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  • Nathaniel Huebsch2017 Jun 22 6:33 p.m. (3 hours ago)

    The original url for downloading the software is no longer active. The correct website for downloading this software is:

    https://gladstone.org/46749d811

    We appreciate everyone who let us know about the weblink being down.

  • Sunil Verma2017 Jun 22 1:20 p.m. (8 hours ago)

    Dear Colleagues,

    This paper had used our Universal Primers (mcb398/mcb869 - US patent 7141364) along with other primers and cited our paper, Verma and Singh 2003, Mol Ecol Notes 3:28–31; therefore, I became interested in it.

    Being the inventor of these primers used in this study, I am aware that our primers CAN NOT establish the identity of the species from "Ash". I was really shocked to read the title, that someone may establish species identity from "Ash" using my primers!

    I was eager that all the scientific queries that I had answered so far in last 20 years, and all the arguments that I have done as wildlife forensic expert in the court of law, that species identity can not be established from "Ash" will prove wrong in the light of this paper.

    After going through the abstract itself, I understood the matter. I also went through the full paper, and understood that the title of the paper is misleading. The authors indeed did not establish the identity from ash but they did it from some partially burnt biological material that was recovered from the scene of crime. Thus, the title of the paper should have NOT been "Molecular identification from ash"

    My scientific view-point is that the title of this paper should be corrected as appropriate and erratum be published in the respective journal.

    Dr Sunil Kumar Verma

    [Sunil Kumar Verma]

  • Robert Insall2017 Jun 20 02:19 a.m. (2 days ago) 1 of 1 people found this helpful

    Professor Sibley's most extensive comments are based around a single paper (Skillman et al. 2013) that concluded the polymerization of Toxoplasma actin uses an isodesmic, rather than a nucleation-based mechanism. While this work was well-executed, and thorough, it is not on its own sufficient to support the level of absolutism that is in evidence in these comments. In particular, results from actin that has been exogenously expressed (in this case, in baculovirus) are less reliable than native apicomplexan actin. The folding of actin is infamously complex, with a full set of specialist chaperones and idiosyncratic N-terminal modifications. Even changes in the translation rate of native actin can affect its function and stability (see for example Zhang, 2010). Exogenously-expressed actin may be fully-folded, but still not representative of the physiological protein. Thus it is not yet appropriate to make dogmatic statements about the mechanism of apicomplexan actin function until native actin has been purified and its polymerization measured. When this occurs, as it surely will soon, stronger rulings may be appropriate.

  • L David Sibley2017 Jun 19 4:20 p.m. (3 days ago)edited 1 of 1 people found this helpful

    Based on the most recent response by Dr. Meissner, it is clear that there is still some confusion about the difference between measuring the kinetics of actin polymerization in vivo vs. monitoring actin dynamics in vivo. These are fundamentally different processes, the former of which cannot be directly inferred from the later. Given this confusion, it is worth reviewing how these two processes are distinct, yet inter-related.

    When referring to the mechanism of actin polymerization in vitro, nucleation is the process of forming stable trimers, which are normally limited by an intrinsic kinetic barrier imposed by unstable dimers. Due to this intrinsic instability, the nucleation step is normally revealed as a pronounced lag phase in the time course of polymerization, after which filaments undergo rapid elongation Pollard TD, 2000. TgACT1 lacks this nucleation step and instead uses a non-cooperative, isodesmic process. The Arp/23 complex facilitates formation of the trimer by acting as the barbed end, thus reducing the lag time and accelerating polymerization, typically by side branching from existing filaments. Toxoplasma has no use for such a step as it would not affect the efficiency of an isodesmic process since dimers and trimers normally form without a lag phase Skillman KM, 2011. By contrast, formins bind to barbed end of existing filaments and promote elongation, both by preventing capping protein from binding and by using profilin to gather actin monomers for addition to the barbed end. Formins may also nucleate F-actin by binding to two monomers to lower the lag phase for trimer formation, thus facilitating elongation, although this role is less well studied. Importantly, formins can act on actins that use either an intrinsic “nucleation-elongation” cooperative mechanism or an isodesmic process, such as that used by Toxoplasma. Hence, the fact that formins function in Toxoplasma has no bearing on the intrinsic polymerization mechanism of TgACT1.

