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  • Evolutionary switches between two serine codon sets are driven by selection.

    Rogozin IB.Proc Natl Acad Sci U S A. 2016.2 commentsDonald Forsdyke also commented

    Joshua L Cherry2017 Jun 23 12:22 p.m. (3 days ago) 1 of 1 people found this helpful


    This article claims that “the great majority of codon set switches proceed by two consecutive nucleotide substitutions…and are driven by selection”. The data in fact support a predominance of simultaneous switches that are not driven by selection. Even if we assume that sequential switches predominate, the implication that selection increases their rate by a factor of ~50 is unjustified. Moreover, selection against the non-serine intermediate is expected to decrease the rate of sequential switches, not to drive them. The authors’ argument to the contrary is analogous to arguing that a mountain range between two locations speeds the journey between them because it accelerates the downhill portion of the trip.

    Inappropriate standard of comparsion

    The usual way to establish that an evolutionary process is driven by selection is show that it is faster than some process that is largely unaffected by selection. The authors instead made comparisons to “expectations” derived from nonsynonymous substitution rates, which are greatly decreased by selection. This comparison cannot establish that switching is driven by selection, and would greatly overestimate any such effect.

    A more appropriate comparison would be to expectations derived from synonymous rates. These are several-fold higher than nonsynonymous rates, and “expectations” involve products of two rates. Thus, the claimed acceleration by selection mostly or entirely disappears with a proper standard of comparison.

    Unjustified rejection of simultaneous switching

    The authors reject a significant role for simultaneous double mutation in switching because the rate of switching is higher than the rate of analogous double changes in noncoding sequences by a factor of 5-10. This argument would be valid if non-coding regions were evolving nearly neutrally, but this is far from the case: rates of non-coding transversions (Fig. 3) are comparable to the rates of some nonsynonymous transversions (Fig 2).

    I have determined that the rates of the relevant synonymous transversions are higher than the corresponding single-base non-coding changes by a factor of >5. This presumably reflects purifying selection in non-coding regions. The effect of selection on simultaneous tandem changes is expected to be larger. Thus, the excess of serine switches over non-coding tandem changes can easily be explained by selection in non-coding regions. Put differently, we can estimate a lower limit on the rate of simultaneous serine switches, and it corresponds to the majority of the observed switches.

    Slow- vs. fast-evolving genes

    The article claims to have shown that the rate of switching is “higher in conserved genes than in nonconserved genes in full agreement with the selection hypothesis”. The results (Fig. 6) in fact demonstrate just the opposite: the rate of switching in “nonconserved” genes (0.0032 or 0.0022) is about three times higher than that in “conserved” genes (0.0010 or 0.0008).

    The authors considered the ratio of the switching rate to a sum of products of nonsynonymous rates. This ratio is higher in “conserved” genes only because the nonsynonymous rates are lower in “conserved” genes. This is true almost by the definition of “conserved” (low dN/dS), and has nothing to do with serine codon switches.

    Theoretical expectation

    Under the simple selection scheme considered by the authors, selection will actually decrease the rate of sequential switching. After fixation of a deleterious Ser->Thr or Ser->Cys mutant, selection will indeed increase the fixation probability of a mutant that restores Ser. However, selection always decreases the fixation probability of the initial deleterious mutant by a larger factor. As illustrated here, the product of the two relative fixation probabilities, and hence the relative probability of a switch during a short interval, is always less than one (selection slows switching) for nonzero s, and it decreases monotonically and approaches zero as the strength of selection (|Nes|) increases.

    The above implicitly assumes weak mutation, but the same conclusion holds outside of this regime (Kimura, 1985).


    Neither the data nor the authors’ model supports the claim that serine codon switching is driven by selection or has an especially “high frequency”. In fact, both data and theory point to the opposite conclusion.


    Kimura, M (1985) The role of compensatory neutral mutations in molecular evolution. Journal of Genetics 64(1):7-19.

  • Randi Pechacek2017 Jun 24 7:30 p.m. (2 days ago)

    Jonathan Eisen wrote a blog post on praising this paper for its careful accuracy.

  • Randi Pechacek2017 Jun 24 7:36 p.m. (2 days ago)edited

    Ashley Ross, first author of this paper, wrote a blog post about this paper on microBEnet describing some of the background.

