On 2014 Jul 25, Serge Ahmed commented:

Thanks for your comments on my previous comments which, contrary to what you claim, were not based on “incorrect assumptions”. I leave the readers to judge for themselves. Here I just want to persist and sign for the sake of clarification. There is no solid evidence for compulsive cocaine use in your study. Mice gave up easily on cocaine and this apparently, I now learn from your comments, regardless of strain differences. I agree that your study was not designed to directly compare the motivation for food with that for cocaine. But still it shows that mice are able to produce hundreds of responses to obtain certain rewards of importance to them. The fact that mice are able to but do not expand a comparable level of effort to obtain cocaine shows that they are not that motivated for cocaine. More specifically, it shows that even after several weeks of cocaine intake, the motivation for cocaine fails to acquire a degree of intensity that approaches that for food in hungry mice. This is perhaps one of the reasons why mice were trained on a FR1 for cocaine in your study and not on a FR10 like for food. Finally, your operational measure of “perseverative responding” is difficult to interpret because the introduction of the drug-off periods also considerably influences cocaine self-administration during the drug-on periods. Judging from Figure 1, cocaine self-administration rapidly becomes very irregular, with short and long inter-injection intervals. As a result, some of the inter-injection intervals during the drug-on periods are much longer than the drug-off periods themselves, making behavior during the latter periods quite difficult to interpret!

On 2014 Jul 22, Veronica Alvarez commented:

Thanks for your interest in our study. These are interesting comments but based on incorrect assumptions. First, the behavioral score is a combination of breakpoint and responding during the time out period and thus it asses/compounds the individual variability in the incentive motivation for cocaine and the inability to restrain responding when the drug is not available (even when it is an anticipatory response and subjects “are unable to wait until the end of the drug period”). Second, the comparison of breakpoint for cocaine and food is an unfair comparison under these conditions. Mice responding for food were under caloric restriction and must work during the session to earn their remaining daily caloric requirement. This is a very different situation than for cocaine and the incentive motivation for food and cocaine are just incomparable. In addition, mice trained on FR1 for cocaine and FR10 for food which will influence the breakpoint values. The study was not designed to compare breakpoints for cocaine and food. Third, there is no effect of CNO per se. The effect seen corresponds to activation of Gi-coupled DREADDS in indirect-pathway D2-receptor expressing neurons in the NAc which is shown to inhibit the output (GABA transmission) of these neurons and increase the breakpoint for cocaine compared to days the same mice received saline (Fig. 4). The difference in responding between mice in Fig. 1-3 and mice in Fig. 4 and 5 is due to the fact that these are two different strains of genetically engineered mice. Experiments in Fig. 1-3 utilized Drd1a-GFP mice (Swiss Webster background) to identity direct- and indirect-pathway neurons while experiments in Fig. 4 and 5 used Adora2a-Cre-/+ mice (C57Bl6 background) to selectively manipulate the activity of indirect pathway neurons using DREADDs or ChR2. Contrary to the comment’s statement, responding was higher for mice of C57Bl6 background (35±9.5 mg/kg/day) than mice of SW background (18.7±6.1 mg/k/day) but the rate of responding might not be so different are the sessions were longer for C57Bl6 mice and there was no drug-off period.

On 2014 Jan 28, Serge Ahmed commented:

This study is very interesting but it also has several potential problems that may limit its relevance to understand the neural basis of compulsive cocaine use. First, there is no solid evidence for compulsive cocaine use in this study. For instance, mice were clearly able to inhibit cocaine seeking at the onset of the signaled drug-off periods. What they seemed unable to do was to wait until the end of the drug-off periods before resuming responding. This behavior may merely reflect an anticipation of cocaine availability at the end of the drug-off periods and not a compulsion (see: http://www.ncbi.nlm.nih.gov/pubmed/22985696). In addition, PR responding for cocaine was very low in this study and, in fact, 10-30 times lower that PR responding for food (i.e., about 50 versus 1000 responses per PR sessions for cocaine and food, respectively). In other words, mice gave up quickly on cocaine, without spending much effort. Calling this behavior “compulsive” or even “compulsive-like” is excessive. Finally, the facilitatory effects of CNO-induced inhibition of D2-MSNs on PR responding for cocaine should be interpreted with caution. These effects were seen in a small group of mice (n = 6) that had an atypical low baseline (i.e., saline) level of PR responding compared to the other groups from this study (i.e., 18.7±6.1 versus 35±9.5, 41.5±18.2 or 59.2±24.1). In fact, PR responding following CNO-induced inhibition of D2-MSNs did not differ from baseline responding in these other groups.

On 2013 Jun 29, Andries Zijlstra commented:

In addition to the specific experimental findings, this article demonstrates that: 1) Regulating a molecular mechanism of migration can be an independent predictor of patient survival. 2) For molecular mechanism that drive cancer, the protein availability and function and not gene-expression correlate to disease progression and patient outcome.

On 2013 Oct 24, Tom Kindlon commented:

None

On 2013 Oct 24, Tom Kindlon commented:

(rest of references)

7 Moss-Morris R, Sharon C, Tobin R, Baldi JC. A randomized controlled graded exercise trial for chronic fatigue syndrome: outcomes and mechanisms of change. Journal of Health Psychology 2005; 10:245-59.

8 White P, Goldsmith K, Johnson A, Potts L, Walwyn R, Decesare J, et al. Comparison of adaptive pacing therapy, cognitive behaviour therapy, graded exercise therapy, and specialist medical care for chronic fatigue syndrome (PACE): a randomised trial. Lancet 2011; 377:823–36.

9 McCrone P, Sharpe M, Chalder T, Knapp M, Johnson AL, Goldsmith KA, et al. Adaptive Pacing, Cognitive Behaviour Therapy, Graded Exercise, and Specialist Medical Care for Chronic Fatigue Syndrome: A Cost-Effectiveness Analysis. PLoS ONE 2012; 7(8):e40808.

10 Haywood KL, Staniszewska S, Chapman S. Quality and acceptability of patient-reported outcome measures used in chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME): a systematic review. Quality of Life Research 2012; 21:35-52.

11 Collin SM, Crawley E, May M, Sterne JA, Hollingworth W, UK CFS/ME National Outcomes Database ME. The impact of CFS/ME on employment and productivity in the UK: a cross-sectional study based on the CFS/ME National Outcomes Database. BMC Health Services Research 2011; 11:217.

(By the way, the reason I deleted another comment on this paper was simply because PubMed Commons had posted the same comment by me twice)

On 2013 Oct 24, Tom Kindlon commented:

Objective outcome data from the chronic fatigue syndrome specialist services would have been interesting

It is disappointing that Crawley et al.¹ presented no objective outcome data from the six chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) specialist services; an audit of Belgian CFS rehabilitation centres that used cognitive behavioural therapy (CBT) and graded exercise therapy (GET) found that, while improvements were reported with various subjective measures, no change was noted with exercise testing.^2-4 Moreover, there was actually a decrease in average hours in paid employment, from 18.3% of a 38 hour-working week to 14.9%.

Discrepancies between subjective and objective measures have also been found in trials of both CBT^5,6 and GET for CFS.⁷ Indeed, in the PACE Trial (which compared to the specialist services reported similar improvements on the Chalder Fatigue questionnaire (CFQ) but better SF-36 physical functioning (PF) scores), although moderate sized improvements were found with self-report instruments like the CFQ and SF-36 PF, differences with more objective measures were smaller, if they existed at all.^8,9 For example, there was no difference between the CBT and specialist medical care only (SMC) groups on the six minute walking test.⁸ And no difference between SMC and both CBT and GET in terms of employment losses, overall service costs, welfare benefits or other financial payments.⁹

Research on patient-reported outcome measures, and measures of improvement that CFS patients consider important, is underdeveloped.¹⁰ Given the known considerable impact of CFS/ME on productivity and employment in those attending such services, it is reasonable to speculate that many patients, and indeed other taxpayers, would find employment data of more interest.¹¹

References:

1 Crawley E, Collin SM, White PD, Rimes K, Sterne JAC, May MT, et al. Treatment outcome in adults with chronic fatigue syndrome: a prospective study in England based on the CFS/ME National Outcomes Database. QJM first published online March 28, 2013 doi:10.1093/qjmed/hct061

2 Rapport d’évaluation (2002-2004) portant sur l’exécution des conventions de rééducation entre le Comité de l’assurance soins de santé (INAMI) et les Centres de référence pour le Syndrome de fatigue chronique (SFC). 2006. http://www.inami.fgov.be/care/fr/revalidatie/general-information/studies/study-sfc-cvs/pdf/rapport.pdf. Accessed March 30, 2013 (French language edition)

3 Evaluatierapport (2002-2004) met betrekking tot de uitvoering van de revalidatieovereenkomsten tussen het Comité van de verzekering voor geneeskundige verzorging (ingesteld bij het Rijksinstituut voor Ziekte- en invaliditeitsverzekering) en de Referentiecentra voor het Chronisch vermoeidheidssyndroom (CVS). 2006. Available online: http://www.inami.fgov.be/care/nl/revalidatie/general-information/studies/study-sfc-cvs/pdf/rapport.pdf. Accessed March 30, 2013 (Dutch language version)

4 Stordeur S, Thiry N, Eyssen M. Chronisch Vermoeidheidssyndroom: diagnose, behandeling en zorgorganisatie. Health Services Research (HSR). Brussel: Federaal Kenniscentrum voor de Gezondheidszorg (KCE); 2008. KCE reports 88A (D/2008/10.273/58) https://kce.fgov.be/sites/default/files/page_documents/d20081027358.pdf Accessed March 30, 2013

5 Wiborg JF, Knoop H, Stulemeijer M, Prins JB, Bleijenberg G. How does cognitive behaviour therapy reduce fatigue in patients with chronic fatigue syndrome? The role of physical activity. Psychological Medicine 2010; 40:1281–1287.

6 Knoop H, Prins JB, Stulemeijer M, van der Meer JW, Bleijenberg G. The effect of cognitive behaviour therapy for chronic fatigue syndrome on self-reported cognitive impairments and neuropsychological test performance. Journal of Neurology, Neurosurgery & Psychiatry 2007; 78:434-446.

(references continue in next message)

On 2013 Jul 10, Julia Gilden commented:

This is a nice little paper that might explain the frustrations of researchers having trouble with consistent ectopic expression. The authors speculate in the discussion that reduced RNA Polymerase I transcription at high densities may reflect a developmental transition toward the short stumpy form of the parasite. It is not clear, however, what the authors think this reduced RNA Pol I activity might accomplish, particularly because (as they point out) procyclin transcription is RNA Pol I-dependent, and a switch to the procyclin coat must be induced upon uptake by the fly.

On 2014 Jul 12, Jorge H Ramírez commented:

The overall quality of this randomized clinical trial is poor:

• Limited time of follow-up (12 weeks) to determine the safety and effectiveness of an active pharmacological principle or a fixed-dose combination (i.e., solifenacin plus tamsulosin).

• No appropriate description of the interventions in each study arm

• The study evaluates surrogate instead of definitive outcomes.

• No description of allocation concealment mechanisms.

• No description of sample size calculation.

• The full text article does not specify dates for patient recruitment and follow-up

• No description of methods used to generate the random sequence used in the randomization of patients to each study arms

• The article does not describe methods for the implementation of the random sequence.

• No appropriate description of blindings in the study.

• No intention-to-treat analysis

• Cardiovascular adverse events were not appropriately described in this article.

• Haemodynamic variables were not included as primary or secondary outcomes in this study.

Tamsulosin is a nonuroselective alpha blocker associated with severe hypotension in men.(1,2) Moreover, over three quarters of the studies with tamsulosin in humans are unpublished.(3)

References

1. Bird Steven T, Delaney Joseph A C, Brophy James M, Etminan Mahyar, Skeldon Sean C, Hartzema Abraham G et al. Tamsulosin treatment for benign prostatic hyperplasia and risk of severe hypotension in men aged 40-85 years in the United States: risk window analyses using between and within patient methodology BMJ 2013; 347:f6320 http://www.bmj.com/content/347/bmj.f6320

2. Ramirez Jorge. Severe hypotension associated with α blocker tamsulosin BMJ 2013; 347:f6492 http://www.bmj.com/content/347/bmj.f6492

3. Ramirez, Jorge H (2014): Expression of concern about tamsulosin: over three quarters of human studies are unpublished. figshare. http://dx.doi.org/10.6084/m9.figshare.1094338 URL:http://figshare.com/articles/Expression_of_concern_about_tamsulosin_over_three_quarters_of_human_studies_are_unpublished/1094338

On 2013 Jul 17, James Troendle commented:

The section on the importance of interim monitoring leaves out one important drawback to sequential studies when the sequential design is motivated by reducing the sample size. Yes, sequential studies can on average reduce the sample size. However, they do that by delivering reduced precision of the effect estimate. This reduction in precision of the final estimate is often ignored when a decision is made about whether to adopt a sequential or fixed design.

On 2014 Jan 01, José Morales Roselló commented:

Like many other psicological disorders in the past, science goes behind reality. In this syndrome, children have an absolutely normal father (or mother) but have been programmed to hate him. The hate and the attitude of the children have no correlation with the father behaviour, and have more to do with a subtle denigration campaign with a mother (or father) origin. The original syndrome descriptions made by Gardner are easily found in most of the cases.

On 2014 Jun 20, Christos Chinopoulos commented:

Could it be that LPS-induced succinate over-production is due to itaconate formation? LPS induces Irg1, a gene

coding for cis-aconitate decarboxylase, specifically expressed in cells of macrophage lineage Proc Natl Acad Sci

U S A. 2013 May 7;110(19):7820-5. doi: 10.1073/pnas.1218599110, yielding itaconate. Itaconate is preferentially

used by succinyl CoA ligase forming itaconyl CoA, thus generating an accumulation of succinate when the ligase

operates towards succinyl CoA formation ADLER J, WANG SF, & Lardy HA (1957) The metabolism of itaconic acid by

liver mitochondria. J. Biol. Chem, 229, 865-879, and WANG SF, ADLER J, & Lardy HA (1961) The pathway of

itaconate metabolism by liver mitochondria. J. Biol. Chem, 236, 26-30.

On 2016 Sep 27, Andrea Messori commented:

Modeling the economic consequences of inhibitor development in previously untreated patients with severe hemophilia A

By Andrea Messori, HTA Unit, Regional Health System, 50100 Firenze (Italy)

Background

If one performs a PubMed search using the keywords “Markov AND (haemophilia OR hemophilia) AND inhibitors”, only 4 articles are retrieved (published in 2013, 2003, 2003, and 2000). This demonstrates that, despite its potential advantages, the use of Markov models in haemophilia patients is still very limited. In the past 10 years, the study by Farrugia and co-workers (Haemophilia. 2013 Jul;19:e228-38.) is the only example of a Markov model designed to handle the data of patients with severe disease and inhibitors. Quite recently, we have developed a similar model that has not yet been formally published. Hence, this Comment has the purpose to describe the main characteristics of our model.