    Once the above definitions are clearly understood, it becomes apparent why the isodesmic process of actin nucleation used by Toxoplasma is fully compatible with both the short filament, rapid turnover dynamics that have been described previously Sahoo N, 2006, Skillman KM, 2011, Wetzel DM, 2003, and the new findings of long-stable filaments described in the present paper Periz J, 2017. These different states of actin polymerization represent dynamics that are driven by the combination of the intrinsic polymerization mechanism and various actin-binding proteins that modulate this process. However, the dynamic processes that affect the status of G and F-actin in vivo cannot be used to infer anything about the intrinsic mechanism of actin polymerization as it occurs in solution. As such, we strongly disagree that there is an issue to resolve regarding the intrinsic mechanism of actin polymerization in Toxoplasma nor do any of the studies in the present report address this point. Our data on the in vitro polymerization kinetics of TgACT1 clearly fit an isodesmic process Skillman KM, 2013 and we are unaware of any data that demonstrates otherwise. Hence we fail to see why this conclusion is controversial and find it surprising that these authors continue to question this point in their present work Periz J, 2017, previous report Whitelaw JA, 2017, and comments by Dr. Meissner. As it is not possible to predict the intrinsic mechanism of actin polymerization from the behavior observed in vivo, these comments are erroneous and misleading. On the other hand, if these authors have new data that speaks directly to the topic of the intrinsic polymerization mechanism of TgACT1, we would welcome them to provide it for discussion.

    Although we disagree with the authors on the above points, we do agree that the fact that actin filaments can be visualized in Toxoplasma for the first time is interesting and certainly in contrast to previous studies. For example, previous studies failed to reveal such filaments using YFP-ACT1, despite the fact that this tagged form of actin is readily incorporated into Jasplakinolide-stabilized filaments Rosenberg P, 1989. As well, filaments have not been seen by CryoEM tomography Paredes-Santos TC, 2012 or by many studies using conventional transmission EM. This raises some concern that the use of chromobodies (Cb) that react to F-actin may stabilize filaments and thus affect dynamics. Although the authors make some attempt to monitor this in transfected cells, it is very difficult rule out that Cb are in fact enhancing filament formation. One example of this is seen in Figure 6 A, where in a transiently transfect cell, actin filaments are seen with both the Cb-staining and anti-actin, while in the non-transfected cell, it is much less clear that filaments are detected with anti-actin Periz J, 2017. Instead the pattern looks more like punctate clusters that concentrate at the posterior pole or residual body. Thus while we would agree that the Cb-stained filaments also stain with antibodies ot F-actin, it is much less clear that they exist in the absence of Cb expression. It would thus be nice to see these findings independently reproduced with another technique. It would also be appropriate to test the influence of Cb on TgACT1 in vitro to determine if it stabilizes filaments. There are published methods to express Toxoplasma actin in a functional state and so this could easily be tested Skillman KM, 2013. Given the isodesmic mechanism used by TgACT1, it is very likely that any F-actin binding protein would increase the stability of the short filaments that normally form spontaneously, thus leading to longer, more stable filaments. This effect is likely to be less pronounced when using yeast or mammalian actins as they intrinsically form stable filaments above their critical concentration. Testing the effects of Cb on TgACT1 polymerization in vitro would provide a much more sensitive readout than has been provided here, and would help address the question of whether expression of Cb alters in vivo actin dynamics.

    In summary, we find the reported findings of interest, but do not agree that they change the view of how actin polymerization operates in Toxoplasma at the level of the intrinsic mechanism. They instead reveal an important aspect of in vivo dynamics and it will be import to determine what factors regulate this process in future studies.

