <link rel="preconnect" href="https://fonts.gstatic.com" crossorigin> <link href="https://fonts.googleapis.com/css2?family=Poppins:wght@400;600&display=swap" rel="stylesheet"> <style> .services { background-color: #000000; /* Here I changed the background color using Hexadecimal value */ color: white; } </style> </head> <body> <header> <a href="index.html" ><img src="images/hairpins-salon-logo.png" alt="hairpins Logo" width="300" ></a> <nav> <ul> <li><a href="index.html">Home</a></li> <li><a href="services.html">Services</a></li> <li><a href="contact.html">Contact Us</a></li> </ul> </nav> </header> <main> <h1>Welcome to Hairpins Boutique Salon</h1> <p> Located in the heart of the Comox Valley, Hairpins Boutique Salon offers a high-end experience with competitive prices, top of the line products and a warm, welcoming atmosphere. </p> <figure> <img src="./images/the-hairpins-salon.jpeg" alt="salon image"> <figcaption> The Hairpins hairdressing salon in Courtenay, BC, Canada. </figcaption> </figure> <p> Stylist and owner, Staysea Brown has been overwhelmed by the success Hairpins has received over the past 10 years and is ever grateful to the Comox Valley community for all the support. With over a decade of industry experience, Staysea has the knowledge and drive to run a successful business that's hard to forget. Pop on by! </p> <p class="services"> Time for a new do? <a href="./services.html">Check out our services</a> </p> <h2>Meet the Team</h2> <p> Offering talented stylists with varied personalities, outgoing customer service, and an eclectic, fun atmosphere, Hairpins is striving to be one of a kind. <br> <br> By evolving with their clientele and constantly offering the latest trends and services, they are ensuring every visit is a unique one. Hairpins is filled with its own special brand of magic. Come in and sit down, the Hairpins' Girls are waiting for you! </p> <figure> <img src="./images/the-hairpins-team.jpeg" alt="team members"> <figcaption> We are incredibly proud of our diverse team of stylists who greet each client with a smile. We prioritize inclusivity, community, and sustainability, and make sure that everyone who walks through our door feels welcome. </figcaption> </figure> <a href="./contact.html">Contact us today to book an appointment!</a> </main> <footer> <p> Content taken from <a href="https://www.hairpins.ca/">https://www.hairpins.ca/</a> Used for educational purposes only. </p> </footer> </body> </html> Copy lines Copy permalink View git blame Reference in new issue Go Footer © 2022 GitHub, Inc. Footer navigation Terms Privacy Security Status Docs Contact GitHub Pricing API Training Blog About You can’t perform that action at this time. You signed in with another tab or window. Reload to refresh your session. You signed out in another tab or window. Reload to refresh your session. .user-mention[href$="/caitgarland"] { color: var(--color-user-mention-fg); background-color: var(--color-user-mention-bg); border-radius: 2px; margin-left: -2px; margin-right: -2px; padding: 0 2px; } assignment-c-dlu-RaviPunia/index.html at main · nic-dgl103-f22/assignment-c-dlu-RaviPunia

      I don't see any issues. Great job, Ravi.

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    1. caitlandia 20 Oct 2022
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RaviPunia Final code Latest commit 3fef99d 4 days ago History 2 contributors Users who have contributed to this file 110 lines (102 sloc) 3.88 KB Raw Blame Edit this file E Open in github.dev . Open in GitHub Desktop Open with Desktop View raw Copy raw contents Copy raw contents Copy raw contents Copy raw contents View blame <!DOCTYPE html> <html lang="en"> <head> <meta charset="UTF-8"> <meta http-equiv="X-UA-Compatible" content="IE=edge"> <meta name="viewport" content="width=device-width, initial-scale=1.0"> <link rel="stylesheet" href="./style.css"> <link rel="icon" type="image/x-icon" href="images/favicon.ico"> <title>Services - Hairpins Boutique Salon</title> <link rel="preconnect" href="https://fonts.googleapis.com"> <link rel="preconnect" href="https://fonts.gstatic.com" crossorigin> <link href="https://fonts.googleapis.com/css2?family=Poppins:wght@400;600&display=swap" rel="stylesheet"> <style> main li{ list-style: none; /* Here I removed the bullets from list items */ } </style> </head> <body> <header> <a href="index.html" ><img src="images/hairpins-salon-logo.png" alt="hairpins Logo" width="300" ></a> <nav> <ul> <li><a href="index.html">Home</a></li> <li><a href="services.html">Services</a></li> <li><a href="contact.html">Contact Us</a></li> </ul> </nav> </header> <main> <h1>Our Services</h1> <h2>Below is a list of services we proudly offer.</h2> <p> At Hairpins, we care about the environment and recognize the impact we all have on it. We are continually making strides to reduce where we can and have only aligned ourselves with companies and products we believe in. We are proud to be a CERTIFIED GREEN CIRCLE SALON and through that partnership are able to divert 95% of our salon waste from landfills. Get in touch if you have any questions or want to learn more about the programs and charities we are focusing our efforts on. </p> <h3>Cuts</h3> <p> Range from 45 minutes to 90 minutes. Please call us at 250-338-7467 (PINS) to book a shorter appointment for Kid's Cuts, Dry Cuts, Fringe Trims, Neck trims, or Clipper Cut maintenance.</p> <ul> <li>47.00 = 45 Minute Clipper Cuts and Short Fine Hair</li> <li>$61.00 = 60 Minute Cut for Fine to Medium Hair</li> <li>$76.00 = 75 Minute Cut for Medium to Thick Hair</li> <li>$90.00 = 90 Minute Cut for THICK THICK Hair, you know who are :)</li> </ul> <p> All cuts include shampoo, scalp massage, blowdry, and style. </p> <h3>Colours</h3> <ul> <li>FULL FOIL: $188/$219 with cut</li> <li>3/4 FOIL: $172/$203 with cut</li> <li>1/2 FOIL: $158/$189 with cut</li> <li>1/4 FOIL: $144/$175 with cut</li> </ul> <h3>Styling</h3> <ul> <li>BLOWOUTS ~ 30 mins: $40 - $45</li> <li>BLOWOUTS ~ 45 mins: $47 - $52</li> <li>BLOWOUTS ~ 1 hour: $60 - $67</li> </ul> <br> <h3><strong>* * 48 Hour Cancellation Required * *</strong></h3> <p> We require 48 hours' notice for any cancellations. </p> <br> <ul> <li>If you are a “no show”, you will be required to pay for your missed service in full in order to rebook.</li> <li>If you cancel with less than 48 hours' notice, you will be required to pay for 1/2 of the service you canceled in order to rebook.</li> </ul> <p> We understand that last-minute things happen! We will address each situation on a case-by-case basis. Please communicate with us and we will try our best to help. We appreciate your understanding. </p> <p> <a href="./contact.html">Contact us today to book an appointment!</a> </p> </main> <footer> <p> Content taken from <a href="https://www.hairpins.ca/">https://www.hairpins.ca/</a> Used for educational purposes only. </p> </footer> </body> </html> Copy lines Copy permalink View git blame Reference in new issue Go Footer © 2022 GitHub, Inc. Footer navigation Terms Privacy Security Status Docs Contact GitHub Pricing API Training Blog About You can’t perform that action at this time. You signed in with another tab or window. Reload to refresh your session. You signed out in another tab or window. 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      Looks great!

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  101. uta.pressbooks.pub uta.pressbooks.pub
    1.4 What is Distant Reading?
    1
    1. jennikolova 20 Oct 2022
      To discover the themes, a user could create a separate document of each of the duo’s albums, upload the corpus to Topic Modeling Tool, and interpret the string of words that the tool finds to be most prominent.

      This could also be a way in which artists could strategize the use of specific words in songs to attract a larger audience. They could look at the similarities between top hits and find certain words that were used in all of them and then include it when advertising the music. For example, they could use it as a tag when posting on instagram or twitter and it may attract more attention.

      engl201week5
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  102. github.com github.com
    nic-dgl103-f22/assignment-d-dlu-chelsieadam: assignment-d-dlu-chelsieadam created by GitHub Classroom
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    assignment-c-cvs1-Manjeet-momi/contact.html at main · nic-dgl103-f22/assignment-c-cvs1-Manjeet-momi
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    1. Shyam___ 19 Oct 2022
      <ul type="square"> If you're looking to make an appointment online, please do so here. <li>Monday ~ Closed</li> <li>Tuesday 9:00am ~ 5:00pm</li> <li>Wednesday 9:00am ~ 8:00pm</li> <li>Thursday 9:00am ~ 8:00pm</li> <li>Friday 9:00am ~ 5:00pm</li> <li>Saturday 9:00am ~ 4:00pm</li> <li>Sunday ~ Closed</li> </ul> <h2>Hairpins Boutique Salon</h2> #4 - 224 6th Street<br> Courtenay, BC V9N 1M1<br> Check us out on Google Maps <a href="http://www.cariboucreative.ca/">http://www.cariboucreative.ca/</a><br> Tel: (250) 338-7467 (add telephone link)<br> Email: <a href="salon.hairpins@gmail.com">salon.hairpins@gmail.com</a> Contact us today to book an appointment!<a href="https://www.hairpins.ca/contact">https://www.hairpins.ca/contact</a></footer>

      You can use div tag as well

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  104. github.com github.com
    assignment-c-dlu-melissajuneau/services.html at main · nic-dgl103-f22/assignment-c-dlu-melissajuneau
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    nic-dgl103-f22/assignment-c-dlu-melissajuneau: assignment-c-dlu-melissajuneau created by GitHub Classroom
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  106. www.reddit.com www.reddit.com
    r/antinet - Worried about paper cards being lost or destroyed
    1
    1. chrisaldrich 18 Oct 2022
      Worried about paper cards being lost or destroyed .t3_y77414._2FCtq-QzlfuN-SwVMUZMM3 { --postTitle-VisitedLinkColor: #9b9b9b; --postTitleLink-VisitedLinkColor: #9b9b9b; --postBodyLink-VisitedLinkColor: #989898; } I am loving using paper index cards. I am, however, worried that something could happen to the cards and I could lose years of work. I did not have this work when my notes were all online. are there any apps that you are using to make a digital copy of the notes? Ideally, I would love to have a digital mirror, but I am not willing to do 2x the work.

      u/LBHO https://www.reddit.com/r/antinet/comments/y77414/worried_about_paper_cards_being_lost_or_destroyed/

      As a firm believer in the programming principle of DRY (Don't Repeat Yourself), I can appreciate the desire not to do the work twice.

      Note card loss and destruction is definitely a thing folks have worried about. The easiest thing may be to spend a minute or two every day and make quick photo back ups of your cards as you make them. Then if things are lost, you'll have a back up from which you can likely find OCR (optical character recognition) software to pull your notes from to recreate them if necessary. I've outlined some details I've used in the past. Incidentally, opening a photo in Google Docs will automatically do a pretty reasonable OCR on it.

      I know some have written about bringing old notes into their (new) zettelkasten practice, and the general advice here has been to only pull in new things as needed or as heavily interested to ease the cognitive load of thinking you need to do everything at once. If you did lose everything and had to restore from back up, I suspect this would probably be the best advice for proceeding as well.

      Historically many have worried about loss, but the only actual example of loss I've run across is that of Hans Blumenberg whose zettelkasten from the early 1940s was lost during the war, but he continued apace in another dating from 1947 accumulating over 30,000 cards at the rate of about 1.5 per day over 50 some odd years.

      reply note collection loss and damage don't repeat yourself zettelkasten Hans Blumenberg's zettelkasten OCR
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  107. github.com github.com
    assignment-c-cvs1-2001sukh/index.html at main · nic-dgl103-f22/assignment-c-cvs1-2001sukh
    6
    4. dhuds1 16 Oct 2022
      <p> FULL FOIL: $188/$219 with cut<p></p> <p> 3/4 FOIL: $172/$203 with cut</p> <p>1/2 FOIL: $158/$189 with cut</p> <p>1/4 FOIL: $144/$175 with cut</p>

      don't make each list item a paragraph, add a list tag

    6. dhuds1 16 Oct 2022
      <h1><u>Our Services</u></h1> <h2><li> Below is a list of services we proudly offer.</li></h2> <p> At Hairpins, we care about the environment and recognize the impact we all have on it. We are continually making strides to reduce where we can and have only aligned ourselves with companies and products we believe in.<p></p> <br> <p> We are proud to be a CERTIFIED GREEN CIRCLE SALON and through that partnership are able to divert 95% of our salon waste from landfills. Get in touch if you have any questions or want to learn more about the programs and charities we are focusing our efforts on.</p> <h3><li>Cuts</li></h3> <p>Range from 45 minutes to 90 minutes</p>

      <ul> is a parent tag of <li> you need to add a <ul> Additionally, in this situation I would recomment not using a list, instead just use the header and paragraph tags.