Model description

Our model is represented in Figure 1 (link to display this figure: http://www.osservatorioinnovazione.net/papers/markov-hemophilia-figure1.gif ). A commercial software (TreeagePro, Treeage Software Inc., 2011 version, Williamstown, MA, USA) has been employed for its development. Briefly, the core of the model is a decision node (not shown in Figure 1) from which two branches originate, the first describing the patients assigned to recombinant factor VIII (Panel A in Figure 1) and the second those assigned to plasma-derived factor VIII (Panel B in Figure 1). A total of 10 health states are included in the Markov model (see the Appendix reported below for further details). The purpose of the model is to carry out a budget impact analysis in which the replacement therapy and the treatment of inhibitors are the main cost items. The transition probabilities that manage how patients move across the health states are also presented in Figure 1 (Panels A and B). Probabilities with values of 0 or 1 are self-explanatory; accordin to the syntax of TreeagePro, the symbol “#” identifies a probability equal to the value needed to reach 100% after taking into account the other probability/probabilities directly expressed in numerical form and assigned to the other branch(es) of the same node. In each of the two main sections of the model (i.e. recombinant factor VIII [Panel A] and plasma-derived factor VIII [Panel B]), the Markov analysis incorporates the adjustment for annual discount rates and traces the number of cycles evaluated in the iterative process. According to the Markov approach, costs incurred in the model are iteratively summed upon each cycle. Three items participate in the cost analysis, namely the annual cost per patient treated with recombinant factor VIII (denoted as “annualcostric”), the annual cost per patient treated with plasma-derived factor VIII (denoted as “annualcostpd”), and the cost per patient of immunotolerance therapy (denoted as “costofIT”). As regards the syntax of the Treeage software, cost data are handled as “incremental rewards” (denoted as “Incr Rwd”). In other words, the variable “Rewards” was used to cumulate the various cost data at each cycle. The TreeagePro code needed for running the model is available from th author upon request.

Appendix. Detailed description of the 10 health states included in the Markov model

The main health states included in the recombinant branch of the model (ordered bottom-up according to Panel A of Figure 1) are the following: a) state denoted as “Without” in which patients assigned to heath-state ( c) are assumed not to develop high-titer inhibitors and then move to the health state “all-people-final-ric”; b) state denoted as “With” in which patients assigned to heath-state ( c) are assumed to develop high-titer inhibitors and then move to the health state “IT-ric”; c) state denoted as “all people ric” in which patients treated with recombinant factor VIII proceed in their Markov iterative process and are exposed to the risk of developing high-titer inhibitors; d) state denoted as “all-people-final-ric” in which patients treated with recombinant factor VIII (irrespective of whether they have developed or not high-titer inhibitors) proceed in their Markov iterative process until the end of the time horizon without being any longer exposed to the risk of developing high-titer inhibitors; e) state denoted as “IT-ric” in which patients treated with recombinant factor VIII who have developed high-titer inhibitors receive immune-tolerance therapy and move to the health state “post-IT-ric” and then to the health state “all-people-final-ric”. The main health states included in the plasma-derived branch of the model (ordered bottom-up according to Panel B of Figure 1) are the following: f) state denoted as “Without” in which patients assigned to heath-state (h) are assumed not to develop high-titer inhibitors and then move to the health state “all-people-final-pd”; g) state denoted as “With” in which patients assigned to heath-state (h) are assumed to develop high-titer inhibitors and then move to the health state “IT-pd”; h) state denoted as “all people ric” in which patients treated with recombinant factor VIII proceed in their Markov iterative process and are exposed to the risk of developing high-titer inhibitors; i) state denoted as “all-people-final-pd” in which patients treated with recombinant factor VIII (irrespective of whether they have developed or not high-titer inhibitors) proceed in their Markov iterative process until the end of the time horizon without being any longer exposed to the risk of developing high-titer inhibitors. j) state denoted as “IT-pd” in which patients treated with recombinant factor VIII who have developed high-titer inhibitors receive immune-tolerance therapy and move to the health state “post-IT-pd” and then to the health state “all-people-final-pd”. The model was designed to predict in these cohorts the expected expenditures, that were quantified on the basis of the (different) cumulative incidence of high-titer inhibitors and the (different) cost of the replacement products between the cohorts. It should be noted that some health states assigned, in Figure 1, to the first-level branches of both Panels A and B (governed by the Markov node) are not reachable at the first cycle of the Markov process (i.e. probability = 0 at this cycle), but are designed to be reached from the second-level branches, associated with the development or not of high-titer inhibitors. Finally, although the Treaage software allows to manage other variables participating in the calculation of rewards (namely: initial reward denoted as “Init Rwd”, which is summed upon entry in the health state; and: final reward denoted as “Fin Rwd”, which is summed upon exit from the health state), these were not necessary for the purposes of our model and were therefore kept at 0 (as illustrated in the two panels of Figure 1).

On 2014 Jul 20, Chris Carter commented:

The T.Gondii/host interactome Regularly updated

On 2013 Jun 14, Sebastian Schneeweiss commented:

Nice summary of assumptions and the issues arising when doing 1:1:1 matching with testing of several algorithms. If you plan PS matching of several treatment groups this is worth reading.

On 2014 Jul 19, Torsten Seemann commented:

The URL in the abstract no longer exists, it has been moved to http://ccb.jhu.edu/software/EDGE-pro/

On 2016 Jun 11, Daniel Schwartz commented:

The HOSPITAL score can be used online and in mobile apps here: http://qxmd.com/calculate/calculator_353/hospital-score

Conflict of interest: Medical Director, QxMD

On 2014 Jan 31, Huiqi Qu commented:

Evolutionary selection evidence on synonymous codon usage was observed in this study. Email: huiqi.qu@gmail.com

On 2014 Jun 16, Allison Stelling commented:

I did a Spotlight Interview for this paper in Spectroscopy Online: Using Infrared Spectroscopy for Real-Time Diagnostics During Brain Surgery.

On 2015 Mar 29, Harri Hemila commented:

Impure placebo as an unsound concept and other problems in the paper by Howick et al. (2013).

A number of problems in the Howick et al. paper are described at the PLoS ONE comment page and the comments are also available as a separate document with A4 layout.

The above mentioned criticism was formulated to a regular paper by Louhiala P, 2015.

On 2016 Sep 27, UFRJ Neurobiology and Reproducibility Journal Club commented:

We’d like to point out that in the legends of all figures in these article (except for Figure 7), the authors mention that data is presented as mean +- s.e.m.; nevertheless, in some of these figures (e.g. 2F, 3B, 3C and 3F), the error bars are clearly asymmetric. As standard errors of the mean are symmetric by definition, this seems to indicate that the data does not match the description in the legend. Therefore, the authors should clarify what the error bars presented stand for, as different kinds of error bars represent very different things and can lead to different inferences about the data (Cumming G, 2007; Krzywinski M, 2013).

On 2014 Dec 17, Sean Ekins commented:

some slides on this work http://www.slideshare.net/ekinssean/dual-event

On 2015 Jan 31, George McNamara commented:

I hope the US patent office rejects their patent application based on prior art - MVI has been selling DarkLite adapter since 2000 or earlier (possibly a decade earlier), see http://www.mvi-inc.com/products/microscopy-accessories/darklite-for-autoradiography/

On 2016 Sep 27, Andrea Messori commented:

Comment deleted.... Please refer to: https://www.ncbi.nlm.nih.gov/pubmed/23534877#cm23534877_30166

On 2014 Mar 07, Arnaud Chiolero MD PhD commented:

A strong argumentation against the idea that having free access to all kind of (big-)data will lead eventually to better research

On 2016 Apr 03, Ariel Fernandez commented:

Protein folding in vitro is a spontaneous process [1]. This means it is irreversible in the thermodynamic sense [2]. The endpoints of the folding process, the native state and the denatured ensemble, may be recovered by restoring renaturing or denaturing conditions, respectively. Thus, at a variance with thermodynamics, this is what protein scientists usually mean when they claim that protein folding is reversible [1]. On the other hand, according to the tenets of thermodynamics, the folding and unfolding pathways themselves are untraceable and irreproducible [2]. In fact the very notion of “pathway” for a spontaneous process is thermodynamically meaningless or at least questionable. Dissipative forces intervene in the folding process producing a net increase in the entropy of the universe, and this is the hallmark of irreversibility. Therefore, the intermediate states associated with the folding process are irretrievable, as it would be the case for any spontaneous process in nature or in the lab.

Notwithstanding these thermodynamic considerations, a very active quest for folding intermediates continues to this day [3]. This search may be futile, probably meaningless from a thermodynamic perspective, unless some sort of paradox holds that, at the very least, needs to be properly dispelled. To the best of my knowledge this has not been done. Real folding intermediates are not only very difficult to trap: they do not exist per se, plain and simple. Any claim to the contrary inevitably violates the second law of thermodynamics.

Even more puzzling are the efforts to characterize folding intermediates by denaturing the native state (however carefully [4]). As the second law of thermodynamics tells us, the denaturation process is not the reverse of the folding process: only the endpoints are reversed. To discourage efforts in this direction, it would be more eloquent to resort to an analogy. Imagine you need to describe the way a standing house has been put together. No one in his/her right mind would address the problem by having the house demolished (however carefully), taking snapshots at different stages of demolition and then playing the movie backwards. Yet, intriguingly, a similar reasoning has been assumed to hold mutatis mutandis in the context of protein folding [5]. This accumulation of seeming contradictions may well lead to a paradox - and thermodynamics is full of those - and at the very least the paradox demands proper clarification in thermodynamic terms before the saga for the quest of folding intermediates continues. Ariel Fernandez

References

1. Anfinsen, C.B. Science 181, 223-230 (1973).

2. Planck, M. Treatise on Thermodynamics, 3rd edition, Dover, New York (2010).

3. Vendruscolo M. & Dobson, C.M. Nature Chem. Biol. 9, 216-217 (2013).

4. Jaremko, M., Jaremko, Ł., Kim, H.Y., Cho, M.K., Schwieters, C.D., Giller, K., Becker, S. & Zweckstetter, M. Nature Chem. Biol. 9, 264-270 (2013).

5. Fernandez, A. Biomolecular Interfaces (ISBN 978-3-319-16849-4), Springer, Berlin (2015).

On 2014 Nov 14, Christopher Southan commented:

While they followed Roche into BACE2-inhibition patents for diabetes, Novartis have since published a abstract contradicting the target status http://cdsouthan.blogspot.se/2014/12/bace2-as-diabetes-target-or-maybe-not.html

On 2014 Feb 22, Christopher Southan commented:

The 46 PubChem structures refered to in this article can be retrieved via http://www.ncbi.nlm.nih.gov/sites/myncbi/collections/public/1zuPd27ihNyflkXH7XHUWAU5L/ The CIDs are specified in the text, with activity values aligned to the example numbers. Note SureChEMBL SIDs may connect to the patents where addtional SAR data can be found. An open version of the PMID:23506624 text can be found at http://figshare.com/articles/BACE2_as_a_new_diabetes_target_a_patent_review_2010_2012_/977844

On 2017 Jul 31, Boris Barbour commented:

The restrictive journal correspondence format might allow the impression that there remains some uncertainty about these issues, but in my opinion there is none. I have added my viewpoint under the original commentary.

https://www.ncbi.nlm.nih.gov/pubmed/22572946#cm22572946_73372

On 2017 Aug 05, Fernando Castro-Chavez commented:

Dear Reader, in this work I had the opportunity to use the novel technology of 3-D magnetic levitation to produce human Vascular Smooth Muscle Cells (VSMC) calcifying after the addition of Lyso Phosphatidyl Choline (LPC), it took me a couple of years to optimize every detail to obtain the most amazing images of those spherical clusters secreting the calcium Hydroxyl Apatite (HA) in an ordered and organized manner; so, we saw that the LPC (a traveler on the surface of the LDL) is the main culprit of Atherosclerosis, because it acts as detergent of the VSMC, so, actually their efforts at calcification, trans-differentiating into osteogenic cells is their strategy of survival to continue alive. The SHN3 slightly delayed the process. Att., Fernando Castro-Chavez.

On 2014 Jan 15, Hirotaka Shoji commented:

"ImageLD/EP/TM", image analysis application softwares used for behavioral analysis in this article, are now freely available from http://www.mouse-phenotype.org/software.html.

On 2013 Oct 31, John Cannell commented:

The authors reported that immune system dysregulation is common in autism spectrum disorder (ASD”. Vitamin D deficiency produces very similar immune dysregulation.

Prietl B, 2013

Kamen DL, 2010

Yang CY, 2013

Baeke F, 2010

Thus the vitamin D theory of ASD (vitamin D deficiency being the environmental risk factor for this highly heritable disorder) is consistent with the authors work. Three recent studies, using community controls, have found 25(OH)D levels are significantly lower in children with ASD. Two of the studies below (Mostafa et al and Gong et al) also found ASD severity, as rated on standard ASD rating scales, is inversely correlated with 25(OH)D levels. Mostafa et al found an R value of -.86 for the association of serum 25(OH)D with ASD severity.

Gong ZL, Luo CM, Wang L, Shen L, Wei F, Tong RJ, Liu Y. Serum 25-hydroxyvitamin D levels in Chinese children with autism spectrum disorders. Neuroreport. 2013 Oct 1. Gong ZL, 2014

Meguid NA, Hashish AF, Anwar M, Sidhom G. Reduced serum levels of 25-hydroxy and 1,25-dihydroxy vitamin D in Egyptian children with autism. J Altern Complement Med. 2010 Jun;16(6):641-5. Meguid NA, 2010

Mostafa GA, Al-Ayadhi LY.Reduced serum concentrations of 25-hydroxy vitamin D in children with autism: relation to autoimmunity. J Neuroinflammation. 2012 Aug 17;9:201. Mostafa GA, 2012

There is a plethora of basic science explaining why low gestational or early childhood 25(OH)D levels would adversely effect brain development.

Eyles DW, Feron F, Cui X, Kesby JP, Harms LH, Ko P, McGrath JJ, Burne TH. Developmental vitamin D deficiency causes abnormal brain development. Psychoneuroendocrinology. 2009 Dec;34 Suppl 1:S247-57. doi: 10.1016/j.psyneuen.2009.04.015. Epub . Review. Eyles DW, 2009

DeLuca GC, Kimball SM, Kolasinski J, Ramagopalan SV, Ebers GC. Review: the role of vitamin D in nervous system health and disease. Neuropathol Appl Neurobiol. 2013 Aug;39(5):458-84. doi: 10.1111/nan.12020. DeLuca GC, 2013

Eyles DW, Burne TH, McGrath JJ. Vitamin D, effects on brain development, adult brain function and the links between low levels of vitamin D and neuropsychiatric disease. Front Neuroendocrinol. 2013 Jan;34(1):47-64. doi: 10.1016/j.yfrne.2012.07.001. Epub 2012 Jul 11. Review. Eyles DW, 2013

Furthermore, the vitamin D theory of autism explains most of the epidemiological facts of ASD.