    The above statement reflect the joint opinions of: John Cooper (Washington University), Dave Sept (University of Michigan) and David Sibley (Washington University).

  • Markus Meissner2017 Jun 19 8:37 p.m. (3 days ago) 1 of 1 people found this helpful

    We thank David Sibley for his last comment. As we mentioned previously, it was not the aim of this study to prove or disprove isodesmic polymerisation. We highlighted the current discussion in the field regarding isodesmic polymerisation (see previous comments). It is contra productive to turn the comments on this paper into a discussion on Skillmann et al., 2013, which is seen with great scepticism in the field. We made our views clear in previous responses and we hope that future results will help to clarify this issue. However, we find it concerning (and distracting) that– in contrast to his earlier comments, according to which our data can be consolidated with isodesmic polymerisation -David Sibley is now doubting the validity of our data, mentioning that CB might affect actin dynamics. This is certainly the case, as shown in the study and as is the case with most actin binding proteins used to measure actin dynamics in eukaryotic cells. This issue was discussed at length in the manuscript, by the reviewers comments and authors response, which can all be easily accessed: https://elifesciences.org/articles/24119 The above statement reflect the joint opinions of: Markus Meissner (University of Glasgow), Aoife Heaslip (University of Conneticut) and Robert Insall (Beatson Institute, Glasgow).

  • In reply to a comment by L David Sibley2017 Jun 14 10:52 a.m.

    Markus Meissner2017 Jun 16 06:20 a.m. (6 days ago)

    Thank you for your comment which appears to be only a slight update of the comments already made on the eLIFE website and it would be helpful for all readers who wish to follow this discussion if we could stick to the website where the discussion started (see: https://elifesciences.org/articles/24119).

    Regarding the second comment of David Sibley: It is good to see that the authors of the Skillmann paper (Skilmann et al., 2013) are able to reconcile our data with their unusual, isodesmic polymerisation model, despite their initial interpretations that clearly states that “…an isodesmic mechanism results in a distribution of SMALL OLIGOMERS, which explains why TgACTI only sediments efficiently at higher g force. Our findings also explain why long TgACTI filaments have not been observed in parasites by any method, including EM, fluorescence imaging of GFP–TgACTI and Ph staining." While it appears that we will need a lengthy discussion about Skillmann et al., 2013 or even better more reliable assays to answer the question of isodesmic vs cooperative polymerisation, our study did not aim to answer this open issue that we briefly introduced in Periz et al., 2017 to give a more complete picture of the open questions regarding apicomplexan actin. As soon as more convincing evidences are available for cooperative or isodesmic polymerisation of apicomplexan actin, we will be happy to integrate it in our interpretation. Meanwhile we remain of the opinion that our in vivo data (see also Whitelaw et al., 2017) best reflects the known behaviours of canonical actin. While it seems that under the conditions used by Skillmann et al., 2013 apicomplexan actin polymerizes in an isodemic manner, in the in vivo situation F-actin behaviour appears very similar to other, well characterised model systems. However, we would like to point out that a major argument in the interpretation of Skillmann et al., 2013 for isodesmic polymerisation is that “This discovery explains previous differences from conventional actins and offers insight into the behaviour of the parasite in vivo. First, nucleation is not rate limiting, so that T.gondii does not need nucleation-promoting factors. Indeed, homologs of actin nucleating proteins, such as Arp2/3 complex have not been identified within apicomplexan genomes”. This statement is oversimplified and cannot be reconciled with the literature on eukaryotic actin. For example, Arp2/3 knockouts have been produced in various cell lines (and obviously their actin doesn’t switch to an isodesmic polymerisation process). Instead, within cells, regulated actin assembly is initiated by two major classes of actin nucleators, the Arp2/3 complex and the formins (Butler and Cooper, 2012). Therefore, we thought it is necessary to mention in Periz et al., 2017 that apicomplexans do possess nucleators, such as formins. Several studies agree, that apicomplexan formins efficiently NUCLEATE actin in vitro, both rabbit and apicomplexan actin (Skillmann et al., 2012, Daher et al., 2010 and Baum et al., 2008). In summary we agree that future experiments will be required to solve this issue and we are glad that David Sibley agrees with the primary findings of our study. We hope that future in vitro studies will help to solve the question of isodesmic vs cooperative polymerisation mechanism in the case of apicomplexan actin so that a better integration of in vivo and in vitro data will be possible.