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  • Heat-Not-Burn Tobacco Cigarettes: Smoke by Any Other Name.

    Auer R.JAMA Intern Med. 2017.2 commentsManuel Peitsch also commented

    Valerio Cozzani2017 Jun 26 4:56 p.m. (7 hours ago)

    Classification of Aerosols formed in the operation of Heat-not-Burn tobacco products.

    “Smoke” is nowadays a term frequently used in colloquial non-technical language to indicate a wide number of different aerosols originated by the combustion or pyrolysis of a material. However, from a technical point of view, due to its importance for fire science, several definitions were adopted to clarify what aerosols should be classified as “smoke” (e.g. see the comprehensive discussions provided by Gross et al. (1967), Mulholland (2008), and Drysdale (2011) to have some examples). A comprehensive definition is given by the National Fire Protection Association (See NFPA 921): smoke is composed of airborne solid and liquid particulates and gases evolved when a material undergoes pyrolysis or combustion, together with the quantity of air that is entrained or otherwise mixed into the mass. Moreover, the above definition clearly remarks that smoke, as evident from the literature, may have a very different chemical and physical nature compared to other aerosols:

    1) from a physical point of view it may be composed of solid and liquid particles with different size distributions and concentrations;

    2) from a chemical point of view, smoke components may be: i) condensed, liquid combustion products, and/or ii) condensed, liquid products of partial combustion (as high molecular weight and/or low vapor pressure organic liquids formed during fuel primary pyrolysis and volatile emission), and/or iii) solid combustion products (mainly graphitic carbon particles as soot, and inorganic fly ashes), iv) and/or unburned or partially oxidized solid or liquid fuel particles.

    Smoke from tobacco combustion has specific features, as reported in reference publications (e.g. see Baker, 2006), and has a very complex composition, including all the above cited chemical components such as: condensed liquid drops of volatiles (tar), soot, and ashes. There is no doubt that the aerosol stream produced by a burning cigarette may be classified as “smoke” according to its definitions reported in the scientific and technical literature.

    Recently, Philip Morris International (PMI) developed a heat-not-burn tobacco product, that operates with very different modalities with respect to conventional cigarettes. Even if also in the operation of the heat-not-burn tobacco product an aerosol stream is formed, its classification as “smoke” is not appropriate. The aerosol generated in the PMI heat-not-burn tobacco product is very different in the chemical composition from the smoke formed by the self-sustained smoldering combustion of tobacco in cigarettes and more in general from smoke formed in combustion processes. The aerosol generated in the heat-not-burn tobacco product is composed mainly of water and of products deriving from the evaporation, in the absence of chemical reactions, of substances present in the original tobacco substrate present in the heat-not-burn tobacco product. Even applying the more comprehensive definitions of smoke reported in the literature, these do not apply to the aerosol produced in the operation of such devices, since:

    i) experimental data have confirmed that combustion processes are absent in the PMI tobacco product when heated in the heat-not-burn device.

    ii) the aerosol produced by the heat-not-burn tobacco product during operation is formed mostly by vaporization phenomena, as proven by the experimental data showing its chemical characterization

    iii) very limited low temperature pyrolysis phenomena may be present in the tobacco substrate present in the device during the operation of the heat-not-burn tobacco product (temperatures during operation are lower than 350°C)

    Nevertheless, it should be remarked that the above only concerns the correct scientific definition of “smoke” and its applicability to heat-not-burn tobacco products, and that in no way what is written above addresses health issues related to the inhalation of such aerosols.


    Baker R.R., Smoke generation inside cigarette: Modifying combustion to develop cigarettes that may be less hazardous to health, Progress in Energy and Combustion Science, 32, 373-385, 2006.

    Drysdale D., Introduction to Fire Dynamics, 3rd Edition, J.Wiley & Sons Ltd, UK, 2011

    Gross D., J.J. Loftus, A.F. Robertson, Method for measuring smoke from burning materials. Symposium on Fire Test Methods – Restraint and Smoke, 1966 ASTM STP 422 (ed. A.F. Robertson), pp. 166–204. American Society for Testing and Materials, Philadelphia, PA.

    Mulholland G.W., Smoke production and properties, SFPE Handbook of Fire Protection Engineering, 4th Ed. (Eds Di Nenno et al.), pp. 2.291–2.302. National Fire Protection Association, Quincy, MA, 2008.