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  108. github.com github.com
    assignment-d-cvs1-Jewellsey/style.css at main · nic-dgl103-f22/assignment-d-cvs1-Jewellsey
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    assignment-c-cvs1-JosmiJose14/index.html at main · nic-dgl103-f22/assignment-c-cvs1-JosmiJose14
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    assignment-d-cvs1-Jan-elle-Chan/index.html at main · nic-dgl103-f22/assignment-d-cvs1-Jan-elle-Chan
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    nic-dgl103-f22/assignment-d-cvs1-yonishi1: assignment-d-cvs1-yonishi1 created by GitHub Classroom
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  113. github.com github.com
    assignment-c-cvs1-Manjeet-momi/index.html at main · nic-dgl103-f22/assignment-c-cvs1-Manjeet-momi
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    1. JanElleChan 17 Oct 2022
      </small></p>

      Perhaps small tag could have been used for a smaller amount of text placed on a separate line instead of an entire paragraph for the sake of readability.

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  114. www.biorxiv.org www.biorxiv.org
    Direct Cell Extraction of Membrane Proteins for Structure-Function Analysis
    1
    1. ASAPbio_crowd_preprint_review 17 Oct 2022

      Review coordinated via ASAPbio’s crowd preprint review

      This review reflects comments and contributions by Ruchika Bajaj, Sree Rama Chaitanya Sridhara and Sara El Zahed. Review synthesized by Ruchika Bajaj.

      This study has developed a novel one-step methodology for the incorporation of membrane proteins from cells to lipid Salipro nanoparticles for structure-function studies using surface plasmon resonance (SPR) and single-particle cryoelectron microscopy (cryo-EM), which is a profound technology in the field of membrane protein structural biology. We raise some points that may strengthen the manuscript below:

      • Main section, 4th paragraph “resuspended in digitoxin-containing buffer”- Does the sentence mean that membrane proteins were solubilized by detergent before reconstitution into salipro particles? Are salipro and digitoxin added at the same step? If this is the case, it is unclear how one can distinguish between the step wise solubilization and reconstitution or direct reconstitution into salipro particles. Further discussion on the mechanism of reconstitution would be helpful. In the same paragraph, the fragment “to increase membrane fluidity and render lipids” raises the question of whether the concentration of digitonin was optimized to balance the increase in membrane fluidity but not rendering the solubilization of membrane proteins.
      • Main section, 4th paragraph, “the formation of saponin-containing mPANX1-GFP particles was assessed by analytical size exclusion chromatography using fluorescence detector” - It is assumed that fluorescence is detected from GFP. As the construct expressed is PANX1-GFP, GFP fluorescence signal will be received from reconstituted as well as not reconstituted PANX1. Is saponin specific signal being used as a signal for measuring the reconstitution of PANX1-GFP? In the same paragraph, “PreScission protease for on-column cleavage” is mentioned. Is GFP still intact in the expressed PANX-1 or is it cleaved? A diagram of these procedures showing the various steps will be helpful for readers.
      • Main section, 4th paragraph “SDS-PAGE revealed the formation of pure and homogeneous Salipro-mPANX1 nanoparticles”- However, extra bands are present above the major band in Figure 1E, can some comment be provided on this point. Possible explanations for the additional bands could be post translational modifications or degradation of mPANX1.
      • Methodology section, “membrane protein reconstitution screening using fluorescence-detection size exclusion chromatography (FSEC)” - The amount of salipro is given in ug. A comment on the ratio of protein to salipro particles would be important to decide the concentration of salipro with respect to the mass of the cell pellet.
      • Figure 1G: The molecular weight of Salipro-mPANX1 particles is mentioned to be approximately 466kD. mPANX1 weighs about 48kD and heptamer will be 336kDa. A discussion on comparison of experimental and actual molecular weight would be interesting.
      • hPANX1 was expressed in sf9 insect cells. A description regarding trials of expression of this construct in expi293 cells would be informative.
      • Supplemental Figure 1B: The gel is overloaded and shows multiple bands for hPANX1, recommend selecting an alternative image for hPANX.
      • Paragraph 6A phrase, “challenged with bezoylbenzoyl-ATP(bzATP), spironolactone and cabenoxolone” - Please explain the meaning of ‘challenged’ here.
      • Supplementary Figure 2: Paragraph 6 mentions “binding constant could not be determined”. Please provide an explanation for this. Is it about the saturation phase not being approachable because of the feasibility of the binding experiment at higher concentration of cabenoxolone?
      • The last summary sentence in Paragraph 6 is not clear, recommend rephrasing it.
      • Figure 2A shows that Salipro particles have His tag. This suggests that an additional step of affinity purification with His tag could have been used to distinguish or separate reconstituted and un-reconstituted PANX1.
      • Supplementary figure 4: Please explain whether the datasets for samples in the presence and absence of fluorinated lipids were combined together.
      • Paragraph 8, “intracellular helices were not well resolved” - Please comment on a possible explanation. Does the Salipro scaffold contribute to the resolution? Please mention any future possibilities regarding improving the resolution by modifying the salipro scaffold or alternative scaffold. In the same paragraph, rmsd is mentioned at promoter level, please comment on how this value changes at heptamer level and why is it important to report the rmdd value to appreciate the direct reconstitution methodology.
      • Last paragraph 10, “future membrane protein research” - Please comment on the utility of this methodology on prokaryotic membrane proteins, bacterial outer or inner membrane proteins or eukaryotic membrane proteins. Some more examples of reconstitution with the same method will support the applicability of this methodology on diverse kinds of membrane proteins. A discussion section comparing this methodology to other methods would also be useful for readers.
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  115. github.com github.com
    assignment-c-cvs1-2001sukh/index.html at main · nic-dgl103-f22/assignment-c-cvs1-2001sukh
    1
    1. dhuds1 16 Oct 2022
      We are incredibly proud of our diverse team of stylists who greet each client with a smile. We prioritize inclusivity, community, and sustainability, and make sure that everyone who walks through our door feels welcome.<br><br>

      needs a tag

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  116. github.com github.com
    nic-dgl103-f22/assignment-c-cvs1-2001sukh: assignment-c-cvs1-2001sukh created by GitHub Classroom
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    1. dhuds1 16 Oct 2022
      Tel:<a href="(250) 338-7467 ">(250) 338-7467 </a> <br><br> Email: <a href=" salon.hairpins@gmail.com "> salon.hairpins@gmail.com </a><br><br>

      needs to have a tag. <a href="tel:+2503387467">to add a telephone, <a href="mailto:salon.hairpins@gmail.com"> to add email

    2. dhuds1 16 Oct 2022
      Hairpins Boutique Salon #4 - 224 6th Street Courtenay, BC V9N 1M1 Check us out on Google Maps <a href="https://www.google.com/maps/place/Hairpins+Boutique+Salon/@49.6904218,-124.9971279,15z/data=!4m2!3m1!1s0x0:0x859b2cfce3bc31ea?sa=X&ved=2ahUKEwiYi_2Ex6T6AhXNMjQIHYkoCUUQ_BJ6BAhSEAc)">https://www.google.com/maps/place/Hairpins+Boutique+Salon/@49.6904218,-124.9971279,15z/data=!4m2!3m1!1s0x0:0x859b2cfce3bc31ea?sa=X&ved=2ahUKEwiYi_2Ex6T6AhXNMjQIHYkoCUUQ_BJ6BAhSEAc)</a>

      needs to have a tag

    4. dhuds1 16 Oct 2022
      Questions, comments, ready for a new do? We look forward to hearing from you! If you're looking to make an appointment online, please do so here.

      needs to have a tag.

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  117. github.com github.com
    assignment-d-dlu-trinitybabichuk/index.html at main · nic-dgl103-f22/assignment-d-dlu-trinitybabichuk
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    assignment-c-dlu-trinitybabichuk/contact.html at main · nic-dgl103-f22/assignment-c-dlu-trinitybabichuk
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    1. abbeygirvan 14 Oct 2022
      <!--Receiving an error for my h2 closing tag, unsure-->

      might be because you've closed your h2 before your div. remember opens and closes should mirror eachother. ex) h2, div, content, /div, /h2

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    nic-dgl103-f22/assignment-d-cvs2-Anmoldeep06: assignment-d-cvs2-Anmoldeep06 created by GitHub Classroom
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  121. www.biorxiv.org www.biorxiv.org
    Magnesium modulates Bacillus subtilis cell division frequency
    2
    1. jkherman.fud 13 Oct 2022
      Read on Arcadia Science

      Oh no! I thought the flag meant post & accidentally reported your thoughtful feedback to moderators instead of replying. Hopefully they figure it out (no obvious way to contact them or "unflag"

      Comment Figure 2C → please include indication of statistical significance Figure 3C → please include indication **of statistical significance Figure 6A → please include indication of statistical significance Figure 8B → please include indication of statistical significance Figure S1B → please include indication of statistical significance Figure S3B → please include indication of statistical significance

      Response Easy to add

      Comment For your overexpression experiments, do the overexpressed proteins have a tag? It would be helpful to have Western blot data showing that the particular proteins are actually being overexpressed. I think the phenotypes that you observe are very compelling so I don’t doubt the conclusions. Western blot data would just provide some additional confirmation that you are actually achieving overexpression of UppS, MraY, and BcrC.

      Response The proteins are untagged. For the UppS and BcrC the cell shortening occurs with addition of inducer, , so strong indication expression is occurring. A western would provide information about degree of overexpression, but we don’t think is necessary to support conclusion drawn. Do you think there is an alternative possibility that needs to be excluded? We note that in another preprint (https://www.biorxiv.org/content/10.1101/2022.02.03.479048v1) the authors delete the native uppS in their inducible Phy-uppS strain (Fig S4) and at 100 uM IPTG (10X less than what we used in experiment) the cells have wt growth on LB plates, so we at least know the Phy-uppS is functional and made (or they would die!). We are introducing the uppS deletion into our strain to see if we can identify a concentration of IPTG that doesn’t affect cell growth but still induces shortening.

      For MraY, the result is negative, so you are spot on – it is impossible to tell if due to lack of overexpression from data shown. We only know the strain is correctly made from sequencing. We will investigate if there is an antibody or functional fusion available. The reason we were not sure was worth doing is because the MraY reaction is reversible (15131133). This means that without a phenotype, there is no simple way to know the reaction can even be pushed forward even if the overexpression is confirmed (more negative data). We actually overexpressed some other proteins that act downstream (MraY, MurJ, AmJ) and they were also negative for shortening. Probably we should remove the negative data or reword to make the caveats of the negative result clear.

      Question Based on your data, there are definitely differences in gene expression when you compare cells grown in media with and without magnesium. Because the majority in cell length increase occurs in such a short time though (the first 10min), I was wondering if you think that some or most of it is not due to gene expression?

      Response The shortening is even faster than 10 min (not only statistically significant, but also obvious qualitatively if we mount immediately after adding Mg2+ ). We did not include the first timepoint because original purpose was to check everything was ready with microscope – did not expect shortening so fast! We can definitely add that data in. When we saw, we tried to capture the transition on pads, but going from culture to pad seems to stress the cells too much in the small window where the cool stuff happens. Since growth rate doesn’t appear to be a big factor in those initial divisions, we might be able to grow at lower temp and shift to pads for adjustment period before adding Mg2+. Did not play with it much due to lack of resources atm, but a flowcell setup would probably be best. In short, we think rapid divisions right after transition do not require transcription or translation. It really “smells” more like a biophysical thing.

      Question Do you have any hypotheses what is most likely to be affected by magnesium? Do you think if the membrane may be affected?