Cannell JJ. On the aetiology of autism. Acta Paediatr. 2010 Aug;99(8):1128-30. Cannell JJ, 2010

Cannell JJ. Autism and vitamin D. Med Hypotheses. 2008;70(4):750-9. Cannell JJ, 2008

70% of American toddlers do not take the American Pediatric Association's recommended vitamin D supplement of 400 IU/day and few toddlers or pregnant women get any sunshine due to the sun scare. As vitamin D fortified milk consumption and sun exposure has declined, so have toddlers and pregnant women’s vitamin D levels. The dramatic increase in the incidence of ASD occurred during the same time vitamin D levels were falling in toddlers and pregnant women.

Cannell JJ. Autism, will vitamin D treat core symptoms? Medical Hypotheses. 2013 Aug;81(2):195-8. Cannell JJ, 2013

Some autism researchers seem cognizant of the entire body of autism research. For example, a group of well-known European ASD researchers, including Professor Christopher Gillberg of the Gillberg Neuropsychiatric Institute, have recently called for the need for “urgent research” into the vitamin D deficiency theory of ASD.

Kočovská E, Fernell E, Billstedt E, Minnis H, Gillberg C. Vitamin D and autism: clinical review. Res Dev Disabil. 2012 Sep-Oct;33(5):1541-50. doi: 10.1016/j.ridd.2012.02.015. Epub 2012 Apr 21.Kočovská E, 2012

Until all autism researchers become cognizant of the wider body of scientific research, we will continue to wonder how to prevent - and perhaps even treat - this modern day plague.

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2013 Jul 01, lu tian commented:

It is a very interesting idea of using observable short-term event information such as PFS status up to a landmark time point to predict the long term survival status. Predicting the residual life based on most updated current information (including short-term event status) is THE real-life problem. The "baseline" used in traditional survival analysis is somewhat artificial and pertains to the study from which the data is collected.

On the other hand, it will be interesting to study the proposed prediction performance measure such as AUC as a function of time t0, i.e., the landmark time. This can provide useful information on the choice of landmark time to evaluate the short-term event status such as tumor growth to maximize the performance boost in the predicting the long-term event outcome.

On 2015 Oct 09, Simon Young commented:

The authors of this study conclude “there was a statistically significant decrease on depression, stress, and mood disturbances scores among the intervention group”. They attribute this to the nutritional content of the Talbinah. However another explanation is more plausible. During the intervention period the participants received a single serving of Talbinah once a day, and during the control phase they received no nutritional supplement. Even if the participants were not aware of the belief that Talbinah improves mood before the study they would have been informed of this in description of the study in the consent form. Thus participants were fully aware of the fact that they were receiving a nutritional supplement thought to improve mood during one period and nothing in the other period. Given the lack of an active control group the expectations of the participants could have explained the changes seen when ingesting Talbinah. In this type of study a plausible placebo treatment is essential if the effect of an intervention is to be established. The authors discuss three potential mechanisms for the effect of Talbinah. The first is the effect of the carbohydrate in the Talbinah. The supplement contained 21.6g of carbohydrate per dose, which would obviously be a small percentage of the participants’ total daily carbohydrate intake and highly unlikely to alter mood significantly over the period of a day. While the paper includes information, on page 283, on nutrient intakes during intervention and control periods, results are given only for total calories (increased by 9.2% during the intervention period, although the Talbinah alone would have increased it by only 6.5%), zinc and magnesium. Although the authors must have had information on the increase in carbohydrate intake during the intervention, that information, surprisingly, is not included in the paper. The second potential mechanism is an increase in brain tryptophan and serotonin due to the tryptophan content of the Talbinah. The paper documents the fact that the ratio of trp:BCAA in Talbinah is much higher than in either milk or barley. However, the authors are not correct in stating on p.284 that “The amino acid composition of food is associated with brain serotonin levels, especially trp:BCAA and trp:LNCAA” and that “The ratio of trp:BCAA may have increased the tryptophan available to the brain”. As stated in the reference they cite in support of these statements (number 9 in the reference list) Wurtman RJ, 2003 “Brain tryptophan concentrations and the flux of tryptophan from blood to brain, depend, in turn, partly on plasma tryptophan and partly on plasma concentrations of ≥ 6 other large neutral amino acids (LNAAs): tyrosine, phenylalanine, leucine, isoleucine, valine, and methionine, which compete with tryptophan for blood-brain barrier transport”. The ratio of trp:BCAA is irrelevant as the brached chain amino acids are only some of the amino acids that compete with tryptophan for transport from blood to brain. The authors docment on page 284 of the manuscript that Talbinah has a low trp:LNAA ratio of 1:21. Given the low protein content of the Talbinah (1.2 g per dose, Table 1) and the low trp:LNAA ratio the Tablinah could not have increased brain tryptophan and serotonin to any appreciable extent. The third potential mechanism is an increase in zinc levels. Several studies have shown that zinc levels are often low in depressed patients. A recent review includes four studies on the effect of zinc on depressed mood Lai J, 2012. Two studies with a daily dose of 25 mg zinc showed an antidepressant effect in patients with major depression, while two other studies, one with a dose of 7 mg per day and the other with doses of 10.1 mg per day and 20.2 mg per day in different groups, did not reliably show an effect on mood in healthy people. In this paper the Tablinah increased mean zinc intake from 3.7 mg per day to 8.7 mg per day. There is no evidence that a supplemental dose of 5 mg per day of zinc can influence mood. In conclusion there is no plausible bioligcal mechanism for an effect of Talbinah, at the dose given, on mood. Given the way the study was designed the most plausible explanation for the effects on mood was a psychological mechanism, the expections of the participants.

On 2013 Dec 23, guoan zhang commented:

this study is very helpful to clarify the role of aspirin in cancer prevention because of its big sample and long follow-up time.

On 2013 Oct 31, John Cannell commented:

The authors reported that immune system dysregulation is common in autism spectrum disorder (ASD”. Vitamin D deficiency produces very similar immune dysregulation.

Prietl B, 2013

Kamen DL, 2010

Yang CY, 2013

Baeke F, 2010

Thus the vitamin D theory of ASD (vitamin D deficiency being the environmental risk factor for this highly heritable disorder) is consistent with the authors work. Three recent studies, using community controls, have found 25(OH)D levels are significantly lower in children with ASD. Two of the studies below (Mostafa et al and Gong et al) also found ASD severity, as rated on standard ASD rating scales, is inversely correlated with 25(OH)D levels. Mostafa et al found an R value of -.86 for the association of serum 25(OH)D with ASD severity.

Gong ZL, Luo CM, Wang L, Shen L, Wei F, Tong RJ, Liu Y. Serum 25-hydroxyvitamin D levels in Chinese children with autism spectrum disorders. Neuroreport. 2013 Oct 1. Gong ZL, 2014

Meguid NA, Hashish AF, Anwar M, Sidhom G. Reduced serum levels of 25-hydroxy and 1,25-dihydroxy vitamin D in Egyptian children with autism. J Altern Complement Med. 2010 Jun;16(6):641-5. Meguid NA, 2010

Mostafa GA, Al-Ayadhi LY.Reduced serum concentrations of 25-hydroxy vitamin D in children with autism: relation to autoimmunity. J Neuroinflammation. 2012 Aug 17;9:201. Mostafa GA, 2012

There is a plethora of basic science explaining why low gestational or early childhood 25(OH)D levels would adversely effect brain development.

Eyles DW, Feron F, Cui X, Kesby JP, Harms LH, Ko P, McGrath JJ, Burne TH. Developmental vitamin D deficiency causes abnormal brain development. Psychoneuroendocrinology. 2009 Dec;34 Suppl 1:S247-57. doi: 10.1016/j.psyneuen.2009.04.015. Epub . Review. Eyles DW, 2009

DeLuca GC, Kimball SM, Kolasinski J, Ramagopalan SV, Ebers GC. Review: the role of vitamin D in nervous system health and disease. Neuropathol Appl Neurobiol. 2013 Aug;39(5):458-84. doi: 10.1111/nan.12020. DeLuca GC, 2013

Eyles DW, Burne TH, McGrath JJ. Vitamin D, effects on brain development, adult brain function and the links between low levels of vitamin D and neuropsychiatric disease. Front Neuroendocrinol. 2013 Jan;34(1):47-64. doi: 10.1016/j.yfrne.2012.07.001. Epub 2012 Jul 11. Review. Eyles DW, 2013

Furthermore, the vitamin D theory of autism explains most of the epidemiological facts of ASD.

Cannell JJ. On the aetiology of autism. Acta Paediatr. 2010 Aug;99(8):1128-30. Cannell JJ, 2010

Cannell JJ. Autism and vitamin D. Med Hypotheses. 2008;70(4):750-9. Cannell JJ, 2008

70% of American toddlers do not take the American Pediatric Association's recommended vitamin D supplement of 400 IU/day and few toddlers or pregnant women get any sunshine due to the sun scare. As vitamin D fortified milk consumption and sun exposure has declined, so have toddlers and pregnant women’s vitamin D levels. The dramatic increase in the incidence of ASD occurred during the same time vitamin D levels were falling in toddlers and pregnant women.

Cannell JJ. Autism, will vitamin D treat core symptoms? Medical Hypotheses. 2013 Aug;81(2):195-8. Cannell JJ, 2013

Some autism researchers seem cognizant of the entire body of autism research. For example, a group of well-known European ASD researchers, including Professor Christopher Gillberg of the Gillberg Neuropsychiatric Institute, have recently called for the need for “urgent research” into the vitamin D deficiency theory of ASD.

Kočovská E, Fernell E, Billstedt E, Minnis H, Gillberg C. Vitamin D and autism: clinical review. Res Dev Disabil. 2012 Sep-Oct;33(5):1541-50. doi: 10.1016/j.ridd.2012.02.015. Epub 2012 Apr 21.Kočovská E, 2012

Until all autism researchers become cognizant of the wider body of scientific research, we will continue to wonder how to prevent - and perhaps even treat - this modern day plague.

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2014 Jul 25, Claudiu Bandea commented:

Everlasting confusion on ‘functional DNA’ and ‘junk DNA’

As discussed by Doolittle (1), addressing and defining the functionality of genomic DNA can be challenging. However, our common sense combined with our philosophical instinct - we know a function when we see one - should allow us to sensibly address the biology of our genome. After all, the ENCODE ‘function fiasco’ was not the result of misunderstanding the concept of biological function, nor was it due to scientific incompetence as suggested by others (2). On the contrary, because it conflicted with some of the project’s objectives and with its significance, there was a concerted effort not to bring this concept forward (3); indeed, as clearly shown in a recent ENCODE publication (4), at least some ENCODE members seem well aware of the scientific rationale and criteria for addressing putative biological functions for genomic DNA.

Nevertheless, as concluded by Doolittle (1), poor use of words in communicating scientific observations, and lack of attention to detail have led to significant misrepresentations and confusion. Here are a few examples spanning more than four decades.

In a recent study entitled “Multiple knockout mouse models reveal lncRNAs are required for life and brain,” which addressed putative biological functions of long noncoding RNAs (lncRNAs), it was concluded that “This study demonstrates that lncRNAs play critical roles in vivo…” (5). Unfortunately, both the title and the conclusion misrepresent the results; an accurate interpretation of the results is that “Multiple knockout mouse models reveal that some (or a few) lncRNAs are required for life and brain,” and that “This study demonstrates that some (or a few) lncRNAs play critical roles in vivo….” As a matter of fact, based on the results of the study, which showed that only 5 of the 18 lncRNAs, which have been selected from hundreds sequences as the best candidates for being functional, a more appropriate scientific interpretation would be: “This study demonstrates that most lncRNAs (i.e. 13 out of 18) do not appear to play critical roles in vivo….” Moreover, if the goal is to evaluate this study at the highest scientific standards (as presumably required by the journal eLife, where it was published), then, I would suggest that, due to lack of appropriate control experiments such as duplicated knockout experiments, we don’t know if the observed dysfunctions were associated with specific lncRNAs, or they were caused by untargeted genome modifications introduced accidentally during the procedure of generating knockout mice, which in fact questions the validly of the entire study. As previously noted (3), all these problems reflect the deficiencies of the current limited and closed peer review system; indeed, a document reviewed by a few peers (usually, 2 or 3) can hardly be considered peer-reviewed.

The next three examples illustrate some of the confusion surrounding the concepts of biological function and junk DNA (jDNA), albeit in a more subtle way.

In one of the first papers addressing the notion of jDNA (6), David Comings writes: “These considerations suggest that up to 20% of the genome is actively used and the remaining 80+% is junk. But being junk doesn’t mean it is entirely useless. Common sense suggests that anything that is completely useless would be discarded.”

In their iconic paper “Selfish DNA: the ultimate parasite” (7), Leslie Orgel and Francis Crick, summarize their thoughts as follows: “The DNA of higher organisms usually falls into two classes, one specific and the other comparatively nonspecific. It seems plausible that most of the latter originated by the spreading of sequences which had little or no effect on the phenotype.”

More recently, in a publication addressing the ENCODE project (8), Sean Eddy, remarked: “These data support a view that eukaryotic genomes contain a substantial fraction of DNA that serves little useful purpose for the organism, much of which has originated from the replication of transposable (selfish) elements.”

Although seemingly innocent, these citations are relevant examples of poor use of words. Some of this confusion might dissipate by recognizing that: (i) if genomic sequences are not entirely useless, (ii) if they have little effect on the phenotype, or (iii) if they serve little useful purpose for the organism, then, these DNA sequences are functional, period.

In the next example, the protagonists are Michael Eisen, the host of a popular blog suggestively named “it is NOT junk”, and Ryan Gregory, the host of another popular blog “Genomicron”. Interestingly, Eisen was the PNAS editor in charge of Doolittle’s article on jDNA (1), and, according to Doolittle, Gregory is “now the principal C-value theorist” (1). Undoubtedly, Eisen and Gregory are among the most knowledgeable and versed communicators on genome biology and jDNA and, therefore, their ‘words’ are representative of the thinking in the field.

Immediately after the publication of ENCODE’s flurry of articles and the associated publicity stunt orchestrated by a few ENCODE scientists, both Eisen and Gregory reacted forcefully, but apparently from opposite perspectives. In a post in which he blasts ENCODE as “a carefully orchestrated spectacle” (9), Eisen writes: “nobody actually thinks that non-coding DNA is ‘junk’ any more. It’s an idea that pretty much only appears in the popular press… It is dishonest – nobody can credibly claim this to be a finding of ENCODE….”

In a prompt response (10), entitled “Michael Eisen’s take on ENCODE — there’s no junk?”, Gregory goes into a detailed and cynical questioning of Eisen’s perspective. Fortunately, due to the open communication platform offered by these blogs, this scientific ‘drama’ ended within hours, when Eisen apparently clarified: “I was not saying that everybody knows that 100% of the genome is functional! I was saying that nobody thinks that 100% of non-coding DNA is non-functional” (10).