    National Fire Protection Association, NFPA Glossary of Terms, 2016 Edition, Updated September 23rd 2016, 2016; p.1336. Last accessed June 26th, 2017.

    Valerio Cozzani is Professor of Chemical Engineering at University of Bologna, Italy. This comment is provided on the basis of the results of a scientific evaluation of PMI’s heat-not-burn device committed by PMI

  • Guangcun Huang2017 Jun 26 10:57 a.m. (13 hours ago)edited

    The third author's name, Guangcun Huang, has been incorrectly spelled as Quangcun Huang.

  • David Marks2017 Jun 26 06:25 a.m. (18 hours ago)

    This trial was neither randomised, nor controlled, and needs to be retracted. The trial did not compare an abrupt method to a gradual method, as stated, it compared an abrupt method with a mixed bag of complicated gradual methods about which it is impossible to draw solid conclusions. The lead authors have either declared significant conflicts of interest in products used in the research (Aveyard and West) or the ICMJE Form for Disclosure of Potential Conflicts of Interest is incorrect, being the form for the wrong study(Michie). The conclusion that abrupt cessation produces superior cessation rates to gradual cessation cannot be maintained on the basis of this flawed trial for the reasons given below. 1) The gradual-cessation group received short-acting nicotine replacement therapy (NRT) and nicotine patches before the quit day. The abrupt-cessation group received only nicotine patches before the quit day. The treatments are therefore confounded with different pre-exposure levels of NRT. 2) Eligible smokers were booked for an appointment with a research nurse during which the study was explained, eligibility was confirmed, and written informed consent was obtained. What research training did the so-called 'research nurses' receive or were the key study personnel basic grade practice nurses given the task of running the trial? 3) The gradual-cessation group were were supposed to reduce smoking to half of the baseline amount by the end of the first week (known as visit −1) and to a quarter of the baseline amount at the end of the second week (visit 0) in daily increments using a complex variety of procedures that were likely to have been confusing and difficult to follow. 4) The nurses provided the gradual-cessation group with nicotine patches, 21 mg/d, and a choice of short-acting NRT products (gum, lozenges, nasal spray, sublingual tablets, inhalator, or mouth spray) during the reduction period. For such products as gum and lozenges, the instruction was to use 1 dose per cigarette missed. Again, apart from the confounding, and different pre-exposure for the gradual-reduction group, the procedure is unnecessarily complex. 5) Before quitting, participants in the abrupt-cessation group were asked to use nicotine patches, 21 mg/d, but no short-acting NRT. Nicotine patches were used in this group before the quit day, a protocol that aimed to balance the effect between groups. Instead of aiming to balance the effect between groups, there should have been precise balancing, otherwise the trial cannot be described as 'controlled'. 6) Allocation of participants was the responsibility of the so-called 'research nurse' who put patients into blocks of 2, 4, and 6. This allocation was manifestly non-random: “After the participant granted consent, the research nurse opened sealed, numbered envelopes in turn. However, for pairs (for example, husband and wife), one person was allocated randomly and the other was allocated to the same group”. This was a clustered method of allocation, not randomisation. 7) The loss of 300 potential participants also raises questions about how the remaining 697 differed from the original applicants. 8) Unsurprisingly, given their complicated and non-matched treatment, significantly fewer participants in the gradual-cessation group attended visit 0, (67.0% [229 of 342] vs. 83.4% [296 of 355] in the abrupt-cessation group; P < 0.001). Fewer participants in the gradual-cessation group (61.4% [210 of 342]) than the abrupt-cessation group (71% [252 of 355]) (P = 0.007) made a quit attempt. In sum, the trial was a mish-mash of umatched 'treatments', one of which was actually three different treatments counted as one, both confounded by differing pre-cessation exposure to a variety of NRT products chosen by the participants themselves. The trial was carried by 'research nurses' working in GP practices using a batch method for allocating participants. A deliberately uncontrolled, improperly randomised trial, confounded by different NRT products between groups.