  • Serious Bacterial Infections Acquired During Treatment of Patients Given a Diagnosis of Chronic Lyme Disease - United States.

    Marzec NS.MMWR Morb Mortal Wkly Rep. 2017.4 commentsRaphael Stricker and Sin Hang Lee also commented

    L Ide2017 Jun 19 5:47 p.m. (3 days ago)

    Sin Hang Lee, please read the NEJM. Longterm antibiotics are rubbish. I hope you 're not in favor of the not evidence-based test elispot? Thank you Marzec et al. for your article.

  • Martine Crasnier-Mednansky2017 Jun 19 11:44 a.m. (3 days ago)

    Novick A, 1957 reaffirmed a fully induced culture could be maintained fully induced at low inducer concentrations. In this paper, the authors reported preinduced cells with melibiose do not maintain induction of the melibiose (mel) operon in the presence of 1 mM TMG. However, experimental conditions and data interpretation are both questionable in view of the following.

    The authors used a lacY strain whose percentage of induction by 1 mM TMG is less than 0.2%, 100% being for melibiose as the inducer (calculated from data in Table 1 and 3). They transfer the cells from a minimal-medium-melibiose to a minimal-medium-glycerol supplemented with 1 mM TMG. The cells therefore have to 'enzymatically adapt' to glycerol while facing pyrimidine starvation (Jensen KF, 1993, Soupene E, 2003). Under these conditions, cells are unlikely to maintain induction of the mel operon (even if they could, see below) because uninduced cells have a significant growth advantage over induced cells. Incidentally, Novick A, 1957 noted, "the fact that a maximally induced culture can be maintained maximally induced for many generations [by using a maintenance concentration of inducer] shows that the chance of a bacterium becoming uninduced under these conditions is very small. Were any uninduced organisms to appear, they would be selected for by their more rapid growth". Advancing further, the percentage of induction by TMG for the mel operon in a wild type strain (lacY+) is 16% (calculated as above). This induction is due mostly to TMG transport by LacY considering the sharp decrease in the percentage of induction with a lacY strain (to <0.2%). Consequently, in the presence of TMG, any uninduced lacY cells remain uninduced. Thus, it appears a population of uninduced cells is likely to 'take over' rapidly under the present experimental conditions.

    In the presence of LacY, the internal TMG concentration is about 100 times the medium one, and under these conditions, induction of the mel operon by TMG is only 16%. Therefore, the cells could not possibly maintain their full level of induction simply because TMG is a relatively poor inducer of the mel operon. It seems the rationale behind this experiment does not make much sense.

    Note: The maintenance concentration of inducer is the concentration of inducer added to the medium of fully induced cells and allowing maintenance of the enzyme level for at least 25 generations (Figure 3 in Novick A, 1957). It is not the intracellular level of inducer, as used in this paper.

  • Jan Tunér2017 Jun 19 11:22 a.m. (3 days ago)edited

    The authors have used 780 nm, 20 mW, 0.04 cm2, 10 seconds, 0.2 J per point, 1.8 J per session. This is a very low energy. Energy (J) and dose (J/cm2) both have to be within the therapeutic window. By using a thin probe, a high dose can easily be reached but the energy here is much too low in my opinion. The authors quote Kymplova (2003) as having success with these parameters, but this is not correct. The multimode approach of Kymplova was as follows: The light sources were as follows: a laser of a wave length 670 nm, power 20 mW, with continuous alternations of frequencies 10 Hz, 25 Hz, and 50 Hz, a polarized light source of a 400-2,000 nm wavelength in an interval of power 20 mW and frequency 100 Hz and a monochromatic light source of a 660 nm wavelength and power 40 mW, with simultaneous application of a magnetic field at an induction 8 mT.