Is there an end to this distressing confusion in the field of genome biology? Unlikely, if the funding system continues to focus primarily on generating data and observations, while neglecting their interpretation and integration into productive conceptual frameworks; in other words, you only get what you pay for.

References

(1) Doolittle WF. 2013. Is junk DNA bunk? A critique of ENCODE. Proc Natl Acad Sci USA., 110:5294-300. Doolittle WF, 2013

(2) Graur D et al., 2013. On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE. Genome Biol Evol., 5:578-90. Graur D, 2013

(3) Bandea CI. 2014. Closing the gap between ‘words’ and ‘facts’ in evaluating genome biology and the ENCODE project. PubMed Commons (National Library of Medicine; Bethesda, MD). Comment on: Doolittle WF, 2013

(4) Kellis M. et al., 2014. Defining functional DNA elements in the human genome. Proc Natl Acad Sci USA., 111:6131-8. Kellis M, 2014

(5) Sauvageau M. et al., 2014. Multiple knockout mouse models reveal lincRNAs are required for life and brain development. eLife (DOI: 10.7554/eLife.01749) Sauvageau M, 2013

(6) Comings DE.1972. The structure and function of chromatin. Adv Hum Genet. 3:237-431. Comings DE, 1972

(7) Orgel LE, Crick FH. 1980. Selfish DNA: the ultimate parasite. Nature. 284:604-7. Orgel LE, 1980

(8) Eddy SR. 2012. The C-value paradox, junk DNA and ENCODE. Curr Biol. 22:898-9. Eddy SR, 2012

(9) Eisen M. 2012. This 100,000 word post on the ENCODE media bonanza will cure cancer. Blog: it is NOT junk. http://www.michaeleisen.org/blog/?p=1167

(10) Gregory TR. 2012. Michael Eisen’s take on ENCODE — there’s no junk? Blog: Genomicron. http://www.genomicron.evolverzone.com/2012/09/michael-eisens-take-on-encode-theres-no-junk/

On 2014 Jul 16, Claudiu Bandea commented:

Closing the gap between ‘words’ and ‘facts’ in evaluating genome biology and the ENCODE project

When I referred to Ford Doolittle’s article (1) as “7 pages of small print text” (2), I meant it both, literary and figuratively. Indeed, Doolittle’s paper is a remarkable example of fine print, which is likely to induce heated arguments for a long time, just as the author concluded: “many of the most heated arguments in biology are not about facts at all but rather about the words that we use to describe what we think the facts might be.”

Remarkably, more than 40 years ago, Susumu Ohno started his famous paper on junk DNA (3) with a related statement: “Over the years, I have learned that there is no such thing as a fact. What passes for a fact is in truth a set of observations and its interpretation. Therefore, the interpretation is just as important to a fact as the observation itself.”

In my previous mini-essay (2) on Doolittle’s article (1), I made 3 main points:

(i) Ever since the notion of ‘junk DNA’ (jDNA) was introduced as a metaphor for presumably non-functional genomic DNA in species with relatively high C-value, it has been clearly used by the scientists in the field of genome biology and evolution to denote genomic DNA that has no biological function at all, whether informational (iDNA) or non-informational (niDNA), and implying otherwise is nonsensical.

(ii) The two main theories on putative non-informational functions for the so called jDNA, the nucleo-skeletal and the nucleotypic functions, which “describe genome size variation as the outcome of selection via the intermediate of cell size” (4), and which Doolittle uses as pillars for his theoretical framework on genome evolution and biology, do not explain the C-value paradox.

(iii) Apparently, Doolittle is not aware of the theory that most genomic niDNA, redefined as symbiotic DNA (sDNA), functions as a protective mechanism (adaptive genomic immunity) against deleterious insertional mutagenesis by endogenous and exogenous inserting elements, such as retroviruses, and that this theory is fully supported by the current data and observations and it explains the C-value paradox (5, 6).

Here, I attempt to further close the gap between 'words' and 'facts' in addressing the genome biology and in evaluating the ENCODE project.

In the introductory section, Doolittle outlines the premise for his paper: “a flurry of articles and letters”, published in Nature in other journals under the umbrella of the ENCODE project, “collectively claim function for the majority of the 3.2 Gb human genome”, which, if true, would debunk the notion of jDNA (1). The problem with Doolittle’s premise, however, is that it is not based on facts; indeed, the “flurry of ENCODE publications” did not claim that the majority of the human genome is functional (7). On the contrary, in what seems to have been a concerted, but tacit ‘silence policy,’ the ENCODE authors went out of their way not to address the ‘functionally’ of the human genome in their publications. In light of this fact, Doolittle had no choice but to build the premise for his paper on secondary sources offered by various science writers (8-10), who were apparently caught into a publicity stunt orchestrated on the side by a few of ENCODE scientists. Whether Doolittle’s approach of using secondary sources, which is a strong departure from conventional academic standards, sets up a hasty precedent for the scientific literature remains to be seen.

So, why wasn’t the ENCODE project designed in context of the fundamental issues and knowledge about genome biology and evolution, such as the C-value paradox, limited sequence conservation among closely related species, mutational load, and the evolutionary origin of most genomic sequences from transposable elements? And, why did the ENCODE researchers choose not to address these fundamental issues in their official publications? Obviously, this makes no sense considering that their massive and expensive project was funded specifically to annotate the ‘functional sequences’ of the human genome.

Fully addressing these questions might take us deep into the science of human behavior, and might highlight deep deficiencies in our current system of funding science, which relies on a weak and closed peer review system (parenthetically, a sensible solution would be to replace this system, which is vulnerable to abuse, with a stronger and true peer review system that is open to all peers).

Nevertheless, it is inconceivable that the ENCODE leaders, who represent some of the finest science institutions in the world, were scientifically incompetent, as suggested by some critics of ENCODE (10), and were not aware of these fundamental issues on genome biology and evolution. On the contrary, it was the appreciation for this fundamental knowledge that prompted them to be silent, as this knowledge is in conflict with some of their study objectives and raises inconvenient questions about the relevance of their study and results.

However, as illustrated by Doolittle’s article, the full significance of this fundamental knowledge on genome biology and evolution is not clearly recognized in the field, which has led to tremendous confusion and has allowed projects such as ENCODE to flourish. Indeed, unfortunately, the knowledge on genome biology and evolution has yet to crystalize in clear facts. However, here is one (in large print): based on the C-value paradox, limited sequence conservation, mutational load, and the evolutionary origin of most genomic sequences from transposable elements, it is clear that MOST OF THE HUMAN GENOME CANNOT HAVE INFORMATIONAL FUNCTIONS, period.

Now that we have cracked ENCODE’s ‘code of silence’, reset some of Doolittle’s small print, and crystalized the fact that only a small fraction of the human genome has informational functions, it is time to focus on the major question remaining in the field of genome evolution and biology:

Does most of the genome in organisms with relatively high C-value have non-informational functions, or most of it is non-functional, metaphorically speaking junk?

References

(1) Doolittle WF. 2013. Is junk DNA bunk? A critique of ENCODE. Proc Natl Acad Sci USA., 110:5294-300. Doolittle WF, 2013

(2) Bandea CI. 2013. Junk DNA is bunk, but not as suggested by ENCODE or Doolittle. PubMed Commons (National Library of Medicine; Bethesda, MD). Comment on: Doolittle WF, 2013

(3) Ohno S. 1973. Evolutional reason for having so much junk DNA. In Modern Aspects of Cytogenetics: Constitutive Heterochromatin in Man (ed. R.A. Pfeiffer), pp. 169-173. F.K. Schattauer Verlag, Stuttgart, Germany.

(4) Gregory TR. 2004. Insertion-deletion biases and the evolution of genome size. Gene, 324:15-34. Gregory TR, 2004

(5) Bandea CI. 1990. A protective function for noncoding, or secondary DNA. Med. Hypoth., 31:33-4. Bandea CI, 1990

(6) Bandea CI. 2013. On the concept of biological function, junk DNA and the gospels of ENCODE and Graur et al. bioRxiv doi: 10.1101/000588; http://biorxiv.org/content/early/2013/11/18/000588

(7) ENCODE Project Consortium. 2012. An integrated encyclopedia of DNA elements in the human genome. Nature, 489:57–74. ENCODE Project Consortium., 2012

(8) Kolata G. 2012 (September 5). Bits of mystery DNA, far from ‘junk’, play crucial role. The New York Times, Section A, p. 1.

(9) Anonymous, 2012. Cracking ENCODE. Lancet, 380:950. Anonymous, 2012

(10) Pennisi E. 2012. Genomics. ENCODE project writes eulogy for junk DNA. Science, 337:1159–1161. Pennisi E, 2012

(11) Graur D et al., 2013. On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE. Genome Biol Evol., 5(3):578-90.Graur D, 2013

On 2013 Dec 08, Claudiu Bandea commented:

Junk DNA is bunk, but not as suggested by ENCODE or Doolittle

“Do data from the Encyclopedia Of DNA Elements (ENCODE) project render the notion of junk DNA obsolete?” (1). Echoing much of the response by evolutionary biologists to ENCODE’s suggestion that more than 80% of the human genome is functional (2), Ford Doolittle’s answer is clearly ‘No’ (1).

However, Doolittle did not write 7 pages of small print text in PNAS (1) to discredit ENCODE’s questionable suggestion; many people only needed a paragraph or two (3; but see Ref 4). Instead, the author uses the ENCODE momentum to cover half of century of research and thinking on the evolution of genome size, junk DNA (jDNA) and the C-value enigma in search of solutions to these top remaining puzzles in genome biology. Doolittle navigates through deep conceptual gaps left open by decades of neglect in defining even the most basic notions, such as the meaning of biological function, and concludes his epic journey with a sensible prescription: “A larger theoretical framework, embracing informational and structural roles for DNA, neutral as well as adaptive causes of complexity, and selection as a multilevel phenomenon, is needed” and that, by building this theoretical framework, “Much that we now call junk could then become functional” (1).

Like other scholars in the field of genome evolution (see, for example, Refs 3 and 4), Doolittle starts building his theoretical framework by outlining convincing data and arguments that exclude informational roles for most jDNA. Addressing non-informational roles for jDNA, however, is a much more complex and confusing issue, to the extent that the author’s narrative gets entangled as it navigates very close to a school of red herrings: “I submit that, up until now, junk has been used to denote DNA whose presence cannot reasonably be explained by natural selection at the level of the organism for encoded informational roles” (bold added) (1).

However, ever since the term ‘junk DNA’ was introduced a few decades ago (apparently, in the 1960’s, not in the 70’s as previously assumed; see Ref. 5) as jargon for presumably non-functional genomic DNA in species with high C-value, it denoted DNA whose presence could not be reasonably explained by natural selection at the level of the organism for both informational and non-informational roles, and implying otherwise is, well, fishy. Moreover, saying that “junk advocates have to date generally considered that even DNA fulfilling bulk structural roles remains, in terms of encoded information, just junk” (1) is also deceiving considering that the nucleo-skeletal and nucleotypic theories, which “describe genome size variation as the outcome of selection via intermediate of cell size” (6) have dominated the thinking in field of genome size evolution for decades; obviously, it would be equally nonsensical to state that from the perspective of non-informational functions of jDNA, the informational DNA is just junk.

Nevertheless, it is admirable that Doolittle embraces the nucleo-skeletal and nucleotypic theories as pillars of his theoretical framework on genome size evolution and jDNA, because, as stated in the following excerpt, the nucleotypic theory was presented as a substitute for his previous ideas (7) about jDNA as ‘selfish DNA’: “Although some researchers continue to characterize much variation in genome size as a mere by-product of an intragenomic selfish DNA "free-for-all" there is increasing evidence for the primacy of selection in molding genome sizes via impacts on cell size and division rates” (8).

So, is Doolittle’s suggestion valid? I don’t think so, not until the nucleo-skeletal and nucleotypic hypotheses or other hierarchical selection theories clearly explain the C-value enigma, and not before they pass the formidable ‘onion test’ (see Ref 4). As previously suggested (9,10), the data and observations interpreted as evidence for these theories can be explained simply as accommodating or adaptive responses by the hosts to the presence of large quantities of genomic jDNA sequences, which are there for other reasons.

Is jDNA bunk? As proposed almost a quarter century ago (9) and re-emphasized more recently (10), there is strong evidence that jDNA serves as an adaptive defensive mechanism against insertional mutagenesis (in both germline and somatic cells) by endogenous and exogenous inserting elements, such as retroviruses, which in humans and other multicellular species can lead to a high incidence of uncontrolled cellular proliferation, or cancer. Expectedly, as an adaptive, genomic defense mechanism, the amount of protective jDNA varies from one species to another based on insertional mutagenesis activity and evolutionary constraints on genome size, which might explain the evolution of genome size and C-value enigma.

References

(1) Doolittle WF. 2013. Is junk DNA bunk? A critique of ENCODE. Proc Natl Acad Sci USA., 110:5294-300. Doolittle WF, 2013

(2) ENCODE Project Consortium. 2012. An integrated encyclopedia of DNA elements in the human genome. Nature, 489:57–74. ENCODE Project Consortium., 2012

(3) Eddy SR. 2012. The C-value paradox, junk DNA and ENCODE. Curr Biol. 6;22(21):R898-9; Eddy SR, 2012

(4) Graur D, Zheng Y, Price N, Azevedo RB, Zufall RA, Elhaik E. 2013. On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE. Genome Biol Evol., 5(3):578-90.Graur D, 2013

(5) Graur D. 2013. The Origin of Junk DNA: A Historical Whodunnit. Judge Starling; http://judgestarling.tumblr.com/post/64504735261/the-origin-of-junk-dna-a-historical-whodunnit

(6) Gregory TR. 2004. Insertion-deletion biases and the evolution of genome size. Gene, 324:15-34). Gregory TR, 2004

(7) Doolittle WF, Sapienza C. 1980. Selfish genes, the phenotype paradigm and genome evolution. Nature. 284(5757):601-3. Doolittle WF, 1980

(8) Gregory TR, Hebert PD. 1999. The modulation of DNA content: proximate causes and ultimate consequences. Genome Res. 9(4):317-24. Gregory TR, 1999

(9) Bandea CI. 1990. A protective function for noncoding, or secondary DNA. Med. Hypoth., 31:33-4. Bandea CI, 1990

(10) Bandea CI. 2013. On the concept of biological function, junk DNA and the gospels of ENCODE and Graur et al. bioRxiv doi: 10.1101/000588; http://biorxiv.org/content/early/2013/11/18/000588

On 2017 Sep 27, Seán Turner commented:

According to the BCRC online catalog of strains, the type strain of Chiayiivirga flava is 80274, not 80273. Strain BCRC 80273 is assigned as the type strain of Azospirillum formosense Lin et al. 2012 (PubMed Id 21742820).