      Response We have a lot of hypotheses, but all speculative. There could be an extracytoplasmic enzyme involved in envelope synthesis is sensitive to Mg2+ availability, and that at lower concentrations, its activity is affected. There is some old literature with membrane preps that suggests PG synthesis requires higher Mg2+ than teichoic acid synthesis. If Und-P is limiting, higher Mg2+ may shift make the pool more available to make the septum. Tingfeng initially hypothesized there might be a receptor/signal mechanism but has not been able to identify one. Und-P seems to be important, but “availability” is not just pool, but how fast (and where!) the flipping across the membrane occurs. If Und-PP needs to be dephosphorylated to Und-P before being flipped back to cytoplasmic side, anything that effects the PP/Pi equilibrium would be predicted to affect the reaction rate, with lower Pi (in periplasm or pseudoperiplasm in case of G+) favoring the dephosphorylation. Cell wall associated Mg2+ could shift equilibrium to be more favorable for a Und-PP phosphatase more closely associated with the divisome. I could go all day… In short, we don’t know enough!

      Question Why do you think less magnesium activates this program of less division and more elongation? Additionally why is abundant magnesium activating a program of increased cell division and less elongation? Do you think there is some evolutionary advantage, especially considering how important magnesium is for ATP production?

      Response In the window we looked at, the elongation rate is constant (not less or more) and only the division frequency changes. Some bacteria (like Caulobacter and to lesser extent E. coli) clearly elongate and divide simultaneously, so model there is some competition for substrate (like Lipid II) makes sense. Septators like Bacillus seem to delineate the two processes more, though we have found conditions where even Bacillus invaginates during division, so it’s not absolute. Like eukaryotic cells, bacterial undoubtedly have mechanisms not only commit to a round of DNA replication when there is some signal that resources are sufficient. Clearly with some bacteria, this is not the case with cell division. The alternative would be that every cell cycle there is an opportunity to divide if some threshold of something(s) is reached. There is a hypothesis from Mtb literature that it may be GTP, but it’s not at all clear that is sufficient. In yeast, size at cell division is affected by perturbing 1-C pool.

      Question Related to this previous question, I also wonder if this magnesium-dependent phenotype would extend to other unicellular organisms, may be protists or algae? That would be a really exciting direction to explore!

      Response It’s a great question – lots to do! We didn’t even look at another Gram-positive, but we plan to. It’s trickier to limit Mg2+ in Gram-negatives (see 27471053 – we tried Bsub homolog for those wondering – it’s not responsible for phenotype we see).

      Question Regarding the zinc and manganese experiments, why do you think they lead to additional phenotypes compared to magnesium? Do you have any hypotheses?

      Response We have hypotheses, but if my (Jen’s @rosh_ba) twitter engagement is any indication, way too speculative for public consumption at present. Need acquire preliminary data/write grant.

      Question Regarding your results that Lipid I availability may be a major a problem for the cell division in the absence of magnesium, do you think that is due to effects magnesium has on the enzymes directly, or do you think magnesium affects the substrate availability/conformation by coordinating the phosphate groups? Or something else, may be membrane conformation?

      Response Several proteins involved in envelope synthesis (like UppS) are Mg2+ dependent enzymes. But at least for intracellular players, levels of Mg2+ should be more than high enough to support enzyme activity (0.8 – 3.0 mM is Bsub range I recall off top of head). Could have impact extracytoplasmically by lowering pool sponged into the cell wall, but intuition (for what that is worth) is that it is not the coordination of an enzyme with a metal that is impacted rather the equilibrium with other ions like Pi and H+ and their impact on net ATP synthesis. Lots to think about and do, and no simple answers. When Tingfeng started project idea was to find mechanism – didn’t realize we were asking “How does the cell work?” Turned out to be a bit much for a dissertation project :)

    2. AtanasRadkov 07 Oct 2022

      General comments:

      This study carefully delineates the role of magnesium in cell division versus cell elongation. The results are really important specifically for rod-shaped bacteria and also an important contribution to the broader field of understanding cell shape. Specifically, I love that they are distinguishing between labile and non-labile intracellular magnesium pools, as well as extracellular magnesium! These three pools are really challenging to separate but I commend them on engaging with this topic and using it to provide alternative explanations for their observations!

      A major contribution to prior findings on the effects of magnesium is the author’s ability to visualize the number of septa in the elongating cells in the absence of magnesium. This is novel information and I think the field will benefit from the microscopy data shown here.

      I completely agree with the authors that we need to be more careful when using rich media such as LB. It is particularly sad that we may be missing really interesting biology because of that! It’s worth moving away from such media or at least being more careful about batch to batch variability. Batch to batch variability is not as well appreciated in microbiology as it is for growing other cell types (for example, mammalian cells and insect cells).

      For me, the most exciting finding was that a large part of the cell length changes within the first 10min after adding magnesium. The authors do speculate in the discussion that this is likely happening because of biophysical or enzymatic effects, and I hope they explore this further in the future!

      I love how the paper reads like a novel! Congratulations on a very well-written paper!

      Kudos to the authors for providing many alternative explanations for their results. It demonstrates critical thinking and an open-mind to finding the truth.

      Specific comments:

      Figure 2C → please include indication of statistical significance

      Figure 3C → please include indication of statistical significance

      Figure 6A → please include indication of statistical significance

      Figure 8B → please include indication of statistical significance

      Figure S1B → please include indication of statistical significance

      Figure S3B → please include indication of statistical significance

      For your overexpression experiments, do the overexpressed proteins have a tag? It would be helpful to have Western blot data showing that the particular proteins are actually being overexpressed. I think the phenotypes that you observe are very compelling so I don’t doubt the conclusions. Western blot data would just provide some additional confirmation that you are actually achieving overexpression of UppS, MraY, and BcrC.

      Questions:

      Based on your data, there are definitely differences in gene expression when you compare cells grown in media with and without magnesium. Because the majority in cell length increase occurs in such a short time though (the first 10min), I was wondering if you think that some or most of it is not due to gene expression? Do you have any hypotheses what is most likely to be affected by magnesium? Do you think if the membrane may be affected?

      Why do you think less magnesium activates this program of less division and more elongation? Additionally why is abundant magnesium activating a program of increased cell division and less elongation? Do you think there is some evolutionary advantage, especially considering how important magnesium is for ATP production?

      Related to this previous question, I also wonder if this magnesium-dependent phenotype would extend to other unicellular organisms, may be protists or algae? That would be a really exciting direction to explore!

      Regarding the zinc and manganese experiments, why do you think they lead to additional phenotypes compared to magnesium? Do you have any hypotheses?

      Regarding your results that Lipid I availability may be a major a problem for the cell division in the absence of magnesium, do you think that is due to effects magnesium has on the enzymes directly, or do you think magnesium affects the substrate availability/conformation by coordinating the phosphate groups? Or something else, may be membrane conformation?

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    biorxiv.org/content/10.1101/2022.10.01.510448v1
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    Genome-wide Profiling of Histone Modifications in Plasmodium falciparum using CUT&RUN.
    2
    1. EMBOpress 13 Oct 2022

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      Reply to the reviewers

      Reviewer #2 (Evidence, reproducibility and clarity (Required)):

      This paper provides a detailed step by step protocol of the CUT&RUN technique, which enables high-resolution chromatin mapping and probing, adapted to the malaria parasite Plasmodium falciparum. In particular, Kafsack and colleagues apply the CUT&RUN protocol to infected red blood cells from in-vitro culture and obtain very good quality genome-wide profiles of two histone modifications, H3K4me3 and H3K9me3. The results are congruent with previous ChIP-seq data with a substantial improvement in terms of coverage and chip-to-input noise. The protocol is very detailed and the figures are great.

      Major comments: 1. Authors successfully adapted the CUT&RUN protocol in P. falciparum. First, the binding profiles obtained by CUT&RUN for H3K4me3 and H3K9me3 are very similar to those reported by previous ChIP-seq studies. Secondly, by down-sampling 4X and 16X the test samples, authors demonstrate that 1M PE reads of sequencing depth would be enough to obtain accurate profiling of these histone modifications.

      Despite this data is convincing, only one region in chr. 8 is shown as an example in figures 2, 3 and 4.

      Different regions should be included, at least as supplementary figures, to reinforce their conclusions.

      __Response: __We chose that locus on chromosome 8 to provide a gene-level resolution view at a locus encompassing genes in both eu- and heterochromatin states. We have now included Supplementary Figure 1, which shows these tracks for full length chromosomes 4 and 7. Additionally, genome-wide enrichment tracks for all data sets in this study are available for download at NCBI Gene Expression Omnibus under accession number GSE210062.

      Related to this, there is evidence of the impact of chromatin structure on ChIP-seq analysis. Specifically, heterochromatin is typically depleted in ChIP input controls because of technical and experimental issues and this can result in a false enrichment of heterochromatic regions in the tested sample. How represented is heterochromatin (i.e. sub-telomeric and telomeric regions) in the test and control samples using the cut&run protocol?

      __Response: __The reviewer is correct that chromatin structure may alter accessibility which may bias absolute measurements but since the accessibility biases based to chromatin-structure are identical for both the histone PTM-specific antibody and the isotype control and cancel out in the enrichment score.

      How biased is the cut&run sample compare to the ChIP-seq sample?

      __Response: __We have included this in Supplementary Figure 1. The H3K4me3 and H3K9me3 enrichment scores are strongly correlated both between CUT&RUN replicates and between CUT&RUN and previously published ChIP-seq results.

      In this sense, it would be desirable if authors provide more information about the quality analysis results, for example the chip to input signal ratio and the coverage for heterochromatic (telomeric, centromeric and subtelomeric) regions.

      __Response: __We agree that this would be of interest to the reader. For this reason, the full genome-wide enrichment tracks were made available for all datasets in this study. We have added language to draw further attention to this availability.

      Additionally, loci typically biased in ChIP-seq samples, i.e. clonally variant gene families in sub-telomeric regions, should be shown as examples.

      __Response: __We chose that locus on chromosome 8 to provide a gene-level resolution view at a locus encompassing genes in both eu- and heterochromatin states, including 2 var genes (PF3D7_0808600 and PF3D7_0808700) and two rifin genes (PF3D7_0808800 and PF3D7_0808900). We have now included Supplementary Figure 1, which shows these tracks for full length chromosomes 4 and 7, which also include subtelomeric and non-subtelomeric heterochromatin loci containing these genes. Additionally, genome-wide enrichment tracks for all data sets in this study are available for download at NCBI Gene Expression Omnibus under accession number GSE210062.

      1. For P. falciparum WGS a PCR-free library preparation is strongly recommended. We wonder if it would be possible to try to integrate this step in their CUT&RUN protocol.

      __Response: __Since such biases are sequence dependent, they would impact raw coverage but cancel out in the enrichment plots since the sequence-based biases are identical in both samples. While PCR-free amplification may be desirable for some applications, we feel this is outside the scope of this study to implement these changes.

      It would have been desirable to have tried the CUT&RUN protocol on other type of proteins, different to hPTMs which are highly abundant, for example one of the Pf Api-AP2 transcription factors. Assaying the CUT&RUN protocol on a different type of protein shouldn't be cost/time consuming and would provide evidence of the versatility of the approach.

      Response: As indicated by the title, this protocol was optimized specifically for profiling of histone modifications. CUT&RUN has been used in other systems to profile genome-wide binding of other proteins but this was not our aim and outside the scope of this study.

      1. The step by step protocol is very detailed, however there are some parts that need to be better explained:

      In the section "Binding cells to Concanavalin A-coated beads": it's not mentioned the harvest time and the stage of the parasites used. In addition, several methods are proposed for iRBCs enrichment, but is not mentioned which method was used and the life stage of the parasites. In this part of the protocol authors state "resuspend cells containing 1-5x107 nuclei to a cell density to 1x107 cells/mL".

      According to our calculations, to guarantee this nuclei number it would be necessary to enrich in iRBCs and late stages. Otherwise the red blood cells density should be much larger. Could you please clarify this point?

      Response: For this study we enriched for trophozoites using a percoll/sorbitol density gradient, which we have now clarified in step 8. However, whether and which enrichment strategy is employed will vary based on the desired parasite stages and experimental design.

      In the section "P. falciparum culturing and synchronization of erythrocytic stages" the authors indicate that the method used for synchronization was double-synchronization with sorbitol treatment to achieve a {plus minus} 6 h synchrony. The details provided appear insufficient to replicate the procedure. E.g. it's not explained how the double step synchronization was performed and for how long the culture was incubated after the synchronization.