  • Stefano Casola2017 Jun 19 10:08 a.m. (3 days ago)edited 1 of 1 people found this helpful

    Authors acknowledge the insightful comments. We would like to draw the attention of Dr Woodgett to the following observations:
    • The antibody used to detect GSK3-beta Serine-9 phosphorylation does not recognize phosphorylated forms of GSK3-alpha (https://www.cellsignal.com/products/primary-antibodies/phospho-gsk-3-beta-ser9-d3a4-rabbit-mab/9322). Thus, whereas our data indicate that loss of the BCR in MYC-driven lymphoma cells leads to a reduction in GSK3-beta Ser-9 phosphorylation, it remains to be investigated whether GSK3-alpha is similarly affected in these cells.

    • GSK3-beta knock-down experiments were performed using six independent shRNAs (referred to in the Methods section of the paper). Data obtained with the two most effective hairpins are shown in Extended Data Figure 5c, d. Importantly, the shRNAs were selected for their ability to target GSK3-beta but sparing GSK3-alpha. Despite only partial GSK3-beta knock-down, lymphoma cells losing BCR expression resisted substantially better to their BCR+ counterparts in competition assays, with the most effective hairpin (shRNA# 2) causing a complete block of their counter selection (Extended Data Figure 5e). These results closely mirror those obtained studying BCR+/BCR- lymphoma cell competitions treated with the GSK3 inhibitor CHIR99021 (Figures 3d and Extended Data Figure 5a).

    Therefore, whereas we cannot exclude a contribution of GSK3-alpha, our data indicate that modest changes in GSK3-beta expression/phosphorylation are sufficient to critically affect BCR-controlled fitness of MYC-driven lymphoma cells.

  • Jim Woodgett2017 Jun 15 2:53 p.m. (7 days ago) 2 of 2 people found this helpful

    This is an interesting paper but from what I can see, the evidence for these effects being dependent on GSK3beta (rather than a combination of GSK-3beta plus GSK-3alpha) is limited to a partial (maximally 35%) knockdown by siRNA in extended data figure 5 - where a marginal effect was observed (partial knockdown of GSK3alpha may have given a similar result). The pharmacological inhibitor used, CHIR99021, has NO significant selectivity for GSK3beta over GSK3alpha (the authors do refer to it being in GSK3 inhibitor in two places). In every example where phosphorylation of Serine 9 of GSK3beta has been examined along with phosphorylation of Serine 21 of GSK3alpha, they are phosphorylated in parallel. There are no kinases that target these sites selectively (not true of a more C-terminal site, Ser389, targeted on GSK3beta by p38 MAPK). Why is this important? Because throughout the paper, there are over 50 mentions of GSK3beta, including the title, yet there was no measurement of GSK3alpha phosphorylation or knockdown.

  • Stuart RAY2017 Jun 19 07:59 a.m. (3 days ago) 1 of 1 people found this helpful

    In response to a query regarding the method used to measure insulin concentration (to calculate HOMA-IR), Dr. Kernan referred me to Viscoli CM, 2014, which states: "The Linco (St. Charles, MO) human insulin-specific radioimmunoassay (RIA) was used at the laboratories in North America and Australia to measure circulating insulin concentrations. Because this assay was not available at the laboratories in Europe and Israel, the Linco animal serum-free enzyme-linked immunosorbent assay was used and results converted to RIA values by means of an internal LINCO correlation equation (insulin RIA [μU/mL] = 1.1056 × (insulin enzyme-linked immunosorbent assay [ulU/mL]) + 2.1494)."