On 2017 Oct 31, Morten Oksvold commented:

Please note that this article contains unreliable data, and should not be cited. The central ethical review board in Sweden found research misconduct in six articles by Macchiarini, including this one.

Please see the report from the Central ethical review board in Sweden: http://www.epn.se/media/2516/pressmeddelande-o-12-2016eng.pdf https://drive.google.com/file/d/0By2HqPi4t2RbYzZweVRieVVMajhJQUM0cmFMekwyRVJTUFVr/view

This information was provided by Leonid Schneider (forbetterscience.com).

On 2013 Oct 18, James C Coyne commented:

The authors had a responsibility to inform readers that patients assigned to the control group had to pay for any care related to diagnosis and treatment of depression. In contrast, patients assigned to the intervention group received free care. More care resulting from care being free might readily explain extraordinary differences between the two groups in receipt of care and in the primary outcome.

For an extended discussion, see my PLOS blog post at Mind the Brain. Coordinating depression treatment from afar: Are results credible?

http://blogs.plos.org/mindthebrain/2013/03/12/coordinating-depression-treatment-from-afar-are-results-credible/

The letter posted below was rejected from JAMA Internal Medicine, eliminating the possibility for post publication correction of the limitations of peer review of this paper.

Effect size in depression trial could be due to inadequacies of control treatment: Comment on Davidson and colleagues (2013) Davidson and colleagues [1] claimed benefits for a collaborative care (CC) intervention for depression that exceed not only previous CC trials, but also effect sizes for a variety of interventions for depression. Strong claims often later prove to be exaggerated or simply false [2] and deserve special scrutiny. We should keep in mind that effect sizes observed in trials are not attributes of interventions, but of comparisons between interventions and control groups. Large effect sizes can simply represent the exceptionally poor outcomes of control groups. Davison et al.’s underspecified “routine” care could simply have been inadequate care. The authors failed to acknowledge that patients in the control group had to pay for any depression treatment, whereas it was provided free to the patients in the intervention group. This might explain that the number of patients in the “routine” care group who received a new prescription of antidepressants increased by only two, versus ten in the intervention group. Similarly, the number of patients in the routine care group that received psychotherapy increased only by seven, versus an increase of 42 in the intervention group. The low rate of increased treatment occurred in the control group despite providers having been informed of patients’ depression scores. Patients were designated as “depressed” based on a self-report questionnaire. Thus, we cannot determine the extent to which patients with heightened depressive symptoms but failing to meet formal criteria for major depression were appropriately not having treatment initiated or inappropriately treated. Overall, we cannot determine whether active treatment or the mere attention and support and awareness of treatment being available free were associated with the greater improvement in the intervention group. Moreover, most patients identified as “depressed” in the intervention group were not in remission at follow up. Difficulty interpreting results could have been anticipated at the time of the study’s design. In short, results of this trial are insufficient to encourage a more ambitious trial with the same basic design, because of a lack of demonstration that any particular elements of centralized care management account for the group differences in improvement in depression that were observed, rather than inadequacies in the care provided to the control group.

1. Davidson, KW, Bigger, JT, Burg, MM, Carney, RM, Chaplin, WF, Czajkowski, S, Dornelas, E, Duer-Hefele, J, Frasure-Smith, N, Freedland, KE; Haas, DC; Allan S. Jaffe, AS,, Ladapo, JA,; Lespe´rance, F, Medina, V, Newman, JD, Osorio, GA Parsons, F, Schwartz, JE, Shaffer, JA Shapiro, PA,. Sheps, DS, Vaccarino, V, Whang, W, Ye, S. Centralized, Stepped, Patient Preference–Based Treatment for Patients With Post–Acute Coronary Syndrome Depression CODIACS Vanguard Randomized Controlled TrialCODIACS Vanguard RCT. JAMA Internal Medicine, 2013, 1-8.

2. Ioannidis, J. P. (2005). Why most published research findings are false. PLoS Medicine,

On 2013 Oct 23, Paul Gardner commented:

This is, in my totally unbiased opinion, a very exciting comparison of transposon mutagenesis libraries from two related species. This has fascinating consequences for pathogen evolution.

On 2015 Apr 04, Arnaud Chiolero MD PhD commented:

A fantastic and useful book to understand what is health risk. Highly recommended for all health scientists and especially epidemiologists lost in their odds ratios

On 2015 May 12, Gareth Pryce commented:

There would appear to be a methodological flaw in this study which makes it's extrapolation to the human situation tenuous. Taken from this article http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1397713 (Food & Drink intake of mouse)the food/fluid intake of a mouse (C57Bl/6 strain used in this study)is 4.4g/day and fluid = 6ml of fluid a day, this study used 1% salt in water = 1g in 100ml and 4% in food = 4g in 100g of food. I have calculated that on average the mice would ingest (before of course they got sick, when they wouldn't ingest any food or water) 236 mg/day for a 25g mouse = 236mg/25g = 9.44g/kg so 660g for a human ie over half a Kg of salt a day. The likelihood of any human ingesting this amount of salt is very unlikely.

Even doing a factor of ten 60g of salt which is 8 times a recommended limit of 8g/day. and twice the 35g/day found in some papers.

There was no dose response done in this paper, or repetition of the result reported. I am surprised that this was not picked up during the refereeing process.

These results need to be taken with a pinch of salt!

On 2015 Jan 05, Alberto Halabe Bucay commented:

As a supplement to this comment, I published this article: http://garj.org/garjpp/pdf/2013/October/Bucay.pdf

On 2017 Jul 13, Robin P Clarke commented:

This is an interesting finding but there is much danger of over-interpreting from it. Urine mercury has long been known to have little relationship to toxicity problems caused by mercury. The mercury causes harm not from being in the urine but from not being in the urine, due to instead getting bound up in the brain and other organs. There is abundant evidence elsewhere of the major harms being caused by mercury, and this study in no way counters that evidence.

On 2015 May 04, Peter Good commented:

I asked Dr. Cynober whether oral arginine, citrulline, or glutamine would be the best source of arginine for brain nitric oxide and creatine in ASD children. He replied it was controversial whether oral arginine or glutamine produces more citrulline in the intestines. Citrulline enters the brain, he said, but whether it generates creatine there, and the balance between production of nitric oxide (NO) and creatine from citrulline-derived arginine, is unclear.[personal communication 2015]

Their 2010 paper [Cynober L, 2010] presented much evidence critical to autistic disorders (ASD). Citrulline (CIT) is not normally present in protein; its usual sources are arginine (ARG) and glutamine in dietary proteins, which produce CIT in the intestines: “CIT is almost absent from natural foods, watermelon being a notable exception.” CIT bypasses the liver and forms ARG in the kidneys, which limits wasting of nitrogen as urea, and provides ARG to many other tissues, including the brain.

“[D]irect supplementation of CIT should be more useful than ARG supplementation, leaving the kidney to convert CIT into ARG, so avoiding heavy first-pass splanchnic extraction of the ARG and the possible harmful effects of an excessive ARG [therefore NO] supply. . . . CIT is able to sustain NO production through eNOS but not iNOS. . . . CIT could also be a safe way to deliver ARG to endothelial and immune cells, and can certainly prevent excessive uncontrolled nitric oxide production. . . . [A]ntioxidant properties, together with the ability to generate NO, make CIT an excellent candidate for the treatment of pathological situations characterized by oxidative stress and decreased arginine availability . . . .”

Because CIT stimulates protein synthesis when dietary proteins (i.e. ARG and glutamine) are low, it should be given in the postabsorptive state (3–5 hrs after meals) or fasted state (before breakfast). 10–15g/day of oral citrulline in healthy adults showed high bioavailability and no adverse effects. Because CIT is synthesized almost exclusively in the intestines, it may also be a useful biomarker of functional gut tissue.[Cynober L, 2010]

In their 2005 paper [Curis E, 2005] Cynober and colleagues discussed other aspects of citrulline metabolism: “Citrulline presents the common reactivity of the α-amino acid family. In particular, it can form peptide bonds; hence it can therefore be present in proteins. However, since there is no known codon in the genetic table for this amino acid, its presence in a protein must always result from a post-translational modification of the protein. . . .

“The main reason for this citrulline metabolism split between two organs [gut and kidney] is related to the efficacy of the capture of arginine by the liver. In fact, without metabolic adaptation, almost all the arginine coming from food supply would be withdrawn from the portal blood by the liver, leaving only very low amounts of available arginine for other organs. . . .

“[M]any cell types which are able to metabolize arginine into NO are able to uptake circulating citrulline, which explains why citrulline induces certain of the NO effects . . . . The figure seems to be even more complex in the brain, since the recycling of citrulline into arginine is split between various cell types, defining a unique inter-cell-type cycle. Indeed, the brain neurones producing NO are not able to reconvert citrulline into arginine since they do not express the [necessary] enzymes. Hence, citrulline is released from the neurons and taken up by surrounding neural cells where return-conversion to arginine is performed.”[Curis E, 2005]

Romero and colleagues presented other valuable information about citrulline [Romero MJ, 2006]: “With development, intestinal synthesis of L-arginine from glutamine decreases and the small intestine gradually becomes the major site of net L-citrulline production. . . . L-citrulline is largely taken up and metabolized by the kidney, which in turn releases arginine equivalent to ~75% of the L-citrulline taken up. Thus, much of the L-citrulline produced by enterocytes reaches the systemic circulation as L-arginine. This L-arginine/L-citrulline homeostasis allows a proper supply of L-arginine for the whole body. About 60% of dietary L-arginine makes it into the hepatic portal circulation, while the rest is metabolized in the intestine. . . . L-citrulline synthesis in many tissues also occurs as a byproduct of NOS activity. . . . Although NOS is widely distributed throughout the body, its activity does not contribute substantially to whole body L-citrulline flux under normal conditions. . . .

“[A]cute oral administration of L-citrulline appears to be considerably more efficient raising plasma levels of L-arginine than L-arginine itself. Additionally, a recent study in children and young adults showed that five oral doses of L-citrulline every 12 hours (1.9 g/m2/dose) for a total dose of 9.5 g/m2 resulted in 57 and 85% increases in mean plasma levels of L-arginine and L-citrulline, respectively. . . . L-citrulline is generally recognized as safe for oral consumption. In fact, L-citrulline can prevent some of the untoward effects of L-arginine supplementation. . . . L-citrulline is a natural and apparently safe means of providing L-arginine for constitutive NOS production of NO.”[my emphasis] [Romero MJ, 2006]

Deutz also presented important observations about plasma concentrations and interorgan transport of glutamine, citrulline, and arginine [Deutz NE, 2008]: “In daily practice, the plasma concentration is usually viewed as a parameter of production. This is not always correct as the plasma concentration can be high due to an increased production of the substrate and/or a reduced capacity of the body to dispose [of] this substrate. This means that the level of plasma concentration can be misleading and does not always give reliable information whether there is actually an intracellular deficiency of a certain substrate. . . .

“The quantitative main production site [of glutamine] in the body is muscle and the main consumption sites are the gut, liver and the kidney. Liver plays a dual role as it can both produce and consume glutamine, depending on the metabolic state (fasting/fed). . . . [A]bout 80–90% of the citrulline is derived from the gut glutamine to citrulline conversion. Therefore, whole body citrulline production is related to the quantity of gut glutamine conversion to citrulline, and is most likely influenced by the amount of active gut tissue.”

Most important conclusions for ASD: (1) oral citrulline bypasses the liver and becomes arginine in the kidneys, making arginine more available to other tissues, including the brain; (2) oral citrulline supports production of constitutive nitric oxide but not inducible nitric oxide; (3) in light of the benefit of casein-free/gluten-free diets in ASD children, citrulline’s stimulation of protein synthesis when dietary protein is low may be invaluable; (4) citrulline synthesis may be a useful marker of functional gut tissue in these children; (5) oral citrulline is safer than oral arginine. The evidence speaks for itself.

Peter Good Autism Studies www.autismstudies.net autismstudies1@gmail.com

Curis E, Nicolis I, Moinard C, et al. Almost all about citrulline in mammals. Amino Acids 2005;29:177–205.

Cynober L, Moinard C, De Bandt J-P. The 2009 ESPEN Sir David Cuthbertson. Citrulline: A new major signaling molecule or just another player in the pharmaconutrition game? Clin Nutrit 2010;29:545–551.

Deutz NEP. The 2007 ESPEN Sir David Cuthbertson Lecture: Amino acids between and within organs. The glutamate-glutamine-citrulline-arginine pathway. Clin Nutrit 2008;27:321–327.

Romero MJ, Platt DH, Caldwell RB, Caldwell RW. Therapeutic use of citrulline in cardiovascular disease. Cardiovasc Drug Rev 2006;24:275–290.

On 2015 Apr 30, Peter Good commented:

None

On 2015 Apr 06, Peter Good commented:

To PubMed Commons: comment to Frye RE, 2013.<br> Frye et al. [2010] previously reported that sapropterin, a synthetic form of tetrahydrobiopterin (BH4), improved behavior in children with autism spectrum disorders (ASD) [Frye RE, 2010]. Their 2013 study [Frye RE, 2013] was intended to test whether sapropterin’s benefit was due to BH4’s role as cofactor for synthesis of the monoamine neurotransmitters dopamine and serotonin (as previous investigators suspected), or BH4’s role as cofactor for nitric oxide synthase (NOS), which produces the critical gaseous molecule nitric oxide (NO). Frye et al. concluded that improvements in communicative language in these children from sapropterin were due to restoration of NOS “coupling” disrupted by lack of BH4, which dysregulated nitric oxide metabolism. In support of their conclusion they cited evidence by Sweeten et al. [Sweeten TL, 2004] and others of high levels of nitric oxide metabolites nitrite and nitrate in blood of ASD children. In their previous study Frye et al. concluded: “[I]t is possible that BH4 in ASD could be depleted by the overactivation of the immune system and inflammatory processes during an excessive production of nitric oxide.” [Frye RE, 2010]

There may, however, be more to this story. A few months after publication of Frye et al. 2013, Stanhewicz et al. reported sapropterin increased reflex vasodilation in aging human skin by increasing release of nitric oxide by endothelial and neuronal nitric oxide synthases [Stanhewicz AE, 2013]. Nitric oxide, the primary dilator of blood vessels in the body, is produced by three different forms of nitric oxide synthase – two constitutive forms present in blood vessel endothelial cells (eNOS) and neurons (nNOS), and a third form (iNOS) induced in brain microglia and other cells of the immune system in response to infections and other agencies. Endothelial nitric oxide maintains the vasodilator tone of blood vessels. Neuronal nitric oxide may be largely responsible for neurovascular coupling – dilation of nearby blood vessels when brain neurons fire. Faraci & Brian: “. . . NO appears to mediate cerebral vasodilatation in response to local neuronal activation.” [Faraci FM, 1994]. Koehler et al.: “. . . NO is required as a mediator of neurovascular coupling in the cerebellum, whereas NO acts as a modulator in the cerebral cortex.” [Koehler RC, 2009]. Inducible nitric oxide is released in large quantities to flush infective agents and toxins, and kill damaged cells.