      The number of parasite cells and the life-stage used is mentioned at the end (in the section of expected outcomes). It would be more useful if this information is specified at the beginning together with the most appropriate procedure to get an iRBC culture well synchronised and enriched in late stages.

      Response: The stage, synchrony and growth conditions are determined by the scientific question the experimenter is asking, not by the assay. For this reason, we provide the number of infected erythrocytes and nuclei used in our studies so that other experimenters can aim for similar numbers regardless of the stage and synchrony. For this study we used asexual blood-stages at 36±4 hp.i. We have clarified this in step 11.

      • With regards to reproducibility, all experiments were done in replicate (3 Rs) and the statistics appear adequate.

      Minor comments: - Abstract. A closing bracket is missing.

      Response: Corrected - Step 11: Split each sample into 1mL aliquots at ?

      Response: Corrected

      • The affinity of proteins A/G to IgG antibodies varies based on host species and IgG subtype (see link). This link does not seem to work

      Response: Corrected

      • Low bind tubes are mentioned several times. Please clarify whether it refers to low bind protein or low bind DNA. Step 77.

      Response: The vendor and catalog number for the low-bind tubes are specified in the Reagents, Materials & Equipment list.

      Which was the desired sequencing depth per library? It could be mentioned here. It is mentioned later in "Quantification and statistical analysis" that the initial desired depth was of 40M read pairs, but what was the real depth obtained? from the Figure 4 seems to be less than 17M read pairs per sample.

      Response: Thank you for catching that error. The target was 10M read pairs per library but since CUT&RUN is so specific the isotype controls release less DNA and the resulting libraries produces fewer clusters than aimed for leading to slight over sequencing of the remaining samples.

      • Step 79. Please clarify/justify why 50 bp paired-end reads were chosen as sequence length. Response: After excluding the telomere repeats 100 bp (50+50) are sufficient for uniquely mapping 98.3% of the nuclear. Paired-end sequencing was chosen over single-end because it provides the actual size of each fragment.

      In the section "Quantification and statistical analysis", references to Figure 3 and 4 are inverted or do not correspond with the actual figures 3 and 4.

      Response: Corrected

      • Figure 2. Among the replicates, sample 2 seems to have higher background, could you comment why? Response: It is inherent in biological replicates that one would have the greatest amount of noise but we unfortunately have no further insight into why Sample 2 had a higher elevated background that Samples 1 and 3. Furthermore, even with this slightly higher background the relative enrichment of signal to noise ratio enrichment peaks are readily identifiable.

      • Below some suggestions that may help the authors improve the presentation of their data and conclusions:

      The limitations and potential shortcomings of the protocol are mentioned along the text (e.g. the use of different antibodies, different targets, weak interactions..), but could be good if they are included in a different section, preferably at the end.

      Response: A “Limitations” section was added.

      Also in this section it would be good if they develop further (or at least speculate) on the differences in the protocol or things to consider if other type of proteins are assayed (i.e. TFs).

      Response: As mentioned above, we have not applied to CUT&RUN to profile chromatin other than Histone PTMs, as this was not the aim of our study. Since chromatin-bound histones always occur within a nucleosomal context, we are hesitant to make claims to the utility of this specific protocol for profiling DNA-binding proteins with smaller DNA-binding footprints. That said, CUT&RUN has been used to great success in other systems to profile a wide range of chromatin-bound proteins. We have included mention of this at the end of the introduction.

      Authors should better comment on the potential impact of chromatin structure and DNA sequence (i.e. AT richness) on the biased representation of heterochromatic regions in the data, the level of background and the peak calling analysis.

      Response: For the enrichment scores, sequence and accessibility biases cancel out since they are the same for both the PTM-specific antibody and the isotype controls.

      The coverage of critical loci, like those belonging to clonally variant gene families, should be calculated and examples of tracks included as supplemental figures.

      Response: Gene Expression Omnibus under accession number GSE210062 as indicated in the Quantification & Statistical Analysis and data availability sections.

      Authors claim that the CUT&RUN protocol has exceptionally low background and has been successfully used to profile chromatin interactions from very small numbers of cells. But it is not specified how many. That is, which is the standard in other fields and how it compares with the number of cells used here.

      Response: As stated in the note following step 10, we did not optimize the minimum number of parasites required in this study since at the 1e7 iRBC required for each sample correspond as little as 1mL of bloodstage culture 2% parasitemia and 5% hematocrit. The down-sampling analysis in figure 3 suggests that the number of input cells can likely be reduced at least 10-fold.

      Information about synchronisation, estimation of iRBCs density and nuclear content appears insufficiently described and has been fragmented in different sections so it is difficult to replicate. For example, within the section "Binding cells to Concanavalin A-coated beads" different alternative protocols for iRBCs synchronisation and enrichment are mentioned but it is not clear whether authors actually perform that step. It could be convenient to describe it and include it in the step-by-step protocol.

      Response: The stage, synchrony and growth conditions are determined by the scientific question the experimenter is asking, not by the assay. For this reason, we provide the number of infected erythrocytes and nuclei used in our studies so that other experimenters can aim for similar numbers regardless of the stage and synchrony. For this study we used asexual blood-stages at 36±4 hp.i. We have clarified this in step 11.

      Significance (Required) The CUT&RUN is a novel technique to profile chromatin modifications genome-wide that has been successfully adapted to P. falciparum by the authors. This technique overcomes important limitations of the traditional ChIP-seq and provides better quality data. First, fewer cells and lower sequencing depths are required which is fundamental for the analysis of certain parasite life stages. Second, the binding step is carried out in-situ using unfixed and intact cells. This allows to avoid crosslinking, which can interfere with target recognition that results in unspecific background, and also avoids the random fragmentation of the chromatin, that can bias in the analysis.

      This work is significant since it represents the first CUT&RUN step by step protocol adapted to P. falciparum. The results are important for researchers from the malaria field and parasitologists in general who could eventually leverage this protocol to other Apicomplexa.

      Our expertise is on transcriptional regulation, molecular parasitology, genomics and epigenomics, of malaria parasites. We hope the comments above will help the authors to improve the ms. Congratulations on the work.

      Reviewer #3 (Evidence, reproducibility and clarity (Required)): ____ In general the paper is very clear and convincing and I have only minor comments for the authors to address.

      Introduction The authors state that 'crosslinking presents another challenge as it can interfere with antibody recognition.' Would it be possible to provide a reference to strengthen this statement? Response: Additional references (Baranello et al, O’Neill et al) were added.

      In the third paragraph the authors mention that CUT&RUN can be used to profile chromatin interactions from very small numbers of cells. This argument would be strengthened by adding references or examples from mammalian systems, and the authors might mention that the slight modification CUT&TAG has been employed for single cell sequencing. https://doi.org/10.1038/s41587-021-00865-z.

      Response: The reference was added.

      Figure 2 A label of Relative fold enrichment should be added to the y axis. This applies also to Figure 4. In the legend, it isn't entirely clear from what control the fold enrichment is being generated. Based on the other figures I assume it's the isotype control, and it would be helpful to state that in the legend.

      Response: Thank you for the suggestion. We have made these changes.

      Figure 3 An HP1 ChIP is included but there is no track for HP1 using CUT&RUN. It isn't entirely clear to me why HP1 is included; is it to make the point that it overlaps with H3K9me3? There is a sentence at the end of the Quantifcation and statistical analysis section that indicates that an HP1 CUT&RUN experiment was performed ('Representative tracks of H3K9me3, H3K4me3, and HP1 produced using CUT&RUN or ChIPseq at chromosome 8 of P. faliciparum are shown in Figure 4), but I don't see an HP1 track for Figure 4 and I don't see CUT&RUN HP1 tracks on Figure 3.

      Response: Correct, no HP1 CUT&RUN was performed, we are just trying to show that H3K9me3 CUT&RUN recapitulates ChIP-seq of both H3K9me3 and HP1, which binds to H3K9me3.

      Figure 4 The downsampling of reads is a nice demonstration that low numbers of reads are required for the CUT&RUN technique. It might be helpful to include downsampling of ChIP-seq reads within this figure to compare the two techniques more directly.

      Response: The reason we included the down-sampling of CUT&RUN sequence reads was to explore whether were over-sequencing our CUT&RUN libraries not to provide a comparison to ChIP-seq. For simplicity we have therefore kept the figure as is.

      DNA purification by Phenol/Chloroform extraction In step 38, I noticed that RNAse was not added at this step, as described in the original paper by Skene et al. Can the authors make a brief note about why they omit this reagent?

      Response: RNAse A is already present since it was included in the STOP buffer at step 35.

      The authors mark the TE buffer in bold, but I don't see a description of its makeup in the buffer section, though possibly I missed it. While this is a pretty standard buffer, it might still be nice to include it for completeness.

      Response: TE buffer recipe was added.

      Clean-up of PCR amplified library Between step 70 and 71 the authors include a warning to not discard the beads. However, this warning is not included in the Post-ligation Clean-up, which involves much the same procedure. Response: Corrected

      Typos and writing It might be helpful to define CUT&RUN in the abstract by spelling out the acronym there. Response: It is defined in the 3rd paragraph of the introduction

      I've mostly seen ChIP-seq with a dash between the IP and the seq.

      Response: Corrected

      Powerful is used twice in consecutive sentences in the first paragraph of the introduction. Consider substituting the words 'important tool' for 'powerful tool.' Response: Corrected

      Figure 2 legend. “(purple) in of three biological replicates” should be “(purple) in three biological replicates” Response: Corrected

      Figure 3 legend Last sentence should include 'to' between the words 'shown' and 'the'. Response: Corrected

      Post-ligation Clean-up “Wash twice with 200µl of 80% Ethanol freshly prepared” should be “Wash twice with 200µl of freshly prepared 80% Ethanol” Response: Corrected

      Library PCR amplification “Fragments are PCR amplified using Kapa polymerase, which it is more efficient” should be “Fragments are PCR amplified using Kapa polymerase, which is more efficient” Response: Corrected

      Figure 7 legend “Indicated in the tope left panel” Should be “Indicated in the top left panel” Response: Corrected

      Expected outcomes “to dismiss any sort of contamination” should be “To dismiss contamination” Response: Corrected

      Potential Solution After the sentence 'Incubation buffer is added.' The next letter 'i' should be capitalized in the word Isolate. Response: Corrected

      CROSS-CONSULTATION COMMENTS Plasmodium is not my model organism, so I'd defer to Reviewer 2 on the comments regarding additional detail for synchronization and Plasmodium culture conditions. I have nothing further to add, and I'm excited to see what experiments come from the addition of this technique to the parasite field.

      Reviewer #3 (Significance (Required)):

      This excellent methods paper describes a detailed protocol for the adaptation of the Cleavage Under Targets & Release Using Nuclease (CUT&RUN) technique to Plasmodium falciparum, the causative agent of malaria. CUT&RUN is an alternative to ChIP-seq, and has the advantage that it does not require crosslinking of targets, which can introduce artifacts and cause issues with antibody recognition. CUT&RUN can also be performed with low numbers of cells and has an excellent signal to noise ratio, which the authors demonstrate by downsampling the number of reads used in their analysis. The authors also clearly demonstrate that profiling of histone modifications using CUT&RUN yields comparable results to ChIP-seq. Because it can be difficult to obtain large numbers of cells from Plasmodium cultures, CUT&RUN is especially useful in this important model system. Publication of a detailed protocol will help other Plasmodium researchers answer important questions regarding genomic localization for their targets of interest.

      PeerReviewed
    2. EMBOpress 13 Oct 2022

      Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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      Referee #3

      Evidence, reproducibility and clarity

      In general the paper is very clear and convincing and I have only minor comments for the authors to address.

      Introduction

      The authors state that 'crosslinking presents another challenge as it can interfere with antibody recognition.' Would it be possible to provide a reference to strengthen this statement?

      In the third paragraph the authors mention that CUT&RUN can be used to profile chromatin interactions from very small numbers of cells. This argument would be strengthened by adding references or examples from mammalian systems, and the authors might mention that the slight modification CUT&TAG has been employed for single cell sequencing. https://doi.org/10.1038/s41587-021-00865-z.