  • Daniele Mengato2017 Jun 19 06:25 a.m. (3 days ago)edited 1 of 1 people found this helpful

    Rituximab biosimilar vs rituximab originator in advanced follicular lymphoma

    by Daniele Mengato Scuola di Specializzazione in Farmacia Ospedaliera, Dipartimento di Scienze del Farmaco, University of Padua, Padova, Italy

    One interesting point of this article by Gao et al. (1) is that the authors have presented a meta-analysis based on the end-point of the overall response rate in which chemotherapy plus rituximab has been compared with chemotherapy alone in patients with advanced follicular lymphoma (AFL). Schulz et al. (2) have conducted a meta-analysis (published in 2007) based on a similar design. In particular, they used the same endpoint obtained from the same RCTs citated in the work by Gao et al. EMA has recently approved a biosimilar of rituximab (called CPT-10) indicated for patients with AFL (3). In documenting the equivalence between CPT-10 and the originator of rituximab, the analysis carried out by EMA has evaluated the randomized trial that directly compared these two agents in the above-mentioned patients. Clinical endpoint investigated in this trial was the same as the one used in the already mentioned meta-analysis: the overall response rate. It has been proposed (4) that an analysis focused on the equivalence between a biosimilar and an originator can be strengthened if a network meta-analysis is performed that includes not only the comparison between biosimilar and originator, but also the comparison between the originator and the old standard of care (i.e. chemotherapy alone). We welcome one such network meta-analysis. For this purpose, as regards the comparison between the originator plus chemotherapy and chemotherapy alone, the 6 trials (5-10) either reported by Gao et al. or by Schulz et al. are suitable for being included in this network meta-analysis.

    References

    1. Gao G, Liang X, Jiang J, Zhou X, et al. A systematic review and meta-analysis of immunochemotherapy with rituximab for B-cell non-Hodgkin's lymphoma. Acta oncologica. 2010 Jan;49(1):3-12.
    2. Schulz H, Bohlius J, Skoetz N, et al. Chemotherapy plus Rituximab versus chemotherapy alone for B-cell non-Hodgkin's lymphoma. The Cochrane database of systematic reviews. 2007 Oct 17(4):Cd003805.
    3. EMA European Medicine Agency - Committee for Medicinal Products for Human Use (CHMP). Truxima : EPAR - Public assessment report. http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Public_assessment_report/human/004112/WC500222695.pdf 2016 May;EMA/CHMP/75695/2017
    4. Messori A, Trippoli S, Marinai C. Network meta-analysis as a tool for improving the effectiveness assessment of biosimilars based on both direct and indirect evidence: application to infliximab in rheumatoid arthritis. Eur J Clin Pharmacol. 2017 Apr;73(4):513-514. doi:10.1007/s00228-016-2177-z. Epub 2016 Dec 14.
    5. Forstpointer R, Dreyling M, Repp R, et al. The addition of rituximab to a combination of fludarabine, cyclophosphamide, mitoxantrone (FCM) significantly increases the response rate and prolongs survival as compared with FCM alone in patients with relapsed and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood 2004;104(10):3064-3071.
    6. Herold M, Pasold R, Srock S, et al. Results of a Prospective Randomised Open Label Phase III Study Comparing Rituximab Plus Mitoxantrone, Chlorambucile, Prednisolone Chemotherapy (R-MCP) Versus MCP Alone in Untreated Advanced Indolent Non-Hodgkin’s Lymphoma (NHL) and MantleCell-Lymphoma (MCL). ASH Annual Meeting Abstracts. 2004; Vol. 104, issue 11:584.
    7. Hiddemann W, Kneba B, Dreyling M, et al. Frontline therapy with rituximab added to the combination of cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) significantly improves the outcome for patients with advanced-stage follicular lymphoma compared with therapy with CHOP alone: results of a prospective randomized study of the German Low-Grade Lymphoma Study Group. Blood 2005;106(12):3725–3732.
    8. Marcus R, Imrie K, Belch A, et al. CVP chemotherapy plus rituximab compared with CVP as first-line treatment for advanced follicular lymphoma. Blood 2005;105(4):1417–1423.
    9. Rivas-Vera S, Baez E, Sobrevilla-Calvo P, et al. Is First Line Single Agent Rituximab the Best Treatment for Indolent Non-Hodgkin’s Lymphoma? Update of a Multicentric Study Comparing Rituximab vs CNOP vs Rituximab Plus CNOP. ASH Annual Meeting Abstracts. 2005; Vol. 106, issue 11:2431.
    10. van Oers MH, Klasa R, Marcus RE, Wolf M, Kimby E, et al. Rituximab maintenance improves clinical outcome of relapsed/resistant follicular non-Hodgkin lymphoma in patients both with and without rituximab during induction: results of a prospective randomized phase 3 intergroup trial.. Blood 2006;108 (10):3295–301.
  • Thomas Heston2017 Jun 18 11:58 a.m. (4 days ago) 1 of 1 people found this helpful