If nitric oxide is too high in autistic disorders, inducible nitric oxide is the form likely responsible, Frye et al. concluded. Sweeten et al. concluded likewise: “[I]t is reasonable to hypothesize that iNOS is involved in the elevated NO production in autism.” [Sweeten TL, 2004]. Yet inducible nitric oxide is often released to compensate deficiencies of constitutive nitric oxide [Hecker M, 1999;Kubes P, 2000]. One indication neuronal nitric oxide is deficient in children with autistic disorders is their failure of neurovascular coupling – their brains are often hyperexcitable, yet brain blood flow is consistently low [e.g. Ohnishi T, 2000]. Nitrite and nitrate also serve as reservoir forms to deliver nitric oxide elsewhere [Dejam A, 2005]. Lundberg & Weitzberg: “[N]itrate and nitrite should probably be viewed as storage pools for NO rather than inert waste products.” [Lundberg JO, 2005].

Did sapropterin increase endothelial and neuronal nitric oxide in the brains of ASD children in Frye et al. 2013? Why would endothelial and neuronal nitric oxide be deficient in these children? One explanation is deficiency of BH4. Another is deficiency of the amino acid arginine – only substrate for nitric oxide [Wiesinger H, 2001]. Frye et al. found higher baseline levels of blood arginine in these children, and higher ratios of arginine to citrulline, were associated with greater improvements in language from sapropterin. They noted blood arginine and the arginine/citrulline ratio did not change significantly during sapropterin treatment – but also stated improvements in language were greater in children with “an attenuated increase in arginine.” [Frye RE, 2013]

Considerable evidence argues that arginine is deficient in ASD children: (a) high levels of inducible nitric oxide; (b) consistently low brain creatine (arginine + glycine) [Friedman SD, 2003]; (c) frequent high blood ammonia [Filipek PA, 2004] which requires arginine to detoxify to urea; and (d) high levels of arginine vasopressin in autistic boys [Carter CS, 2007; Momeni N, 2005]. Furthermore, NOS produces harmful oxidants superoxide and peroxynitrite when NOS “uncouples” from lack of BH4 – or when arginine is deficient [Xia Y, 1996]. Because most supplemental arginine is taken up by the liver (thus unavailable to other tissues), citrulline (arginine’s precursor) or glutamine (citrulline’s precursor) may be better sources of arginine for NOS [Cynober L, 2010]. The evidence speaks for itself.

Peter Good Autism Studies La Pine, OR www.autismstudies.net autismstudies1@gmail.com

Carter CS. Sex differences in oxytocin and vasopressin: implications for autism spectrum disorders? Behav Brain Res 2007;176:170–186.

Cynober L, Moinard C, De Bandt J. The 2009 ESPEN Sir David Cuthbertson. Citrulline: A new major signaling molecule or just another player in the pharmaconutrition game? Clinical Nutrition 2010;29:545–551.

Dejam A, Hunter CJ, Pelletier MM, et al. Erythrocytes are the major intravascular storage sites of nitrite in human blood. Blood 2005;106:734–739.

Faraci FM, Brian Jr. JE. Nitric oxide and the cerebral circulation. Stroke 1994;25:692–703.

Filipek PA, Juranek J, Nguyen MT, et al. Relative carnitine deficiency in autism. J Autism Dev Disord 2004;34:615–623.

Friedman SD, Shaw DW, Artru AA, et al. Regional brain chemical alterations in young children with autism spectrum disorder. Neurology 2003;60:100–107.

Frye RE, Huffman LC, Elliott GR. Tetrahydrobiopterin as a novel therapeutic intervention for autism. Neurotherapeutics. 2010;7(3):241–249.

Hecker M, Cattaruzza M, Wagner AH. Regulation of inducible nitric oxide synthase gene expression in vascular smooth muscle cells. Gen Pharmacol 1999;32:9–16.

Koehler RC, Roman RJ, Harder DR. Astrocytes and the regulation of cerebral blood flow. TINS 2009;32(3):160–169.

Kubes P. Inducible nitric oxide synthase – a little bit of good in all of us. Glia 2000;47:6–9.

Lundberg JO, Weitzberg E. NO generation from nitrite and its role in vascular control. Arterioscler Thromb Vasc Biol 2005;25:915–922.

Momeni N, Nordström BM, Horstmann V, et al. Alterations of prolyl endopeptidase activity in the plasma of children with autistic spectrum disorders. BMC Psychiatry 2005;5:27–32.

Ohnishi T, Matsuda H, Hashimoto T, et al. Abnormal regional cerebral blood flow in childhood autism. Brain 2000;123(Pt. 9):1838–1844.

Stanhewicz AE, Alexander LM, Kenney WL. Oral sapropterin acutely augments reflex vasodilation in aged human skin through nitric oxide-dependent mechanisms. J Appl Physiol 2013:115:972–978.

Sweeten TL, Posey DJ, Shankar S, McDougle CJ. High nitric oxide production in autistic disorder: a possible role for interferon-gamma. Biol Psychiatry 2004;55(4):434–437.

Wiesinger H. Arginine metabolism and the synthesis of nitric oxide in the nervous system. Prog Neurobiol 2001;64(4):365–391.

Xia Y, Dawson VL, Dawson TM, et al. Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Proc Natl Acad Sci USA 1996;93:6770–6774.

On 2013 Oct 24, Andrew M Leifer commented:

Full text pre-print is freely available at http://arxiv.org/abs/1303.2876

On 2015 Oct 06, Jan Egger commented:

A videos demonstrating the semi-automatic GBM segmentation can be found here: https://www.youtube.com/watch?v=z0f9RFRnC3g

Best wishes, Jan

On 2014 Mar 08, Iffat Sumia commented:

Love the paper ! I wonder, though, why the β-catenin-Venus fusion protein used has two point mutations in its Armadillo domain. I checked the von Kries et al., 2000 paper that's cited and it didn't help much. Is the reason for these mutations something trivial or purely technical that I missed?

On 2014 Jul 29, David Keller commented:

B vitamins for levodopa-associated peripheral neuropathy

The problem: "Cases of symptomatic peripheral neuropathy [PN], sometimes severe, have been reported in patients receiving LCIG [levodopa/carbidopa intestinal gel]. Cases are generally a sensorimotor polyneuropathy with both subacute and chronic onsets, often associated with vitamin B12 and/or B6 deficiency."

Observation: "Cases of LCIG-associated PN often have responded to vitamin supplementation"

Advice: "It may be advisable to monitor vitamin B12/B6 status before and after patients start LCIG and be vigilant for signs of PN. Prospective, large-scale, long-term studies are needed."

Administration of levodopa is associated with elevation of blood levels of homocysteine, a neurotoxic waste product which consumes vitamins B6, B12 and folic acid during its metabolism (1). Administration of these B-complex vitamins has been demonstrated to lower homocysteine levels, with postulated favorable effects on neurological degeneration (2). However, while I agree with the recommendation to monitor levels of B12 and B6 during levodopa therapy, I would add the suggestion that homocysteine levels be monitored directly as well, since homocysteine is the actual neurotoxin. Preventative addition of a vitamin B-complex supplement if taking levodopa is also reasonable, despite the lack of evidence from a clinical trial yet. By the time homocysteine-mediated peripheral neuropathy is symptomatic, it may be difficult or impossible to reverse.

References

1: Obeid R, Herrmann W. Mechanisms of homocysteine neurotoxicity in neurodegenerative diseases with special reference to dementia. FEBS Lett. 2006 May 29;580(13):2994-3005. Epub 2006 May 6. Review. PubMed PMID: 16697371.

2: Cacciapuoti F. Lowering homocysteine levels with folic acid and B-vitamins do not reduce early atherosclerosis, but could interfere with cognitive decline and Alzheimer's disease. J Thromb Thrombolysis. 2013 Oct;36(3):258-62. doi: 10.1007/s11239-012-0856-x. Review. PubMed PMID: 23224755.

On 2014 Mar 21, Jacob Puliyel commented:

Williams and colleagues have described assessment of AEFI employing the algorithm described by Halsey < PMID: 22507656>.

I have posted two very detailed comments to an article by Tozzi Tozzi AE, 2013 which discusses the same subject of the revised WHO Classification of AEFI. I will not repeat the points I have made there but it may be viewed here.

As this is a matter of patient safety I think it is important that the experts who understand the new scheme must explain why the revision was needed and that it will not miss opportunities of picking up new signals. The question is whether the new scheme would have picked up and flagged the signal of adverse-effects like the RotaShield-reactions, had the scheme been in use in 1999. The purpose of this posting is to invite the learned authors of this article on causality assessment to respond to the issues raised in the postings to the Tozzi article and I propose to flesh out those concerns a little further in the context of the article in Pediatrics by Williams and colleagues.

1) Williams and colleagues Williams SE, 2013 suggest that the first step in the general approach to evaluating serious AEFI is to establish a clear diagnosis using Brighton Collaboration case definitions.

The second step is to consider known biological mechanisms.

Neither of these would have been evident when the intussusceptions signal was picked up by the old scheme (and the vaccine was withdrawn expeditiously preventing unnecessary distress to thousands of babies). Even today although a case definition has been developed for ‘intussusceptions’, the biological mechanism is not clearly defined and so the second step described by Williams et al cannot be completed.

It was reported recently that Pentavalent vaccine (DPT co-administered with measles vaccine (MV) and yellow fever (YF) vaccine) is associated with increased mortality compared to MV + YF alone Fisker AB, 2014. It is pertinent to mention that the biological mechanisms involved are not understood.

Neither is the biological mechanism for increased female mortality in recipients of the high-titer Edmonston-Zagreb vaccine known, although this was first noticed 2 decades ago. < PMID: 8237989>, Aaby P, 1993.

2) It will be instructive to look at how the new algorithm has failed to flag up the deaths following Pentavalent vaccine used in Asia (DPT + Hib + Hepatitis B) and as a result, numerous children continue to be exposed to the risks of this vaccine.

The glossary of the User Manual for the [Revised WHO classification]( who.int/vaccinesafety/publications/aevimanual.pdf) suggests ways and means to rule out a causal association. It defines causal association as a cause-and-effect relationship between the causative factor and a disease with no other factor intervening in the process.

There have been many deaths following use of this Pentavalent vaccine in Sri Lanka. The committee WHO vaccine safety examined 19 deaths in Sri Lanka, 14 of them between 2010 and 2012. In six of the 19, a congenital heart disease was reported.

Does preexisting congenital heart disease rule out a causal association between the vaccine and the deaths? Under this definition the 6 deaths in children with heart disease were not causally related to the vaccination.

The older Advisory Committee on Causality Assessment Collet JP, 2000 looked at the problem more logically and holistically. For example it noted that elderly persons with concomitant or preceding chronic cardiac failure can develop cardiac decompensation after influenza vaccination due to a vaccine-caused elevation in temperature or from stress from a local reaction at the site of vaccinating. The vaccine is considered to have contributed to cardiac failure in this specific situation. It is obvious that with the older method of assessment of AEFI, caution would have been exercised when administering influenza vaccine to persons with preceding chronic cardiac failure, to avoid decompensation.

The deaths in children with heart disease following administration of Pentavalent vaccine could well be due to decompensation. The Pentavalent vaccine must be used with caution in the presence of an underlying heart condition albeit asymptomatic. However detection of asymptomatic heart disease prior to vaccination in developing countries is impractical where the vaccine is administered by health workers who are barely literate. Is it prudent to use the vaccine under these circumstances given the findings of the Sri Lanka investigation? The new system disregards this real danger.

3) Step 2 Checklist 4 of the revised [WHO classification for causality assessment]( who.int/vaccinesafety/publications/aevimanual.pdf) asks to check if the event can occur independently of vaccination (background rate). Thus it seems that until the deaths from vaccine AEFI are frequent enough as to increase the age specific mortality-rate in a statistically significant manner, they are to be ignored.

The question of what background rate to use is not addressed specifically and this can further confound objective assessment of the AEFI. The Pentavalent vaccine in Asia is administered after 6 weeks of age. Would the local post-neonatal infant mortality rate (PN IMR) in the community before introduction of the vaccine be the comparator?

Most of this post-neonatal IMR is made of babies who are very sick with pneumonia, diarrhea, sepsis, meningitis etc. The fact that the AEFI babies were brought by the mother for routine immunization suggests that the child was not sick and the mother did not consider the child was likely to die in the next day or two. The comparator must really be the SIDS rate in the locality for babies of a comparable age.

Deaths in Bhutan were investigated and local newspapers reported on the various official explanations. It was argued that the deaths could have been due to encephalitis although there was little evidence for it. Officials explained that the encephalitis death rate in the years after the vaccine was introduced (even after adding AEFI deaths) had not increase significantly. This was sufficient grounds to accept the ‘coincidental encephalitis’ theory. One cheeky health official however pointed out that there were no cases of meningo-encephalitis reported among children below one year, in the eight months when Pentavalent vaccine was suspended in Bhutan.

4) Another factor related to the deaths following Pentavalent vaccine is that the vast majority have occurred after the first dose and fewer after the second dose. A random event or coincidental SIDS cannot explain these deaths. However the new algorithm does not take this important factor into consideration.

For all these reasons it would appear that the new algorithm is not a comprehensive means to assess serious adverse events. Its use will delay withdrawal of vaccines that result in serious AEFI and in the end it will erode confidence in the entire immunization programme and those who administer it.

Can I suggest that we need to go back use older scheme namely Brighton Classification of AEFI till we find a better method to assess AEFI.

On 2017 Dec 01, Su-Fang Lin commented:

Dear Authors:

There are some discrepancies between Figure S7 legend (p9) and Table S8, could you please clarify those confusion for us? BTW, your work has paved the way for HNC stratification, thank you ALL very much! :-)

1. SCC15 (predicted basal in Fig S7, noted as MS in Table S8)
2. KYSE140 (predicted MS in Fig S7, noted as CL in Table S8)
3. FADU (predicated MS in Fig S7, noted as AT in Table S8)

On 2013 Oct 23, Stephen Turner commented:

This review lays out some of the challenges and recent advances in viral metagenomic sequencing. There is a good discussion of library preparation and how that affects downstream sequencing. Alarmingly, they reference another paper that showed that different amplification methods resulted in detection of a completely different set of viruses (dsDNA viruses with LASL, ssDNA with MDA). The review also discusses many of the data management, analysis, and bioinformatics challenges associated with viral metagenomics.

On 2014 Jan 14, Hilmar Lapp commented:

While I agree with the advantages the author advocates for Git, any distributed version control system (DVCS) can in principle have the same. There are several other mature and widely used DVCSs, in particular Mercurial (Hg) and Bazaar (Bzr), with similarly sophisticated repository hosting platforms and web interfaces available (Bitbucket and Launchpad, respectively). Thus, while Git does have great potential for facilitating reproducibility and transparency in science as the author argues, it is DVCSs in general that do, in contrast to client-server oriented VCSs, and Git is just one example of those, even if a particularly good one.