      Figure 2

      A label of Relative fold enrichment should be added to the y axis. This applies also to Figure 4. In the legend, it isn't entirely clear from what control the fold enrichment is being generated. Based on the other figures I assume it's the isotype control, and it would be helpful to state that in the legend.

      Figure 3

      An HP1 ChIP is included but there is no track for HP1 using CUT&RUN. It isn't entirely clear to me why HP1 is included; is it to make the point that it overlaps with H3K9me3? There is a sentence at the end of the Quantifcation and statistical analysis section that indicates that an HP1 CUT&RUN experiment was performed ('Representative tracks of H3K9me3, H3K4me3, and HP1 produced using CUT&RUN or ChIPseq at chromosome 8 of P. faliciparum are shown in Figure 4), but I don't see an HP1 track for Figure 4 and I don't see CUT&RUN HP1 tracks on Figure 3.

      Figure 4

      The downsampling of reads is a nice demonstration that low numbers of reads are required for the CUT&RUN technique. It might be helpful to include downsampling of ChIP-seq reads within this figure to compare the two techniques more directly.

      DNA purification by Phenol/Chloroform extraction In step 38, I noticed that RNAse was not added at this step, as described in the original paper by Skene et al. Can the authors make a brief note about why they omit this reagent?

      The authors mark the TE buffer in bold, but I don't see a description of its makeup in the buffer section, though possibly I missed it. While this is a pretty standard buffer, it might still be nice to include it for completeness.

      Clean-up of PCR amplified library

      Between step 70 and 71 the authors include a warning to not discard the beads. However, this warning is not included in the Post-ligation Clean-up, which involves much the same procedure.

      Typos and writing

      It might be helpful to define CUT&RUN in the abstract by spelling out the acronym there.

      I've mostly seen ChIP-seq with a dash between the IP and the seq.

      Powerful is used twice in consecutive sentences in the first paragraph of the introduction. Consider substituting the words 'important tool' for 'powerful tool.'

      Figure 2 legend.

      (purple) in of three biological replicates

      Should be

      (purple) in three biological replicates

      Figure 3 legend Last sentence should include 'to' between the words 'shown' and 'the'.

      Post-ligation Clean-up Wash twice with 200µl of 80% Ethanol freshly prepared

      Should be

      Wash twice with 200µl of freshly prepared 80% Ethanol

      Library PCR amplification Fragments are PCR amplified using Kapa polymerase, which it is more efficient

      Should be

      Fragments are PCR amplified using Kapa polymerase, which is more efficient

      Figure 7 legend Indicated in the tope left panel

      Should be

      Indicated in the top left panel

      Expected outcomes to dismiss any sort of contamination

      Should be

      To dismiss contamination

      Potential Solution After the sentence 'Incubation buffer is added.' The next letter 'i' should be capitalized in the word Isolate.

      CROSS-CONSULTATION COMMENTS

      Plasmodium is not my model organism, so I'd defer to Reviewer 2 on the comments regarding additional detail for synchronization and Plasmodium culture conditions. I have nothing further to add, and I'm excited to see what experiments come from the addition of this technique to the parasite field.

      Significance

      This excellent methods paper describes a detailed protocol for the adaptation of the Cleavage Under Targets & Release Using Nuclease (CUT&RUN) technique to Plasmodium falciparum, the causative agent of malaria. CUT&RUN is an alternative to ChIP-seq, and has the advantage that it does not require crosslinking of targets, which can introduce artifacts and cause issues with antibody recognition. CUT&RUN can also be performed with low numbers of cells and has an excellent signal to noise ratio, which the authors demonstrate by downsampling the number of reads used in their analysis. The authors also clearly demonstrate that profiling of histone modifications using CUT&RUN yields comparable results to ChIP-seq. Because it can be difficult to obtain large numbers of cells from Plasmodium cultures, CUT&RUN is especially useful in this important model system. Publication of a detailed protocol will help other Plasmodium researchers answer important questions regarding genomic localization for their targets of interest.

      PeerReviewed
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    biorxiv.org/content/10.1101/2022.08.01.502378v2
  123. www.biorxiv.org www.biorxiv.org
    New submission 13/10/2022, 16:06:26
    2
    1. Public_Reviews 13 Oct 2022

      Reviewer #2 (Public Review):

      Horton et al combined computational and functional approaches to identify a role for a mouse transposable element (TE) family in the transcriptional response to interferon gamma (IFNG, also known as type II interferon). This paper builds on previous work, some of which was done by the corresponding author, in which TE families have been shown to contribute transcription factor binding sites to genes in a species-specific manner. In the current work, the authors analyzed datasets from mouse primary macrophages that had been stimulated by IFNG to identify TEs that might contribute to the transcriptional response to IFNG treatment. In addition to previously identified endogenous retrovirus subfamilies, the authors identified sites from another TE family, B2_Mm2, that they found contained STAT1 transcription factor binding sites and whose binding by STAT1 was induced following IFNG stimulation. To test the hypothesis that a B2_Mm2 element was providing IFNG-inducibility to an associated gene, the authors chose one of the 699 mouse genes that had nearby (<50 kb) B2_Mm2 elements and was upregulated upon IFNG treatment in previous datasets. The gene they chose was Dicer1, which also is upregulated by IFNG in mouse macrophages but not in human primary macrophages, furthering the hypothesis that the presence of B2_Mm2 in mouse cells may provide IFNG-inducibility to Dicer1. Following KO of a ~500 bp region in two separate clones of immortalized mouse macrophages, the authors show a decrease in basal as well as IFNG-induced expression of Dicer1, providing support for their conclusion that a B2_Mm2 is important for IFNG-inducibility. The authors further show that two nearby genes that are also upregulated by IFNG, Serpina3f and Serpina3g, are also reduced at basal conditions as well as when stimulated with IFNG. The authors use these data to suggest that additional elements in the B2_Mm2 element in the Dicer1 gene, possibly CTCF elements, are have long distance effects on transcription of nearby genes.

      Overall, this is an interesting and well written manuscript. The computational conclusions are supported by their data and add to the growing field of TEs and their role in transcription regulatory network evolution. While the authors do a good job of experimentally validating one example, inclusion of additional data, all of which they already have, as detailed below would substantially increase the applicability of their work and strengthen their conclusions about the broad role of TEs in the IFNG response in mice versus other species.

      1) Following their genome-wide comparisons, the authors hone in on Dicer1 as an interesting example in which they hypothesize that a B2_Mm2 element near the Dicer1 gene could be contributing to the fact that this gene is upregulated by IFNG in mouse cells but not human cells. What would be very useful to the readers of this paper is knowing how many other examples there might be like this one. Adding DEseq values from human RNAseq data the authors already use (current references 10 and/or 37) for identifiable human orthologs to Table S7 would thus strengthen their conclusions. If Dicer1 is unique in this aspect of having (a) a nearby B2_Mm2 element and (b) a binary difference between inducibility in mouse versus human cells, that is interesting. If Dicer1 is not unique, that strengthens the authors' assertion that B2_Mm2 insertions have altered the transcriptional network in a host-specific manner. Either way, the answer is interesting, but without including this analysis, the authors leave out an important aspect of their work and it remains unclear how generalizable their conclusions are.

      2) The results with Serpina3g and Serpina3F gene expression in the authors' knockout cells are very interesting. However, the authors focus almost exclusively on Serpina3g and Serpina3F, which makes it difficult to understand what is happening genome wide. Are other IFNG-induced genes (including those not on chromosome 12) similarly affected at the level of basal or induced transcription? How many genes are different in WT versus KO cells, both at basal and induced states? Does this correlate with their CUT&TAG data shown in Fig. 5? By focusing only on nearby genes (Serpina3g and Serpina3F), the authors hint that this may be a long range regulatory effect, "potentially mediated by the CTCF binding activity of the element" that they removed. But by only focusing on two nearby IFNG-induced genes, their data do not rule out the (also potentially quite interesting) possibility that there may be a more indirect role for this TE site or Dicer1 in basal transcription of IFNG-induced genes or IFNG-mediated gene expression. Providing more data on other genes throughout the genome in WT and KO cells, which the authors have generated but do not include in the manuscript, would help distinguish between these models. While a broader effect of these KOs on IFNG expression, or gene expression in general, would not fit as neatly with their model for local gene regulation, these analyses are needed to understand the effects of TE insertion on gene regulation.

      Review 2
    2. Public_Reviews 13 Oct 2022

      Reviewer #3 (Public Review):

      First of all, I enjoyed the manuscript by Horton et al. In the manuscript, they first re-analyzed published ChIP-seq data for STAT1 binding in INF-activated macrophages and found that a fourth of the >20,000 STAT1 binding sites were in transposable elements. Especially, about 10% of the total STAT1 binding sites were in B2_Mm2, a murine-specific SINE. They showed that these B2 elements are associated with H3K27ac signal upon INF treatment, thus likely serve as an INF-inducible enhancer through STAT1 binding. The authors then focus on the STAT1-bound B2_Mm2 in the Dicer1 gene (designated as B2_Mm2.Dicer1), and demonstrated that deletion of this B2 in a macrophage-like murine cell line resulted in loss of STAT1 binding, H3K27ac, and Dicer1 upregulation upon INF treatment. Their findings suggest that B2 transposition events has altered the transcriptional regulatory network in the innate immune response in the mouse.

      The manuscript is well organized, and the findings are potentially interesting in terms of the evolution of species-specific regulatory networks of the innate immune response. But, I am not convinced with the enhancer role of the B2_Mm2.Dicer1 copy for the Dicer1 expression (see below).

      Major Comments:

      (1) In Fig. 4, the degree of Dicer1 induction by INF was small (1.2-fold or so), and accordingly the effect of the B2 deletion on the Dicer1 induction was also small. In addition, this B2 binds to CTCF, and its deletion should also eliminate CTCF binding. Therefore, it is difficult to conclude from the presented data that this B2 serve as an enhancer for Dicer1. The B2 may increase the frequency of transcription (as suggested by the authors), may serve as an obstacle for transcriptional elongation (via binding to CTCF), or may regulate the splicing efficiency. In Fig.5C, promoter acetylation level does not seem to be affected in KO1. Pol II either does not seem to be affected if the Pol II peak is compared to the background level. Taken together, the enhancer role is not supported by strong evidence.

      (2) On the other hand, the authors discovered that the B2 deletion resulted in the decrease of Serpina3h, Serpina3g, Serpina3i and Serpina3f by >100-fold, which are 500 kb apart from the B2 locus. This is also interesting, and could be evidence for the B2 enhancer. Given that this B2 binds to both STAT1 and CTCF, the locus could interact with the Serpina3 locus to act as an enhancer. Were there STAT1 CUT&TAG peaks around the Serpina3 genes? Did H3K27ac and Pol II ChIP peaks in the Serpina3 promoters disappear in the KO cells? It would be interesting to see the IGV snapshots for H3K27ac, POLR2A and STAT1 ChIP-seq data around Serpina3 genes. In addition, HiC data for activated macrophages, if available, could be supportive evidence for the interaction between B2_Mm2.Dicer1 and the Serpina3 locus.

      Minor Comments:

      (3) Regarding Fig.1C, the authors calculated the B2 expression levels by mRNA-seq and DESeq2 analysis. But it does not accurately give the B2 transcription level, because the method does not discriminate B2 RNAs and B2-containing mRNA (and lncRNA as well). I wonder that the apparent upregulation of STAT1-binding B2 loci is due to the increase of Pol II transcription around the loci, rather than Pol III-mediated B2 transcription. This possibility should be discussed in page 6 after "Taken together, these data indicate that thousands of B2_Mm2 elements show epigenetic and transcriptional evidence of IFNG-inducible regulatory activity in primary murine bone marrow derived macrophages."

      (4) Fig. 2B shows that about 70-80% of B2_Mm2 loci carry the STAT1 motif, whereas only a limited number (2-3%) of B2_Mm2 bind to STAT1. Is this because of differences in their motif sequences, in genomic locations, or in epigenomic environments? For example, do these STAT1-binding loci have a C-to-A mutation at the second last position in the GAS motif (TTCNNGGAA), like B2_Mm2.Dicer1 (shown in Fig. S4)? Can the authors discuss about it? In addition, although the consensus sequence of B2_mm2 has a GAS motif with only a single mismatch, the presence of the STAT1 motif in >70% of B2_Mm2 is surprising, given that their average divergence to the consensus sequence is about 10% (ref. 26 of the manuscript). Is the binding site significantly conserved in compare to the other regions of the B2 sequence?