    This trial basically shows that treating diabetes helps decrease cardiovascular morbidity, which is not a new finding. To determine if canagliflozin has a unique property in decreasing cardiovascular events beyond simply lowering blood sugar, the analysis would have had to match patients by their hemoglobin A1c, then compare outcomes of placebo vs canagliflozin. Amazingly, this was not done. They did not look at cardiovascular events after correcting for hemoglobin A1c levels. Note that this was a pharmaceutical company funded research project, and the conclusion heavily implies that canagliflozin (as opposed to any agent that lowers blood sugar) has a unique quality of lowering cardiovascular events in diabetics. By not separating out the potential unique effects of canagliflozin beyond just lowering blood sugar, the results are basically meaningless and even worse, misleading.

  • Hilda Bastian2017 Jun 18 09:40 a.m. (4 days ago)edited

    An assessment of a critical problem, with important conclusions. It would be helpful, though, if the scope of the 4 guidelines were shown. The inclusion criteria are not very specific on this matter, and the citations of the versions of the 4 included guidelines are not provided.

    In addition to the scope, the dating of the guidelines' last search for evidence (if available) with respect to the dates of the systematic reviews would be valuable. Gauging to what extent systematic reviews were not included because of being out of scope, out of date, or not yet published is important to interpreting these findings. Given how quickly systematic reviews can go out of date (Shojania KG, 2007), the non-inclusion of older systematic reviews may have been deliberate.

    The publisher of the article does not appear to have uploaded Appendix A, which includes the references to the systematic reviews. Further, confusion has been created by linking the citations of the first 44 systematic reviews to the references of the article's texts. The end result is that neither the 4 guidelines nor the 71 systematic reviews are identifiable. It would be helpful if the authors would post these 75 citations here.

    Disclosure: I work on PubMed Health, the PubMed resource on systematic reviews and information based on them.

  • Misha Koksharov2017 Jun 17 7:48 p.m. (5 days ago)

    This is an interesting work on the use of Gaussia luciferase as a recombinant label for immunoassays. Hopefully it will encourage further use and development of new GLuc-based enzymatic tools.

    In that case it will be interesting to try the Gaussia luciferase mutant GlucM23 (Lindberg et al, Chem. Sci., 2013, 4, 4395-4400; Lindberg E, 2013) which appears to be the brightest and the smallest luciferase of all.

  • Randi Pechacek2017 Jun 17 3:20 p.m. (5 days ago)

    Simon Lax, first author of this paper, wrote a brief blog post on microbe.net about the importance of the hospital microbiome in context of this paper.

  • Vigilante Science.

    Blatt MR.Plant Physiol. 2015.26 comments

    In reply to a comment by Gerhard Nebe-von-Caron2015 Dec 15 07:26 a.m.

    Misha Koksharov2017 Jun 17 12:24 p.m. (5 days ago) 1 of 1 people found this helpful

    "Is what we do really science or a glorified marketing exercise?"

    This is indeed a very interesting and controversial question. Opinions vary from "problems are minor" to "iceberg, dead ahead, sir".