  • Thomas Heston2017 Jun 25 6:49 p.m. (yesterday)

    This study performs a meta-analysis in the right way, by following the recommendations from the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Statement. Computer aided diagnosis has been used for many years in radiology prior to the advent of computerized provider order entry incorporation in electronic medical records. Computer aided diagnosis in radiology has consistently been shown to increase diagnostic accuracy and consistency. Computerized provider order entry (CPOE) takes computer assistance beyond diagnosis, and into therapy. The next step for CPOE beyond assistance in medication prescribing, is applying CPOE to order sets. This is commonly done as of 2017. For example, a sepsis protocol order set in the CPOE may generate a dozen or so physician orders. These order sets can decrease further errors that occur when hand writing out orders. The order sets can also decrease errors that occur when the provider has to selecting orders individually from the electronic medical record. What is critically important now is to extend computer aided diagnosis beyond medical imaging into clinical areas. For example, a computerized progam, acting as a digital doctor, could continually survey a hospital's entire electronic medical record database of active patients. This computer program would look at lab results, vital signs, and additional quantitative objective data. These digital doctor programs could then quickly and perhaps more accurately identify significant medical conditions. When a clear diagnosis is identified, the digitial doctor could even initiate order sets automatically. Such a system of computer aided diagnosis in clinical medicine would not only decrease morbidity and mortality, but also decrease healthcare costs. Having common computerized terminology for the variables would allow implementation via a blockchain that would increase interoperability and widespread distribution of these smart applications running on top of electronic medical record systems.

  • Samuel Shor2017 Jun 23 3:24 p.m. (3 days ago)edited

    Marzec, et al (1) described 5 cases of treated chronic Lyme disease that resulted in poor outcomes We are concerned about 3 conclusions: 1. Characterization of chronic Lyme disease as an invalid nebulous condition 2 “…..evidence that the recommended two-tiered serologic testing is actually more sensitive the longer B. burgeorferi infection has been present” 3. “Studies have not shown that such treatments lead to substantial long-term improvements for patients.” We too are concerned about any individual whose outcomes represent complications to well-intentioned intervention. However, there is substantive support in the literature for the existence of 1. Chronic Lyme disease-Our perspective is that this represents the clinical manifestations of ongoing active infection by Borrelia burgdorferi (Bb) sensu latu complex in the setting of either chronic untreated or inadequately treated individuals. The lilkihood of undiagnosed acute Lyme is increased by the infrequency of patients recalling tick bites. In one study representing CDC criteria diagnosed Lyme disease, only 14% had that recollection. (2) Not all cases of acute Lyme are associated with an erythema (EM) rash. Over 15 years, 31% of the reported surveillance cases lacked an EM rash. (3) The ILADS guidelines (4) describe the Lyme post treatment "….persistence of B. burgdorferi in specific individuals and animal models.." The 2012 Embers (5) nonhuman primate and 2014 Hodzic (6) murine studies provide evidence of persistence of Bb infection after MBC adequate courses of antimicrobials. Additional animal and human studies support this concept (references upon request). We want to emphasize that other etiologies may be causal, but that a cohort of these patients likely have a perpetuation of chronic signs and symptoms due to an active Bb infection. 2. Sensitivity of two tiered testing in late Lyme: Based upon a 2008 study by Steere et al (7) “the sensitivity of 2-tier testing in patients with later manifestations of Lyme disease was 100%, and the specificity was 99%” Entrance criteria for late stage Lyme: “In all patients with neurologic, cardiac, or joint involvement, a serologic result positive for B. burgdorferi by ELISA and Western blot was required for case inclusion….” “Because the entrance criteria for the aforementioned analysis REQUIRED positive serologies … by definition, all patients with disseminated or persistent Lyme disease were required to have a positive serologic test result. It is disingenuous to define a condition by a positive test result and then state that the test has 100% sensitivity…” (8) By extension, the concept of seronegativity is well-documented in cases of chronic Lyme disease. (references upon request) 3. “Studies have not shown that such treatments lead to substantial long-term improvements for patients.” A number of studies discount this claim. In 2 of the 4 NIH supported prospective human trials by Fallon (9) and Krupp (10), sub-cohort analysis showed statistically significant benefit to retreatment. In the former study 37 patients who were suspected of having active neuroborreliosis, and were treated with 10 weeks of 2gms/day IV Ceftriaxone. Pain and physical functioning improved at 12 and was sustained at 24 weeks. The authors indicated that “these benefits were felt to be independent of carefully assessed placebo effects.” In the latter study 55 patients who were felt to have active infection by Bb, with persistent severe fatigue of 6 or more months received 28 days of IV Ceftriaxone. A significant improvement in fatigue was sustained at 6 months. Other prospective trials of prolonged antimicrobial treatment were employed that revealed statistically significant improved outcomes. (11-13) In summary, as unfortunate are the 5 cases reported by Marzec, it is this author’s belief that they should not be used to discount a real entity, chronic Lyme disease. Whether due to the lack of timely diagnosis or adequacy of intervention, the literature supports the concept of chronic active Bb infection. That the diagnostic sensitivity of the 2 tiered paradigm is flawed, and seronegative active Bb infection exists. That emphasis should be made to generate a careful differential diagnosis, proactive management with probiotics and careful monitoring in the selective utility of long term antibiotics. As such, these often disabled individuals will more readily have access to the care they deserve, with compassion and empathetic oversight. Samuel Shor, MD, FACP President ILADS [International Lyme and Associated Diseases Society] Associate Clinical Professor George Washington University Health Care Sciences 1. Marzec NS, 2017 2. Berger BW, 1989 3. Bacon RM, 2008 4. Cameron DJ, 2014 5. Embers ME, 2012 6. Hodzic E, 2014 7. Steere AC, 2008 8. Stricker RB, 2008 9. Fallon BA, 2008 10. Krupp LB, 2003 11. Cameron D, 2008 12. Wahlberg P, 1994 13. Oksi J, 1998