      Review 3
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  124. www.flickr.com www.flickr.com
    3. GTD Card
    1
    1. chrisaldrich 13 Oct 2022
      GTD Card Icon : Square (check box)Tag : 4th block. Squared as open-loop first, and filled later as accomplished. The GTD is advanced To-Do system proposed by David Allen. Next action of your project is described and processed through a certain flow. The GTD cards are classified into this class. 4th block is squared as open-loop first, and filled later as accomplished. The percentage of GTD Cards in my dock is less than 5 %.
      GTD Pile of Index Cards to do lists productivity open loops index cards
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  125. www.reddit.com www.reddit.com
    r/antinet - Separate private information from the outline of academic disciplines?
    1
    1. chrisaldrich 13 Oct 2022
      Posted byu/raphaelmustermann9 hours agoSeparate private information from the outline of academic disciplines? .t3_xi63kb._2FCtq-QzlfuN-SwVMUZMM3 { --postTitle-VisitedLinkColor: #9b9b9b; --postTitleLink-VisitedLinkColor: #9b9b9b; --postBodyLink-VisitedLinkColor: #989898; } How does Luhmann deal with private Zettels? Does he store them in a separate category like, 2000 private. Or does he work them out under is topics in the main box.I can´ find informations about that. Anyway, you´re not Luhmann. But any suggestions on how to deal with informations that are private, like Health, Finances ... does not feel right to store them under acadmic disziplines. But maybe it´s right and just a feeling which come´ out how we "normaly" store information.

      I would echo Bob's sentiment here and would recommend you keep that material like this in a separate section or box all together.

      If it helps to have an example, in 2006, Hawk Sugano showed off a version of a method you may be considering which broadly went under the title of Pile of Index Cards (or PoIC) which combined zettelkasten and productivity systems (in his case getting things done or GTD). I don't think he got much (any?!) useful affordances out of mixing the two. In fact, from what I can see looking at later iterations of his work and how he used it, it almost seems like he spent more time and energy later attempting to separate and rearrange them to get use out of the knowledge portions as distinct from the productivity portions.

      I've generally seen people mixing these ideas in the digital space usually to their detriment as well—a practice I call zettelkasten overreach.

      reply zettelkasten overreach GTD Hawk Sugano Pile of Index Cards
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  126. onlinelibrary.wiley.com onlinelibrary.wiley.com
    Characterizing physical habitat preferences and thermal refuge occupancy of brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar) at high river temperatures
    1
    1. Tommywalker 12 Oct 2022
      Snorkelling surveys were conducted at each site to observe fish locations

      Another method with questionable accuracy. A big question that arises is how can you tell different fish apart from one another without a tag and release system?

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  127. www.biorxiv.org www.biorxiv.org
    New submission 12/10/2022, 12:53:39
    2
    1. Public_Reviews 12 Oct 2022

      Reviewer #1 (Public Review):

      In this paper, the authors use purified Xenopus γ-TuRCs and experiments in cell extract combined with cutting edge imaging techniques to investigate whether binding of the γ-TuNA fragment can activate γ-TuRCs. The authors show that γ-TuNA fragments from both humans and Xenopus are obligate dimers and that dimerization is necessary for γ-TuRC binding. They further show, using direct visualisation of microtubule nucleation from individual purified γ-TuRCs, that γ-TuNA binding increases the nucleation efficiency of γ-TuRCs by ~20 fold, helping to overcome negative regulation by Strathmin.

      γ-TuNA, otherwise known as the CM1 domain, CM1 motif or CM1 helix, is well conserved and found within the N-terminal region of proteins across evolution. These proteins bind and recruit γ-TuRCs to MTOCs, such as the centrosome, meaning that γ-TuRC recruitment and activation could be closely linked. Earlier studies had provided strong evidence that binding of γ-TuNA activated γ-TuRCs, hence the name "γ-TuRC mediated nucleation activator" (Choi et al., 2010), and this claim was supported by similar work a few years later (Muroyama et al. 2016). Moreover, several other studies showed that expressing in cells γ-TuNA, or equivalent protein fragments, led to ectopic microtubule nucleation in the cytoplasm, with some of the studies showing that mutations preventing the binding of these fragments to γ-TuRCs ablated this effect (Choi et al., 2010; Lynch et al., 2014; Hanafusa et al., 2015; Cota et al., 2016; Tovey et al., 2021). Collectively, therefore, it was accepted that binding of these fragments somehow activated γ-TuRCs. More recent data, however, including from the authors themselves, had provided evidence that γ-TuNA binding did not activate γ-TuRCs (Liu et al., 2019; Thawani et al., 2020). A major objective of this paper was therefore to help resolve this controversy. The author's data suggest that the ability of these fragments to activate γ-TuRCs depends upon the type and position of tag attached to the N-terminus of the γ-TuNA fragment, with large tags seemingly turning γ-TuNA into a γ-TuRC inhibitor (although they also note that one of the previous studies, which concluded γ-TuNA was an activator, had also used fragments with large N-terminal tags). The authors also insist that the new results benefit from a much-improved γ-TuRC purification protocol that results in higher yield and purity. This purification approach uses the affinity of the γ-TuNA fragment and so could be adopted by others in the field.

      The major strength of this paper is directly showing, using very powerful single molecule imaging and their improved protocols, that γ-TuNA is a γ-TuRC activator, thus resolving the controversy that has existed for the last few years. The weakness is that we still don't learn how γ-TuNA binding activates γ-TuRCs (this has been proposed to be via structural changes but other mechanisms can be considered), and thus there is little conceptual advance from the original Choi et al. 2010 paper, which had already concluded that γ-TuNA binding increased the nucleation efficiency of γ-TuRCs. Moreover, the authors do not include experiments with the other proposed γ-TuRC activator, XMAP215, which they have investigated previously (Thawani et al., 2020), and so we are left wondering whether γ-TuNA and XMAP215 work together or as part of separate activation pathways.

      Overall, this paper is timely as it finally resolves the controversy over γ-TuNA and it is admirable that the authors are willing to directly address and correct their previous conclusion. The data is solid and well-presented and the text is clear and has appropriate citations. In my opinion, papers that clarify the literature are just as important as those that make conceptual advances.

      Review 1
    2. Public_Reviews 12 Oct 2022

      Reviewer #2 (Public Review):

      This is the first report that establishes gamma-TuNA as an activator of gamma-TuRC-dependent microtubule-nucleation, using purified components. This is an in-depth study that establishes experimental conditions under which gamma-TuNA can function as an activator (dimerization of gamma-TuNA, appropriately sized N-terminal tag) and clarifies why similar attempts to study gamma-TuNA have failed in the past. I think that the information in this manuscript will be of immense value to the scientific community, as it resolves a long-standing mystery concerning the function of gamma-TuNA. A key question that still remains unanswered is whether the gamma-TuNA-dependent activation mechanism involves a conformational change of the gamma-TuRC, from an asymmetric to a ring-shaped template structure, but this may be beyond the scope of the present submission.

      Review 2
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  128. github.com github.com
    nic-dgl103-f22/assignment-c-dlu-caitgarland: assignment-c-dlu-caitgarland created by GitHub Classroom
    1
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  129. github.com github.com
    nic-dgl103-f22/assignment-c-dlu-chelsieadam: assignment-c-dlu-chelsieadam created by GitHub Classroom
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    1. caitlandia 10 Oct 2022
      Skip to content In this repository All GitHub ↵ Jump to ↵ No suggested jump to results In this repository All GitHub ↵ Jump to ↵ In this organization All GitHub ↵ Jump to ↵ In this repository All GitHub ↵ Jump to ↵ Dashboard Pull requests Issues Codespaces Marketplace Explore Sponsors Settings caitgarland Sign out New repository Import repository New gist New organization Sorry, something went wrong. /  ...  /   nic-dgl103-f22  /   assignment-c-dlu-...  /   Clear Command Palette Tip: Type # to search pull requests Type ? for help and tips Tip: Type # to search issues Type ? for help and tips Tip: Type # to search discussions Type ? for help and tips Tip: Type ! to search projects Type ? for help and tips Tip: Type @ to search teams Type ? for help and tips Tip: Type @ to search people and organizations Type ? for help and tips Tip: Type > to activate command mode Type ? for help and tips Tip: Go to your accessibility settings to change your keyboard shortcuts Type ? for help and tips Tip: Type author:@me to search your content Type ? for help and tips Tip: Type is:pr to filter to pull requests Type ? for help and tips Tip: Type is:issue to filter to issues Type ? for help and tips Tip: Type is:project to filter to projects Type ? for help and tips Tip: Type is:open to filter to open content Type ? for help and tips We’ve encountered an error and some results aren't available at this time. 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Open in GitHub Desktop Open with Desktop View raw Copy raw contents Copy raw contents Copy raw contents Copy raw contents View blame <!DOCTYPE html> <html lang="en"> <head> <link rel="stylesheet" href="style.css"> <link rel="icon" type="image/x-icon" href="images/favicon.ico"> <meta charset="UTF-8"> <meta http-equiv="X-UA-Compatible" content="IE=edge"> <meta name="viewport" content="width=device-width, initial-scale=1.0"> <link rel="preconnect" href="https://fonts.googleapis.com"> <link rel="preconnect" href="https://fonts.gstatic.com" crossorigin> <link href="https://fonts.googleapis.com/css2?family=Open+Sans&family=Raleway:wght@700&display=swap" rel="stylesheet"> <title>Hairpins Salon Contact Page</title> </head> <body> <header> <a href="index.html"><img src="images/hairpins-salon-logo.png" alt="hairpins Logo" width="300"></a> <nav> <ul> <li><a href="index.html">Home</a></li> <li><a href="services.html">Services</a></li> <li><a href="contact.html">Contact Us</a></li> </ul> </nav> </header> <h1>Contact Us</h1> <br> Questions, comments, ready for a new do?<br> We look forward to hearing from you!<br> If you're looking to make an appointment online, please do so here.<br> <br> Monday ~ Closed<br> Tuesday 9:00am ~ 5:00pm<br> Wednesday 9:00am ~ 8:00pm<br> Thursday 9:00am ~ 8:00pm<br> Friday 9:00am ~ 5:00pm<br> Saturday 9:00am ~ 4:00pm<br> Sunday ~ Closed<br> <br> Hairpins Boutique Salon<br> #4 - 224 6th Street<br> Courtenay, BC V9N 1M1<br> <br> <a href="https://www.google.com/maps/place/Hairpins+Boutique+Salon/@49.6904218,-124.9971279,15z/data=!4m2!3m1!1s0x0:0x859b2cfce3bc31ea?sa=X&ved=2ahUKEwiYi_2Ex6T6AhXNMjQIHYkoCUUQ_BJ6BAhSEAc)">Check us out on Google Maps</a><br> <br> Tel: (250) 338-7467<br> <a href="tel:250-338-7467">Click to Call</a><br> <br> Email: salon.hairpins@gmail.com <br> <a href="mailto:salon.hairpins@gmail.com">Email us</a><br> <br> <a href="contact.html">Conctact us today to book an appointment!</a> <br> <div class="footer"> Content taken from<a href="http://www.hairpins.ca/">https://www.hairpins.ca/</a>Used for educational purposes only. </div> </body> </html> Copy lines Copy permalink View git blame Reference in new issue Go Footer © 2022 GitHub, Inc. Footer navigation Terms Privacy Security Status Docs Contact GitHub Pricing API Training Blog About You can’t perform that action at this time. You signed in with another tab or window. Reload to refresh your session. You signed out in another tab or window. Reload to refresh your session. .user-mention[href$="/caitgarland"] { color: var(--color-user-mention-fg); background-color: var(--color-user-mention-bg); border-radius: 2px; margin-left: -2px; margin-right: -2px; padding: 0 2px; } assignment-c-dlu-chelsieadam/contact.html at main · nic-dgl103-f22/assignment-c-dlu-chelsieadam

      Similar comments - I would use unordered lists and paragraph elements where necessary!