    Yuri Lazebnik wrote a really interesting and thoughtful paper (Lazebnik Y, 2015) trying to dissect this problem.

    At this time it may be even more important than his famous piece ('Can a biologist fix a radio?' Lazebnik Y, 2002) on still current challenges of comprehensive understanding of biological systems.

  • Feng Zhang2017 Jun 15 6:23 p.m. (7 days ago)edited 3 of 3 people found this helpful

    A number of researchers have inquired about the presence of duplicate sgRNAs (same sgRNA for more than one gene) in the GeCKOv2 library (Sanjana et al., Nature Methods 2014) and non-specific sgRNAs that have additional exact matches in the genome. We would like to further clarify the design considerations for GeCKOv2 (Supplementary Methods, Sanjana et al., Nature Methods 2014).

    For the GeCKOv2 libraries we decided to take the “best” sgRNA (i.e. with the fewest off-targets) we could find for a given gene, even if in some cases our “best” sgRNA had more than one targeting location in the genome. This was done to sample as many targets as possible and minimize false negatives, since false positives that are due to an sgRNA with more than one target or off target effects can be easily eliminated in post-screen validation experiments or through a gene-based analysis that selects hits based on the consistent effect of multiple unique sgRNAs. Regardless, each candidate obtained through a GeCKO screen needs to be validated through rigorous experimentation, including testing using new guides targeting each screen hit.

    A special example are gene families with high homology, in such cases our algorithm was not able to find a unique sgRNA targeting a constitutive exon. The approach that we took was to leave in these sgRNAs to give users the greatest range of options (and potential targets) during post-screen validation experiments. For example, in the human GeCKOv2 library, there are 5,664 non-specific sgRNAs. This works out to be ~4% of all guides in the library. Those redundant sgRNAs can always be removed computationally, following a screen, to simplify data analysis. (A table of these sgRNAs and genes targeted with multiple non-specific sgRNAs is available here: [link]). In contrast, GeCKOv1 did not include as many non-specific guides and consequently only targets a smaller number of genes.

    To clarify this and help users in their analysis we previously provided the GeCKOv2 sgRNA database with information about the number of off-target target hits (e.g. [link]). We also have provided an additional sgRNA index for both human and mouse GeCKOv2 libraries that lists only unique sgRNAs such that when multiple genes are targeted all of those are listed under gene_id [link].

    The GeCKOv2 libraries have already been successfully used by many groups to generate a number of interesting biological findings (e.g. Golden et al., Nature, 2017, Erb et al., Nature, 2017; Xu et al., PNAS, 2017; Jain et al., Science, 2016; Marcaeu et al., Nature, 2016; Zhang et al., Nature, 2016; Meitinger et al., JCB, 2016; Wallace et al., PLoS One, 2016; Parnas et al., Cell, 2015; Chen et al., Cell, 2015). In addition to GeCKOv2, there are a number of alternative libraries (e.g. Wang et al., Science, 2015; Doench et al., Nat. Biotechnol., 2016; Hart et al., Cell, 2015), including libraries that were designed to avoid duplicate sgRNAs by targeting fewer genes. A list of different libraries is available on Addgene’s pooled CRISPR libraries page: [link].

    We would like to specifically thank Joey Riepsaame and Timokratis Karamitros for recently bringing this issue to our attention. We also thank the GeCKO users who contacted us through the CRISPR Genome Engineering online forum and by email for additional helpful discussions.

    Neville E. Sanjana (nsanjana at nygenome.org)

    Ophir Shalem (shalemo at email.chop.edu)

    Joey Riepsaame (joey.riepsaame at path.ox.ac.uk)

    Timokratis Karamitros (timokratis.karamitros at zoo.ox.ac.uk)

    Feng Zhang (zhang at broadinstitute.org)

    References Cited

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  • Ahmet Erdemir2017 Jun 15 4:28 p.m. (7 days ago)

    Data on elastomeric foams investigated as part of this study are now available at https://simtk.org/projects/elastomericfoam

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