  • L Ide2017 Jun 19 5:47 p.m. (7 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.

  • Alexander Kraev2017 Jun 23 12:03 p.m. (3 days ago) 1 of 1 people found this helpful

    Regretfully, this article has a misleading title and abstract. The correct title should be "Strenuous exercise triggers a life-threatening response in C57BL/6J mice carrying RYR1 Y522S/WT and CASQ1 null mutations". Besides, the authors never care to state that they analyze the pathogenesis of an experimental disease, without attempting to decide, whether it is closely related to the respective disease of man.

  • Dorothy V M Bishop2017 Jun 23 01:30 a.m. (3 days ago) 1 of 1 people found this helpful

    There is some evidence for diagnostic substitution from developmental language disorder to autism. I suspect, unfortunately, that this might be harder to quantify in public data than the substitution from intellectual disability, unless states have been consistent in terminology used for language disorder. Our study on this topic was just based on an adult follow-up of a small sample of children who had been diagnosed with language disorder, but it was striking how some cases who were identified as cases of language disorder (or specific language impairment) 20-30 yr earlier would nowadays been seen as clearcut cases of ASD - not because of any change in their profiles, but rather because of less restrictive diagnostic criteria for autism. Autism and diagnostic substitution: evidence from a study of adults with a history of developmental language disorder By: Bishop, Dorothy V. M.; Whitehouse, Andrew J. O.; Watt, Helen J.; et al. DEVELOPMENTAL MEDICINE AND CHILD NEUROLOGY Volume: 50 Issue: 5 Pages: 341-345 Published: MAY 2008

  • Nathaniel Huebsch2017 Jun 22 6:33 p.m. (4 days ago) 1 of 1 people found this helpful

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

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

  • Sunil Verma2017 Jun 22 1:20 p.m. (4 days ago) 1 of 1 people found this helpful

    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]

  • Randi Pechacek2017 Jun 21 5:44 p.m. (5 days ago) 1 of 1 people found this helpful

    Elisabeth Bik references this paper on a blog post, discussing Roman culture.

  • Randi Pechacek2017 Jun 21 5:37 p.m. (5 days ago) 2 of 2 people found this helpful

    Elisabeth Bik wrote a blog on, describing this paper to the public.

  • In reply to a comment by Claudiu Bandea2013 Nov 25 5:38 p.m.

  • Robert Insall2017 Jun 20 02:19 a.m. (6 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. (7 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. (7 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: The above statement reflect the joint opinions of: Markus Meissner (University of Glasgow), Aoife Heaslip (University of Conneticut) and Robert Insall (Beatson Institute, Glasgow).

  • Martine Crasnier-Mednansky2017 Jun 19 11:44 a.m. (7 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. (7 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. (7 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 ( 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.

  • Stuart RAY2017 Jun 19 07:59 a.m. (7 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)."

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