      Also, I found there is an < address > tag you could use. Learn more: https://www.w3schools.com/tags/tag_address.asp

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  130. content.time.com content.time.com
    What Is A Life Worth?
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  131. www.biorxiv.org www.biorxiv.org
    Message in a Bottle – Metabarcoding Enables Biodiversity Comparisons Across Ecoregions
    1
    1. GigaScience 10 Oct 2022
      biomonitoring

      Reviewer 4. Christina Lynggaard

      This manuscript assesses the variation in arthropod communities in three ecoregions in Canada. The study is well done, and the sampling was very thorough with a big sampling effort. I only have minor comments. Specially I consider that the aim can be focused on the ecoregions instead of the feasibility of the method, as this has already been shown. In addition, it would be nice to have more details in certain sections in the data analyses and in the results. I have addressed these comments below. -I am not sure why the title "Message in a bottle". -Line 65- Could you specify which indicator species have been targeted? Or cite studies that target those species? - Line 96- Based on the limitations of the ecoregions, it is not clear why ecoregions are an obvious candidate. -In line 104 seems that your aim is to demonstrate how feasible is to use metabarcoding for large-scale monitoring and that you use the ecoregions to prove that. However, showing the feasibility of this method for large-scale studies has already been done (e.g. Svenningsen et al 2021, Detecting flying insects using car nets and DNA metabarcoding; Bush et al 2020, DNA metabarcoding reveals metacommunity dynamics in a threatened boreal wetland wilderness). I suggest keeping it focused on the need to apply this method in different ecoregions. -In the Data description section, you mention that you examined phylogenetic diversity, but in the Analyses section you vaguely mention it. The phylogenetic diversity findings are discussed later on, but it is difficult to follow the discussion when the results were not presented previously. In addition, the authors use the findings in phylogenetic diversity to support the idea of a structure in the ecoregions, so I suggest making more emphasis in this in the results section. -Line 189. I agree that the higher number of BINs could be due to eDNA, but couldn't another reason be that the BINs were oversplit during data analysis? -Line 215-217. Has this been found previously in other studies using Malaise trap? If so, please reference to those findings. -Line222- This is a brief discussion about temporal turnover. However, these results are not presented previously, or at least not clearly enough. -Line 266-267- Yes, you showed compositional shifts using metabarcoding in bulk arthropod samples, but the way this sentence is structured it sounds like you are the first to show this. Compositional shifts in arthropods have been shown previously in other studies using metabarcoding. -Line 321- Did you have negative PCR controls? In line 326 you mention negative controls, but I assume you refer to the extraction negative controls. -Line 340- It is not clear why you queried the data against a bacterial library. -Line 348- What was the reason for choosing "at least three reads"? and the same for line 350 where you cluster sequences with a minimum of 5 reads per cluster. -Line 357- If you see tag switching in your negative controls that means that most likely you have it in the rest of the data. How did you ensure that the rest of the data did not have that? You may have tags switching in sequences not found in the negative controls but found in your samples. -Line 369- As you used the Bray-Curtis index in this metabarcoding data, did you convert your data to presence/absence? It is known that for metabarcoding data the use of read numbers for community analysis is not adequate (see Nichols et al 2018 "Minimizing polymerase biases in metabarcoding") .

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  132. www.reddit.com www.reddit.com
    r/gamedev - A Guide to Marketing your Indie Game! How I got 20,000 Followers on Twitter
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    1. Astro49 09 Oct 2022
      When people first see your Twitter, without even having to scroll down, they should knowThe name of our Game (make this your Twitter tag, not your company name!)Gameplay footage of our game pinned to the top of the feedOur unique selling point/elevator pitchWhat our logo looks likeWhat genre the game isWhere our Discord isWhere our Patreon isWhere our Facebook isOur email address
      Game marketing
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  133. www.kamilgrzybek.com www.kamilgrzybek.com
    Modular Monolith - Kamil Grzybek
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  134. denisetodd.medium.com denisetodd.medium.com
    8 Factors for Effective Use of Obsidian Tags, Links, and Folders
    3
    2. K.nour 08 Oct 2022
      1) Ensure the following Core Obsidian plugins are turned ON: Go to Obsidian Settings > Core plugins, and turn on:✅ Backlinks✅ File Explorer✅ Graph view✅ Quick Switch✅ Slash Command✅ Tag panes

      Done !

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  135. www.biorxiv.org www.biorxiv.org
    Tup1 is Required for Transcriptional Repression Necessary in Quiescence in S. cerevisiae
    1
    1. EMBOpress 08 Oct 2022

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

      Learn more at Review Commons

      Reply to the reviewers

      Thank you for giving us the opportunity to submit a revised draft of the manuscript “Tup1 is Required for Transcriptional Repression Necessary in Quiescence in S. cerevisiae” to Review Commons. We appreciate the time and effort that you and the other reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper. We believe that the experiments suggested in these comments would bring clarity to the manuscript, and wish that we had the ability to perform them all. Unfortunately our lab is closing, and the only remaining lab member is the PI, so we are only able to perform limited experiments to address some of the concerns raised during review. We have incorporated several changes in response to comments from the reviewers. Those changes are highlighted within the manuscript. Please see below, in blue, for a point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the revised manuscript Word file with tracked changes.

      Of particular note is the discussion of cellular morphology of the tup1∆ and sds3∆ strains. We realize that our findings are purely descriptive, and are not surprised that all three reviewers had comments on this data. This was the source of much discussion among the authors and consultation with other labs; we debated even including these observations in the manuscript, since we were unable to figure out the underlying mechanism. Ultimately we decided that it was worth reporting in case other labs may benefit from the knowledge, and we have altered the language in the manuscript (page 6) to better reflect this. However, if the reviewers feel that this observation would be better left out of the manuscript, we would be willing to remove Figure 6 and any discussion of these images.

      Reviewer #1 Comments:1. The authors chose to examine 3-day SP cells to interrogate quiescence because tup1∆ cells are highly flocculant, interfering with the isolation of purified quiescent cells. These cells are a mixture of both nonquiescent and quiescent cells, so it is not correct to state that they represent a quiescent cell population. The addition of EDTA to the gradients used to isolate quiescent cells could eliminate flocculation and permit the isolation of quiescent cells. EDTA is also often added to media in low amounts to reduce flocculation. The authors need to indicate the proportion of quiescent cells in their SP cultures by applying these tools.

      We appreciate the suggestion, but the phenotype of this strain is not typical flocculation (see photo below, also added to the paper as Supplementary Figure 1). We did add EDTA (pH 8.0) to a final concentration of 10 mM to two separate tup1∆ and it did not visibly affect the clumping of cells. Furthermore, changes to the cell wall are a distinct feature of quiescent S. cerevisiae and contribute to the ability to separate different cell types by density-gradient centrifugation, so it is difficult to anticipate how EDTA would affect our ability to isolate Q cells. We have provided more explanation in the manuscript to better explain this (page 3).

      1. The authors reported that while Xpb1 and Tup1 share many overlapping binding sites, but that Xbp1 does not regulate Tup1's binding. What other factors might be responsible for their shared binding? Could histone deacetylation play a role? This could be addressed by a Tup1 ChIP in an sds3∆ mutant.

      This is a good thought; histone acetylation levels may have a role in regulating Tup1 localization and we would have liked to address this if we had more time. Unfortunately, we had some difficulty performing ChIP of Tup1, because initially we used a FLAG tag which caused a phenotype similar to deletion of Tup1, and had to switch to making myc-tagged strains. This delay meant we did not have time to pursue creating myc-tagged Tup1 in an sds3∆ strain, and now we do not have the ability to follow up on this for revisions.

      1. Has PolII occupancy been examined in Log vs SP cells of tup1∆ to determine if Tup1 inhibits PolII association with its genes that are repressed ?

      We did not look at PolII occupancy in our Tup1 deletion, and could not find any existing datasets with this information. It is our hope that another lab is able to carry out this experiment, because it could be very enlightening, but it is beyond the scope of this work.

      1. The observation that tup1∆ cells have several nuclear puncta is intriguing, although the cytological images need to be improved.

      The nuclear puncta we see in the tup1 deletion are definitely a puzzle. We had limited time to investigate this phenomena, and in discussing the matter with some other labs it seemed doubtful that more advanced imaging would yield anything of use to us. We realized that we accidentally omitted important details for this figure and have updated the manuscript to add them. We imaged 2 biological replicates for each strain and imaged many yeast samples for each strain (which has been added to the caption for Figure 6) and found that our findings were statistically significant (p

      Reviewer #2 Comments1. The authors acknowledge that it would be better to work with purified quiescent cells but couldn't isolate pure populations. As a result, a mixture of quiescent and nonquiescent cells are analyzed in stationary phase. They say this is because Tup1 deletion strains are flocculent. But they performed ChIP-Seq on Myc-tagged Tup1 strain. Don't these cells express Tup1? If not, could this be performed in wild-type yeast with Myc-tagged Tup1? It seems important to separate quiescent from nonquiescent yeast for the authors' conclusions.

      It is true that we could have done ChIP-seq for Tup1 in purified Q cells. We considered it, but decided to look at the mixed population so that we could directly compare our RNA-seq results from the tup1∆ strain. It’s a balance between having some results that are specific to quiescence, versus being able to directly compare the effects of deletion of Tup1 at the sites where it binds. We are now unable to perform this experiment, but we have updated the language in the manuscript (page 3) to better reflect this choice.

      1. The Chipseq data in Fig 1B do not have a y axis and it is consequently not clear whether these data are normalized and shown with the same axis.

      Thank you for pointing this out - these data are normalized to RPKM during processing, and we have updated the caption for figure 1 and the methods on page 10 to reflect this information. Normalizing the data in IGB itself, however, causes an adjustment in the y-axis that makes the tracks appear to be inconsistent. In any case, we are not making claims about the relative amount of signal, and as it is common in the field to not include y-axes on IGB tracks, we have opted to keep the y-axis for Figure 1B as-is.

      1. In Fig 2, it seems important to determine how many genes are different between WT and Tup1 deletion strains in log phase. Are just as many genes different? Or is Tup1 more important in diauxic shift and stationary phase than log phase?

      We did intend to focus only on diauxic shift and stationary phase data for this paper, since there has already been so much work on the role of Tup1 in log phase. As mentioned above, comparisons of RNA between log and DS/Q is difficult. We attempted to find a publicly available dataset to perform some analysis for revisions, but unfortunately most previous work on the effect of Tup1 on transcription was performed via tiling arrays, which is not comparable.

      1. Are the genes that are regulated by Tup1 normally regulated during diauxic shift or stationary phase compared with log growth?

      Because there is a massive global decrease in the level of total RNA in diauxic shift and quiescence (McKnight, Boerma, et al., 2015) it is impossible to directly compare transcript levels between these states in our experiments. If there was time, we could have attempted to repeat these experiments with an external spike-in control; this is potentially something another lab could do to follow up on our findings.

      1. What fraction of the genes that are differentially expressed in Tup1 knockout yeast have Tup1 binding at the promoter? Enhancer? What fraction can be explained by Tup1, Hap1, Nrg1, Mig1 individually and together?

      We have added the number of genes that are differentially expressed in Tup1 knockout yeast during DS to the manuscript (page 3). Regarding enhancers, the genome of S. cerevisiae is very compact, and there is not evidence of long-distance activation of genes as seen in metazoans (Dujon, 1996; Dobi & Winston, 2007; Spiegel and Arnone, 2021). Upstream activating sequences (UASs) are generally considered the closest equivalent to enhancers in cerevisiae, and they tend to function within a few hundred base pairs of the promoter. Our analysis only identifies the nearest gene; it would be difficult to parse out locations in the promoter versus a UAS without a more advanced analysis that is beyond our capabilities now.

      As for the effect of Hap1, Nrg1, and Mig1, we were able to look for their motifs in the genes that are differentially expressed in the Tup1 knockout but we do not have binding data for these factors in quiescence or stationary phase so it is impossible to conclusively state what role those TFs play. This would be a very interesting followup to our work, but is outside the scope of this manuscript.

      References:

      Dujon, B. 1996. The Yeast Genome Project: What did we learn? Trends Genet. 12, 263-270.