This is indeed mentioned in the text, albeit only briefly, and is thus easily missed. In particular, I wish the title had a reference to distributed version control, rather than only to a single product that implements DVCS.

On 2014 Mar 18, Willem Schaik commented:

There is a typo in the abstract of our paper. The name of the gene which was deleted is ptsD (not pstD) to indicate that it encodes the IID subunit of the PTS. I apologize for this error.

On 2017 Mar 23, University of Kansas School of Nursing Journal Club commented:

Team Members: Cassie Caedo, Jessica Garcia, Jamie Radakovich, Olivia Kolar, Aaron Rutledge, Courtney Will, Sarah Stokes, Macy McKee. [Class of 2017)

Background

Shared governance is an increasingly prevalent leadership strategy in which nurses are an integral component of decision-making, both on the unit and institutional level. Shared governance gives nurses the power to influence their practice, which can positively affect both nurses’ job satisfaction and patient satisfaction. As we have learned in class, shared governance systems should integrate nursing professionals’ experiences and attitudes to shape nursing practice and improve nurses’ job satisfaction. Shared governance can also be viewed as a source of empowerment for nursing professionals. This literature helps fill the gap in perspectives that were not presented in class. In the presentations from staff nurses who participate in shared governance, they expressed the perspective of staff nurses’ participation in unit based councils and institutional councils. However, this article integrates viewpoints from both the staff nurses and nurse managers who participate in shared governance models. It was explained that shared governance models deviate from classic hierarchal structures of governance because nurses at the bedside can influence clinical practice across the entire institution. The success of shared governance can depend on the personal accountability of the nursing staff involved, and requires the institution to be receptive to the suggestions and ideas brought forth by nursing staff. By utilizing the perspectives of nurses who are directly impacted by institutional and unit policies, shared governance gives nursing staff more control over their practice.

Methods

This particular article was found on PubMed using the search terms “shared governance,” “nursing,” and “satisfaction.” We decided to select this particular article based on the insight that this qualitative study provided. In class, we explore the ways that shared governance can affect nursing staff and inadvertently affect patient outcomes. Nurse burnout, satisfaction, and retention can all be influenced by nursing leadership. The design of this study was a qualitative analysis via interview questions answered by 11 RNs. The study was designed for the purposes of assessing and documenting the attitudes and experiences of both staff RNs and nurse managers in shared governance structures. Specifically, nurses and nurse managers were asked for their opinion of the effect shared governance has on patient outcomes, their experiences with implementing a shared governance model, and describing their perceived roles in a shared governance system. The population of interest focused on staff nurses and nurse managers who implement shared governance models; in this study, this was implemented by utilizing unit practice councils. Evaluating attitudes about shared governance is important in assessing the efficacy of shared governance models and whether or not they have a positive impact on staff nurses’ experiences. Each verbal interview ranged from one minute to a minute and a half, and was recorded for data analysis later. Most nurses were interviewed in pairs by the unit practice councils.

Findings

The nurse managers cited shared decision-making and staff empowerment as integral components of succeeding in shared governance models. Nurses collaborated with clinical staff on the floor, including physicians and nursing administration, and nurse managers acted as facilitators between staff nurses and other professionals within the institution. Nurses who utilized shared governance models also integrated more evidence-based practice and quality improvement to improve processes on the unit. Empowerment by nurse managers fostered better job satisfaction and accountability for the staff nurses. Shared governance was viewed as a way to foster better communication between members of the care team, and encouraged a team-oriented mentality. Overall, shared governance was seen as an acceptable and effective avenue for improving patient outcomes, increasing nurses’ job satisfaction, and building stronger interprofessional relationships. Limitations of the study included small sample size (n=11), including five nurse managers and six staff RNs. Unit councils, as well as the individual participants, varied in shared governance experience but were all involved on their units’ practice councils. The generalizability of the study is limited because of the small sample size, the universal participation in practice councils, and the limited experiences with shared governance.

Nursing Implications

Implementing shared governance is important to nursing professionals because nurse burnout and job satisfaction can be affected based on the amount of influence nurses are able to have on their own practice. This is important on a personal level because nurses should be able to influence the policies and procedures that impact our daily practice. Not only would shared governance improve nursing professionals’ visibility, but it would also promote workplace satisfaction. The health care system stands to benefit from shared governance models because nurses are such a profound aspect of patient care, and their direct participation in how their unit provides care could lead to better patient outcomes and increased patient satisfaction. Nurses have a unique perspective that can identify more efficient procedures, more cost-effective utilization of resources, and promotion of a healing environment. This literature relates to future nursing practice because shared governance offers the promise of improved patient outcomes, higher rates of job satisfaction among nurses, and a method for collaborative improvement of entire institutions.

References

Ott, J., & Ross, C. (2014). The journey toward shared governance: the lived experience of nurse managers and staff nurses. Journal of nursing management, 22(6), 761-768.

On 2014 Aug 19, Qin-hong Zhang commented:

Comment on "Interventions for treating persistent and intractable hiccups in adults"

Zhang QH, Yue JH

Department of Acupuncture and Moxibustion, College of Acupuncture and Moxibustion, Heilongjiang University of Chinese Medicine, Harbin 150040, China.

I read with great interest the published article by Moretto et al., entitled “Interventions for treating persistent and intractable hiccups in adults”[1]. They evaluated the effectiveness of pharmacological and non-pharmacological interventions for treating persistent and intractable hiccups of any aetiology in adults. However, I had a few comments to make regarding the same. The authors only included four Chinese trials in acupuncture. However, they failed to search the Chinese databases, such as Chinese Biomedical Literature Database (CBM), China National Knowledge Infrastructure (which includes the database China Academic Journals) (CNKI), VIP Information (VIP), and Wanfang Data (WAN FANG). Thus, to our best knowledge, the authors far failed to include at least 18 eligible studies (including four randomized controlled trials (RCTs), and 14 quasi-randomized controlled trials (Q-RCTs)). In addition, three of 4 included studies used wrong comparator that means both groups used acupuncture, which could not evaluate the effect of acupuncture [2-4].

References

1.Moretto EN, Wee B, Wiffen PJ, Murchison AG. Interventions for treating persistent and Intractable hiccups in adults. Cochrane Database Syst Rev 2013; 1:CD008768.

2.Bao F, Liang Z, Wang F. Clinical observation on treatment of stubborn hiccup by acupuncture with different needle retaining time. World Journal of Acupuncture-Moxibustion 2003;13(2):50–4.

3.Jiang Feizhou.Stubborn hiccup treated with thumb-tack needles and needle-embedding on otopoint. Journal of Clinical Acupuncture and Moxibustion 2002;18(2):36.

4.Wang B. 80 cases of intractable hiccup treated by acupuncture. Chinese Acupuncture and Moxibustion 2011; 31(2):181–2.

On 2017 Nov 29, Liz Dooley commented:

This Cochrane Review has been superseded. A new author team has taken over and will expand the scope of the review to cover acute upper respiratory tract infections. Information provided by the Cochrane Acute Respiratory Infections Group.

On 2014 Mar 02, Arnaud Chiolero MD PhD commented:

A very good review on a difficult topic

On 2015 May 11, Ivan Oransky commented:

This paper has become involved in three different lawsuits: http://retractionwatch.com/2015/05/08/crossfit-gym-owner-sues-ohio-state-says-fraudulent-data-led-to-273-million-in-nih-grants/

On 2015 Jun 02, thomas samaras commented:

Additional information on height, CHD, and longevity is available from these recent publications.

Samaras TT. Evidence from eight different types of studies showing that smaller body size is related to greater longevity. Journal of Scientific Research & Reports. 2014: 3 (16): 2150-2160, 2014; article no. JSRR.2014.16.003.

Samaras TT. Human Scaling and Body Mass Index. In: Samaras TT (ed): Human Body Size and the Laws of Scaling: Physiological Performance, Growth, Longevity and Ecological Ramifications. New York: Nova Science Pub; 2007: pp 17-32.

He Q, Morris BJ, Grove JS, Petrovitch H, Ross W, Masaki KH, et al. Shorter men live longer: Association of height with longevity and FOXO3 genotype in American men of Japanese ancestry. Plos ONE 9(5): e94385. doi:10.1371/journal.pone.0094385.

Salaris L, Poulain M, Samaras TT. Height and survival at older ages among men born in an inland village in Sardinia (Italy), 1866-2006. Biodemography and Social Biology, 58:1, 1-13.

Bartke A. Healthy Aging: Is Smaller better? A mini-review. Gerontology 2012; 58:337-43.

On 2015 Jun 02, thomas samaras commented:

None

On 2017 Jul 25, Marek Michalak commented:

The full response to Alexander Kraev's comment can be viewed at the following URL: http://journals.plos.org/plosone/article/comments?id=10.1371/journal.pone.0056387

On 2017 Jul 20, Alexander Kraev commented:

Note that this is likely one of about 150 publications affected by the MHC-driven MerCreMer transgene, notoriously prone to induction of dilated cardiomyopathy in the absence of any induced target gene expression, in this case calreticulin. See PMID:27165291 for details. Erratum for this article in PLoS One. 2013;8(11) was made for a different reason.

On 2014 Dec 05, Andrea Messori commented:

Treatments for naive patients with follicular non-Hodgkin lymphoma

The study by Rummel et al. [1] is a potentially important advancement in the treatment of naïve patients with indolent non-Hodgkin’s lymphoma. Since, in this trial, the clinical results with bendamustine plus rituximb were very favourable in the subgroup of patients with follicular lymphoma, it can be worthwhile to compare these findings with those observed in other randomized trials aimed at naïve patients with this disease condition. According to the systematic review by Schulz and co-workers [2], two randomized trials [3,4] have previously been conducted in treatment-naive patients with follicular lymphoma. Another trial (the PRIMA trial[5]) has been published thereafter.

If one employs the end-point of progression-free survival at 2 years, these randomized trials have reported the following results (n/N): a) Hiddeman et al. [3]: R-CHOP, 167/223 (74.9%) vs CHOP, 103/205 (50.2%); b) Marcus et al. [4]: R-CVP, 137/159 (86.2%) vs CVP, 81/162 (50%); c) Rummel et al [1] : bendamustine+ rituximab, 93/139 (66.9%) vs R-CHOP, 63/140 (45%); d) PRIMA trial [5] (subgroup of 1,019 patients who completed the induction treatment): i) First data set (unadjusted)= survival data determined from randomization until last date of the follow-up: induction+rituximab maintenance, 418/505 (82.8%) vs induction+no rituximab maintenance, 345/513 (67.3%); ii) Second data set with empirical adjustment (see FOOTNOTE) = survival data determined from registration until last date of the follow-up: induction+rituximab maintenance, 418/529 (79%) vs induction+no rituximab maintenance, 345/537 (64.2%). With the exception of the first data set of the PRIMA trial, these results selectively refer to treatment-naïve patients with follicular lymphoma. The numerators and denominators of the crude rates reported above were estimated from the Kaplan-Meier curves according to graphical methods [6,7]. In each trial, the following sources of information were used: Hiddeman et al. [3]: Figure 2, Panel A; Marcus et al. [4]: Figure 1; Rummel et al. [1]: Figure 3, Panel A; PRIMA trial [5]: Figure 2, Panel A.

If one analyses the above pair-wise comparisons according to the end-point of achievement of progression-free survival at 2 years, the following values of odds-ratio (OR) can be obtained (random-effect model, Open Meta-analysts software): R-CHOP vs CHOP: 2.953 (95%CI: 1.964 to 4.442); R-CVP vs CVP, OR=6.227 (95%CI: 3.609 to 10.744); bendamustine+rituximab vs R-CHOP, OR=2.47 (95%CI: 1.521 to 4.015); R-CHOP+R-maintanance vs R-CHOP (adjusted data set): 2.303 (95%CI: 1.741 to 3.047).

Interestingly enough, all of these comparisons show a significantly better effectiveness of the first treatment as compared to the second. In this framework, a Bayesian meta-analysis aimed at assessing these data could be useful to synthesise the comparative effectiveness of these treatments.

FOOTNOTE: In the PRIMA trial [5], after a total of 1,202 treatment-naive patients were enrolled and registered, randomization to either rituximab maintenance or no maintenance was applied to the subgroup of 1,019 patients who completed the induction treatment. To make the PFS data of this trial (that refer to the randomised patients) comparable to those of the other trials reported above (that refer to treatment-naïve patients), the rates of progression (or, likewise, the rates of PFS) in the PRIMA trial need to be subjected to an empirical adjustment (increase in the numerator of the progression rate or increase in the denominator of the PFS rate applied to both the maintenance and the no-maintenance arms). This adjustment can be based on further information reported on the same patients in a more recent analysis [8].

In their Figures 1 and 2, Ghesquières and co-workers [8] have provided useful details to compare the PFS data between the subgroup of registered patients (data shown in Figure 1, panels A and B) and the subgroups of randomised patients (data shown in Figure 2, panels AI, AII, BI, and BII). For this purpose, the two following comparisons can be made: i) Comparison A between Panel A of Figure 1 vs. Panels AI and AII of Figure 2; ii) Comparison B between Panel B of Figure 1 vs. Panels BI and BII of Figure 2. Comparison A uses the data of FCGR3A polymorphism, while Comparison B uses the data of FCGR2A polymorphism.

In Comparison A, the crude rate of progression for registered patients (according to Figure 1, panel A) is 171/460 (37.17%) where 171=68+80+23 and 460=177+215+68 while the crude rate of progression for randomised patients (according to Figure 2, panels AI and AII) is 141/397 (35.52%) where 141= 35+45+11+17+26+7 and 397=76+93+27+ 72+98+31. The ratio of these two percent rates of progression (37.17% / 35.52%) is 1.0465; this indicates that, using an acceptable approximation, the rate of progression in registered patients can reasonably be estimated from the rate in randomized patients by applying a 4.65% increase in the number of events found in randomised patients (i.e. by increasing the numerator of the rate without changing the denominator). Likewise, in Comparison B, the crude rate of progression for registered patients (according to Figure 1, panel B) is 168/450 (37.33%) while the same rate for randomised patients (according to Figure 2, panels BI and BII) is 138/387 (35.66%). The ratio of these two percent rates of progression (37.33% / 35.66%) is 1.0468. which is very similar to that estimated from Comparison A.

These two re-analyses (i.e. Comparisons A and B) consistently indicate that the rates of progression in registered patients can be estimated with a very good approximation from the rates found in randomized patients by applying a 4.7% adjustment to the survival data (i.e. an increase in the numerator of the progression rate or an increase in the denominator of the PFS rate).

References

1. Rummel et al. Bendamustine plus rituximab versus CHOP plus rituximab as first-line treatment for patients with indolent and mantle-cell lymphomas: an open-label, multicentre, randomised, phase 3 non-inferiority trial. Lancet. 2013 Apr 6;381(9873):1203-10.