      Dobi, K.C.; Winston, F. 2007. Analysis of Transcriptional Activation at a Distance in Saccharomyces cerevisiae. Mol Cell Biol. 27(15), 5575-5586. https://doi.org/10.1128/MCB.00459-007

      Spiegel, JA; Arnone, J.T. 2021.Transcription at a Distance in the Budding Yeast Saccharomyces cerevisiae. Appl. Microbiol. 1(1), 142-149. https://doi.org/10.3390/applmicrobiol1010011

      1. The methodology used to generate the gene ontology enrichments should be described in the methods.

      Thank you for noticing this omission; we have added the relevant information to the manuscript (page 10) and have also added the related citation (page 11).

      1. The authors should provide genomewide data to support the statement that Tup1 and Rpd3 ChIP datasets have substantial overlap. They should also provide genomewide data to support the statement that there is substantial overlap between Rpd3 and Tup1. How much overlap is observed and how much is expected by chance?

      We have compared the existing ChIP data for Rpd3 binding in quiescent cells to our ChIP data for Tup1 in 3-day cultures and included this in the manuscript (page 4, Supplementary Figure 2B), along with a p-value.

      1. For Sds3, similar to Tup1 inactivation, it would be helpful to know how many genes change in with Sds3 inactivation in log phase in addition to diauxic shift and stationary phase.

      As with our response to comment #3, we focused only on diauxic shift and stationary phase data for this paper, and analysis of this data would be difficult without a spike-in control. While there are some existing datasets for RNA-seq of Rpd3 knockouts, this would include both Rpd3L and Rpd3S activity, rather than just Rpd3L, which is our focus with the Sds3 deletion strains. As such, we did not perform RNA-seq of sds3∆ in log phase.

      1. If the argument is that Sds3 and Xbp1 cooperate with Tup1 to affect gene expression, testing the gene expression changes that are associated with Tup1 in Sds3 or Xbp1 knockout strains would help the authors make this point.

      We do not have tup1∆/sds3∆ or tup1∆/xbp1∆ double knockout strains. We attempted to make these strains but could not, which may indicate that these double deletions are synthetic lethal. Deletion of sds3 alone causes a significant reduction in growth rate, so it is perhaps not surprising that we could not create the double knockouts.

      1. The final phenotype of extra DAPI positive blobs in the nucleus is not very specific or clear.

      We agree, please see our comments at the top of this letter.

      Reviewer #3 (Major comments):Did tup1∆/sds3∆ double mutant show the same phenotype with tup1∆ (or sds3∆) single mutant in G0? If Tup1 actually plays role in tandem with Sds3 in the gene regulation during G0, the epistatic relationship might be estimated.

      We do not have tup1∆/sds3∆ or tup1∆/xbp1∆ double knockout strains. We attempted to make these strains but could not, which may indicate that these double deletions are synthetic lethal. Deletion of sds3 alone causes a significant reduction in growth rate, so it is perhaps not surprising that we could not create the double knockouts.

      The histone acetylation was not synergistically augmented in the above double mutant?

      Please see the response above.

      The authors showed that tup1∆ but not sds3∆ cells contain multiple DAPI signals but sds3∆ cells show abnormal cell shape in G0 phase. These phenotypic abnormalities in these mutants suggest a potential mitotic defect. Both mutants showed very similar abnormalities in H3K23 acetylation and gene expressions in quiescent state. Why these showed distinctly different abnormality in cell morphology during G0?

      Unfortunately we were unable to investigate this further.

      Did iswi2∆ cells also show abnormality in G0 phase?

      No, they did not; thank you for asking, this is a good question. We have added this information to the manuscript (page 6).

      (Minor comments)Supplementary figure1. This data seems to be very important. I recommend to use this data in the main figure with statistical analysis (p-values) to show the significant overlap of Tup1 and Rdp3 distribution.

      We have compared the existing ChIP data for Rpd3 binding in quiescent cells to our ChIP data for Tup1 in 3-day cultures and included this in the manuscript (page 4, Supplementary Figure 2B) . We do feel that this data belong in the supplement, however, because the data is not exactly equivalent to our studies: quiescent cells and 3-day cultures are not the same, and knockout of Rpd3 eliminates function of both Rpd3L and Rpd3S complexes, while knocking out Sds3 targets only the Rpd3L complex.

      Figure 4. Histone acetylation level data in Figure 4A and the data for gene repressions by Tup1 and Sds3 in Figure 4C seem to be very important. However, statistical analysis data (p-values) was not presented. Please show the statistical analysis data (p-values) as in figure 3 to show that the Tup1 and Sds3 contribute similarly in histone deacetylation and repression. The author did not find the significant changes of histone deacetylation in xbp1∆ cells but said that when filtered in Xbp1 binding motif Xbp1 depletion has similar effect on the acetylation level. Please show this data.

      We have added language in the manuscript comparing genes with altered acetylation levels to those that are differentially expressed in our RNA-seq datasets, along with a p-value, to page 5.

      PeerReviewed
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  136. www.reddit.com www.reddit.com
    r/antinet - 2015 exhibition of Roland Barthes' zettelkasten and writing process
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    1. chrisaldrich 08 Oct 2022

      https://www.reddit.com/r/antinet/comments/xyhpq4/2015_exhibition_of_roland_barthes_zettelkasten/

      Johanna Daniel has some interesting reflections (in French) about Barthes' reading, note taking, and writing processes: http://johannadaniel.fr/isidoreganesh/2015/06/archives-roland-barthes/ Perhaps most importantly she's got some photos from an exhibition of his work which includes portions of his note cards and writing. Roland Barthes' note cards on the Sorrows of Young Werther by Goethe. And you thought your makeshift cardboard boxes weren't "enough"? Want more about Barthes' practice? Try my digital notes: https://hypothes.is/users/chrisaldrich?q=tag%3A%22Roland+Barthes%22

      Roland Barthes zettelkasten Roland Barthes' zettelkasten
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  137. github.com github.com
    nic-dgl103-f22/assignment-c-dlu-KrisTessier: assignment-c-dlu-KrisTessier created by GitHub Classroom
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    1. Colefreeman 07 Oct 2022
      <style> body { font-family: "Trirong", serif; font-weight:400;

      I'm pretty sure you can add this to your style.css page as it's considered a css tag?. I think this is mixing html & css into one sheet which can become confusing.

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  138. forgravity.com forgravity.com
    Displaying PDF in Confirmation Message - ForGravity
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    1. kambrose 07 Oct 2022
      You can display the file name and link to your generated PDF in your Gravity Forms confirmation message using merge tags. When editing a confirmation message, there will be a Fillable PDFs group at the bottom of the merge tag drop down. Every configured Fillable PDFs feed will have three merge tags available: one that displays a properly formatted link with the PDF URL and file name, one for only the file name, and one for only the PDF URL.

      The fillable pdf IDs are found in the url for each form on the fillable pdf page

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  139. www.wired.com www.wired.com
    The Return of Fake News—and Lessons From Spam
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    1. cseidel2 07 Oct 2022
      Where does clicktivism end and algorithmic gaming begin?

      This linked article was super interesting to me. I had never heard of the term "clicktivism" before but it made a lot of sense to hear that, because many online seem to feel like helping things to trend is a notable part of activism, and this in turn can lead to behavior paltforms find suspicious. It's true that it does have its share of impact- causes and people in need can reach more people than was ever possible thanks to the internet- but it has also led to some odd cultural quirks online when it comes to giving such issues a platform. Occasionally the goal to "boost awareness" or publicly declare yourself aligned with the "right" or "correct" cause can overpower the actual execution of concrete, meaningful action. The term "performative activism" also encompasses this phenomenon, in which people publicly post a lot about a tag or trend to sort of assure their followers they too are on the right side of affairs. Accounts that are clearly mining activist circles for engagement (even if they are helping to spread awareness of issues) can definitely act similarly to bots or spammers.

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  140. www.biorxiv.org www.biorxiv.org
    MitoStores: Chaperone-controlled protein granules store mitochondrial precursors in the cytosol
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    1. ASAPbio_crowd_preprint_review 07 Oct 2022

      Review coordinated via ASAPbio’s crowd preprint review

      This review reflects comments and contributions by Ruchika Bajaj, Gary McDowell, Sree Rama Chaitanya Sridhara. Review synthesized by Iratxe Puebla.

      The preprint studies the process for mitochondrial targeting of mitochondrial precursor proteins. Using a yeast model, experiments show that the cytosol transiently stores matrix-destined precursors in dedicated granules which the authors name MitoStores. The formation of MitoStores is controlled by the heat shock proteins Hsp42 and Hsp104, and suppresses the toxicity arising from non-imported accumulated mitochondrial precursor proteins.

      The manuscript is clear and well-written. The reviewers raised a few comments and suggestions as outlined below:

      The introduction was extremely clear and provides a good summary of the protein homeostasis dimension of the problem in question. However, there could be a clearer discussion of the processes of import, in particular with respect to the results discussing “clogging”. It is suggested to add a penultimate transitional paragraph in the introduction that facilitates this transition e.g. this could be expansion of the first paragraph in the Results section, moved into the introduction to provide more context about the cloggers, PACE, and the Rpn4-mediated proteasomal regulation.

      Figure 2E and Figure S2 - Can some further explanation be provided about what data belongs to delta-rpn otr WT, or whether the associated fold change is reported - delta-rpn/WT.

      Results ‘while the levels of most chaperones were unaffected or even reduced in Δrpn4 cells, the disaggregase Hsp104 and the small heat shock protein Hsp42 were considerably upregulated (Fig. 2F, G)’ - Suggest adding some further clarification as to why Hsp104 and Hsp42 are selected despite perturbations in other protein partners. Are there other proteins than proteosomes and chaperones which are significantly up- or down-regulated? STRING or cytoscape tools may help with the interactome analysis.

      Figure 3

      • Figure 3A - It seems Δrpn4 cells are bigger in size than control cells, suggest commenting on this point.
      • Figure 3B ‘Hsp104-GFP was purified on nanotrap sepharose’ - Please clarify on which tag the purification was based.
      • ‘grown at the indicated temperatures’ - Please clarify the rationale for using 30 or 40C.
      • ‘SN, supernatant representing the non-bound fraction’ - Please report what is total, wash and elute etc.

      Results ‘protein accumulated at similar levels as Hsp104-GFP in the yeast cytosol (Fig. S4B)’ - Please clarify whether the image reports qualitative or quantitative data, and how the levels of DHFR-GFP and Hsp104-GFP are compared based on S4B.

      ‘Owing to the striking acquisition of nuclear encoded mitochondrial proteins in these structures, we termed them MitoStores’- Suggest providing some discussion about the fraction of Hsp104 that is part of the MitoStores? Does a major portion of Hsp104 in the absence of Rpn4 form MitoStore structures?

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    biorxiv.org/content/10.1101/2022.08.02.502474v1
  141. github.com github.com
    nic-dgl103-f22/assignment-c-cvs2-tatianasuarez23: assignment-c-cvs2-tatianasuarez23 created by GitHub Classroom
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    1. TomioM 06 Oct 2022
      <br>

      I get what you're trying to do here with the "br" tag, but it falls apart when the window is resized. I think wrapping the content in an element and styling it with a max-width property would be better, as this would prevent it from become really long on wide displays while letting it resize dynamically on small displays

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  142. github.com github.com
    assignment-c-cvs2-elizabethajg/style.css at main · nic-dgl103-f22/assignment-c-cvs2-elizabethajg
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    assignment-c-cvs2-tomioM/index.html at main · nic-dgl103-f22/assignment-c-cvs2-tomioM
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  144. github.com github.com
    assignment-c-cvs2-elizabethajg/contact.html at main · nic-dgl103-f22/assignment-c-cvs2-elizabethajg
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    assignment-c-cvs2-pmenezes3094/contact.html at main · nic-dgl103-f22/assignment-c-cvs2-pmenezes3094
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    assignment-c-cvs2-elizabethajg/services.html at main · nic-dgl103-f22/assignment-c-cvs2-elizabethajg
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  147. github.com github.com
    nic-dgl103-f22/assignment-c-cvs2-Anmoldeep06: assignment-c-cvs2-Anmoldeep06 created by GitHub Classroom
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    1. elizabethajg 06 Oct 2022
      <br>Meet the Team<br>

      Breaks make sense but this text is not surrounded by an element tag. Something like: <br>

      Meet the Team

      <br>

      makes it possible to format that line.

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