2. Schulz et al. Chemotherapy plus Rituximab versus chemotherapy alone for B-cell non-Hodgkin's lymphoma. Cochrane Database Syst Rev. 2007 Oct 17;(4):CD003805.

3. Hiddemann 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.

4. Marcus et al. CVP chemotherapy plus rituximab compared with CVP as first-line treatment for advanced follicular lymphoma. Blood 2005;105(4):1417–1423.

5. Salles et al. Rituximab maintenance for 2 years in patients with high tumour burden follicular lymphoma responding to rituximab plus chemotherapy (PRIMA): a phase 3, randomised controlled trial. Lancet. 2011 Jan 1;377(9759):42-51. doi: 10.1016/S0140-6736(10)62175-7.

6. Messori et aL. Survival meta-analysis of individual patient data and survival meta-analysis of published (aggregate) data. Clin Drug Invest 2000;20(5):309-316.

7. Tierney et al. Practical methods for incorporating summary time-to-event data into meta-analysis. Trials. 2007 Jun 7;8:16.

8. Ghesquières et al. Clinical outcome of patients with follicular lymphoma receiving chemoimmunotherapy in the PRIMA study is not affected by FCGR3A and FCGR2A polymorphisms. Blood. 2012 Sep 27;120(13):2650-7.

On 2014 Oct 10, Fernando Castro-Chavez commented:

Dear Researcher,

I think that the PubMed comments are a good added feature for authors to comment and expand on their work, to answer and to get input from the readers, and to make personal comments related the human aspect of their research. For example, in my current work and in the upcoming ones, I aim at taking a computer programmer's perspective upon the genetic code. Here I was able to test its Defragging ability, and in a future article here to appear, I aim to test its File Compression ability.

To develop programming algorithms and languages based on the genetic code, for me seems like a thrilling exercise.

What do you think?

With my best regards,

Fernando Castro-Chavez, PhD.

10/10/2014, 5:34 PM.

Houston, TX.

On 2017 Jun 21, Misha Koksharov commented:

For those curious - an excellent response from Dan Graur: http://judgestarling.tumblr.com/post/67599627086/a-pre-refuted-hypothesis-on-the-subject-of-junk

On 2013 Nov 25, Claudiu Bandea commented:

Junk DNA and the skewed perspective of Graur et al. on biological function and genome size evolution

In their article “On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE” (1), Graur et al. dismantle ENCODE’s evidence and suggestion that 80% of the human genome is functional (2), which would render the traditional concept of junk DNA (jDNA) obsolete (3).

I agree with many assertions made by Graur et al. about the poor rationale behind some of ENCODE’s experimental approaches and the overall interpretation of the results; indeed, it has been known for decades that, by its bare presence in the genome, so called junk DNA (jDNA) gets replicated and undergoes repair, recombination, non-specific transcription, transposition, etc., and that all these biochemical activities involve various DNA-binding proteins.

However, the article by Graur et al. contains assumptions and statements that are questionable. For example, the authors limit their evaluation of genomic DNA’s biological functions to informational roles, which are based on sequence specificity. Although Graur et al. recognize, apparently as an afterthought, that: “It has been pointed to us that…some parts of the genome may be functional but not under constraint with respect to nucleotide composition”, the paper sidesteps the significance of non-informational roles for genomic DNA.

In a recent paper (4), I discussed this and some the other weaknesses of Graur et al. and expanded on an old hypothesis (5) that might explain the evolution of genome size and jDNA, and solve the long-standing C-value enigma.

References

(1) Graur D, Zheng Y, Price N, Azevedo RB, Zufall RA, Elhaik E. 2013. On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE. Genome Biol Evol., 5(3):578-90. Graur D, 2013

(2) ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. 2012. Nature, 489:57–74. ENCODE Project Consortium., 2012

(3) Pennisi E. 2012. ENCODE project writes eulogy for junk DNA. Science, 337:1159–1161. Pennisi E, 2012

(4) Bandea CI. 2013. On the concept of biological function, junk DNA and the gospels of ENCODE and Graur et al. bioRxiv doi: 10.1101/000588 (http://biorxiv.org/content/early/2013/11/18/000588)

(5) Bandea CI. 1990. A protective function for noncoding, or secondary DNA. Med Hypotheses, 31(1):33-4. Bandea CI, 1990

On 2013 Nov 25, Claudiu Bandea commented:

None

On 2016 Aug 23, Ben Goldacre commented:

This trial has the wrong trial registry ID associated with it on PubMed: both in the XML on PubMed, and in the originating journal article. The ID given is NCT0027753. We believe the correct ID, which we have found by hand searching, is NCT00277537.

This comment is being posted as part of the OpenTrials.net project^[1] , an open database threading together all publicly accessible documents and data on each trial, globally. In the course of creating the database, and matching documents and data sources about trials from different locations, we have identified various anomalies in datasets such as PubMed, and in published papers. Alongside documenting the prevalence of problems, we are also attempting to correct these errors and anomalies wherever possible, by feeding back to the originators. We have corrected this data in the OpenTrials.net database; we hope that this trial’s text and metadata can also be corrected at source, in PubMed and in the accompanying paper.

Many thanks,

Jessica Fleminger, Ben Goldacre*

[1] Goldacre, B., Gray, J., 2016. OpenTrials: towards a collaborative open database of all available information on all clinical trials. Trials 17. doi:10.1186/s13063-016-1290-8 PMID: 27056367

* Dr Ben Goldacre BA MA MSc MBBS MRCPsych<br> Senior Clinical Research Fellow<br> ben.goldacre@phc.ox.ac.uk<br> www.ebmDataLab.net<br> Centre for Evidence Based Medicine<br> Department of Primary Care Health Sciences<br> University of Oxford<br> Radcliffe Observatory Quarter<br> Woodstock Road<br> Oxford OX2 6GG

On 2014 Mar 14, Jonathan Eisen commented:

The first author of the paper, Rachel Adams wrote a post about this paper for the "microbiology of the Built Environment network (microBEnet)" blog. See Dispersal in microbes: fungi in indoor air are dominated by outdoor air and show dispersal limitation at short distances

On 2016 Dec 14, Ivan Oransky commented:

The lawsuit mentioned in the above comment was dismissed in September 2016: http://retractionwatch.com/2016/12/14/judge-tosses-case-saying-court-ordered-retractions-not-part-scientific-publication/

On 2015 Apr 20, Ivan Oransky commented:

This paper has become the focus of a lawsuit: http://retractionwatch.com/2015/04/20/former-postdoc-suing-lab-head-for-publishing-paper-he-wrote-without-his-name/

On 2013 Dec 23, Tom Kindlon commented:

ME/CFS Patient Organisations should be free to not promote CBT and GET for ME/CFS and the models underlying them

As somebody involved in running a myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patient organisation which might be seen to have been criticised by this paper, I would like to take the opportunity to respond.

The paper seems to be largely based on an appeal to authority. However, the history of medicine is replete with examples of situations where views on the aetiology and pathophysiology of conditions, and how they should be treated, particularly those that were psychologised, have changed over time. Moreover, there is actually little consensus within the field on the aetiology or pathophysiology of ME/CFS; there are many competing theories (1). Patient organisations should not be compelled to support a particular model for the illness that is the basis for the use of cognitive behaviour therapy (CBT) and graded exercise therapy (GET), or indeed, the therapies themselves. The authors may claim such therapies are safe but this is in dispute, with large percentages of patients reporting deteriorations in their overall health in patient surveys (1,2). Moreover, claims for the efficacy of such therapies are mainly based on subjective outcome measures in non-blinded studies so there are plenty of reasons to be sceptical of such claims (1-3).

The models underlying CBT and GET for ME/CFS may be described as "biopsychosocial" but are not equally "physical" and "psychological". They are based on the view that the symptoms and disability in the condition are reversible using these behavioural methods (4). This is distinctly different from many other conditions where these would be used as adjunctive therapies. I would venture that it is the proponents of such models (i.e. those underlying CBT and GET) who are out of step with the majority of the ME/CFS research community worldwide. For example, there are many studies which suggest the symptoms of the condition are not due to deconditioning (e.g. studies with sedentary controls) and I believe most would not accept the view that all the findings can simply be explained by deconditioning (1,2,5). In such circumstances, patient organisations should be entitled to make their own assessment of the evidence in its various forms rather than be compelled to follow any orthodoxy in a particular country.

References:

[1]. Kindlon T. Reporting of Harms Associated with Graded Exercise Therapy and Cognitive Behavioural Therapy in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Bulletin of the IACFS/ME. 2011;19:59-111.

[2]. Twisk FNM, Maes M. A review on Cognitive Behavorial Therapy (CBT) and Graded Exercise Therapy (GET) in Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS): CBT/GET is not only ineffective and not evidence-based, but also potentially harmful for many patients with ME/CFS. Neuro Endocrinol Lett. 2009;30:284-299.

[3]. Kewley AJ. Does Cognitive Behavioral Therapy or Graded Exercise Therapy Reduce Disability in Chronic Fatigue Syndrome Patients? Objective Measures Are Necessary. Clinical Psychology: Science and Practice. 2013 20;3:321-322 DOI: 10.1111/cpsp.12042

[4]. PACE manuals for Cognitive Behaviour Therapy and Graded Exercise Therapy for CFS/ME. http://www.pacetrial.org/trialinfo/ (last accessed: October 27, 2013)

[5]. Snell CR, Stevens SR, Davenport TE, Van Ness JM. Discriminative Validity of Metabolic and Workload Measurements to Identify Individuals With Chronic Fatigue Syndrome. Phys Ther. 2013 Jun 27.

Conflict of Interest: I am the Assistant Chairperson and Information Officer of the Irish ME/CFS Association. All my work for the Association is unpaid.

On 2015 Aug 18, Bill Cayley commented:

A good example of when "Less" is "More": https://lessismoreebm.wordpress.com/2015/07/31/health-outcomes-of-continuous-positive-airway-pressure-versus-oral-appliance-treatment-for-obstructive-sleep-apnea-a-randomized-controlled-trial/

On 2014 Mar 02, Joel Topf commented:

I am disappointed that they did not provide a intention to treat analysis.

On 2016 May 10, Morten Oksvold commented:

Please pay attention to the following report by ORI (Office of Research Integrity) before reading this article:

https://ori.hhs.gov/content/case-summary-walker-kenneth

On 2017 May 10, Marko Premzl commented:

The comment "Response to Caiati and coauthors' article" was published on The Journal of Neuroscience article's internet site: http://www.jneurosci.org/content/33/7/2973#response-to-caiati-and-coauthors-article (10 May 2013).

Project leader: Marko Premzl PhD, ANU Alumni, 4 Kninski trg Sq., Zagreb, Croatia

E-mail address: Marko.Premzl@alumni.anu.edu.au

Internet: https://www.ncbi.nlm.nih.gov/myncbi/mpremzl/cv/130205/

On 2014 Jan 03, RICHARD NIEDERMAN commented:

This report is both intriguing and vexing.

It is intriguing because it addresses a central issue in oral health care - what is the relative value of prevention. This is of central importance as the global community begins to seriously consider prevention vis-a-vis traditional surgical treatment of caries.

It is vexing because nowhere in the article is there an explicit listing of the 17 reviews or 71 included articles identified in Figure 2, nor is there an appendix or link to the identified publications.

Perhaps the authors might make this information available here?

On 2015 Nov 13, University of Kansas School of Nursing Journal Club commented:

Reviewers: (Team 10) Sydney Jourdan, Lucy Bush, Elijah Penny, Cassidy Playter, Brittany Winter, Alex Noller, Parwana Noori (Senior Nursing Students – Class of 2016)

Background:

Many of our class discussions in our microsystem leadership course focused on methods currently available to accurately measure nurse job satisfaction and the elements needed to contribute to a healthy work environment, such as nursing leadership. Our selected article attests to how there is an increasingly high number of nursing turnover due to a lack of managerial leadership and support. This study found that nurse leaders who practiced authentic leadership created an environment of nurse empowerment regardless of their experience level - this ultimately promotes nurse health and retention. . We believe leadership training for nurse managers should be a requirement in today’s health-care organizations as it will aid in the development of an empowering work environment and attract and retain nurses (Laschinger, Wong & Grau, 2013). Laschinger, Wong, & Grau (2013), examined the impact of authentic leadership, autonomous practice and continuous support on the nursing work environment. Our group felt this article directly correlated to recent class discussions, further exemplifying the impact of authentic leadership on nurse job satisfaction, performance and safety.

Methods:

Our group located this article using the CINAHL database from the Dykes Library Resource Center. The literature was discovered using the search terms “authentic leadership” AND “healthy work environment.” The design of this study included a secondary analysis of data obtained via questionnaire of two study groups from Ontario in an acute care setting. The pattern of relationships regarding emotional exhaustion and cynicism versus authentic leadership and structural empowerment were examined in a group of 342 new graduate nurses with less than two years of nursing experience and in another group of 273 nurses with over two years of work experience (Laschinger et al., 2013). This study targeted both new graduate nurses and experienced nurses in order to examine how each group was affected by a work environment in the acute care setting containing structural empowerment and authentic leadership and how such practices related to group cynicism and burnout.. The acute care patient population has a lower socioeconomic status and higher rate of drug addiction and drug seeking than other healthcare settings, rendering a higher rate of cynicism and burnout amongst its nurses. Not only does this study impact acute care nurses, but their patients as well - the empowered and contented nurse will provide better patient-centered care. Data was obtained via questionnaire. 907 questionnaire packages were sent out to a sample population with a follow-up questionnaire sent three weeks later. Of the 907 questionnaires sent, 342 responded and met the criteria of less than two years nursing experience. 280 responded for the “experienced” portion of the study however, seven cases were removed from the later analysis as these respondents did not have over two years of nursing experience, leaving a sample size of 273 experienced nurse subjects (Laschinger et al., 2013). The questionnaires utilized included the Conditions of Work Effectiveness Questionnaire-II, which examines the four components of structural empowerment and has been previously discussed in our class, the Authentic Leadership Questionnaire, which measures the four components of nurses’ perceptions of their managers’ authentic leadership qualities and the Maslach Burnout Inventory-General Survey, which measures the emotional exhaustion and cynicism of nursing burnout (Laschinger et al., 2013).

Findings:

The results of this study supported a model linking authentic leadership and structural empowerment to nursing burnout (Laschinger et al., 2013). This study is the first to investigate the relationship between authentic leadership and structural empowerment to nursing burnout and cynicism and concluded authentic leadership had both direct and indirect effects on cynicism, a phenomena that is found to be linked to emotional exhaustion related to poor work environments (Laschinger, et al., 2013). Self-awareness and balanced processing are two characteristics of authentic leadership found to be critical to the creation of an empowered work environment. Leaders who were perceived to support employees and provide both positive and negative feedback rendered nurses who reportedly felt empowered to perform quality patient care in a meaningful way. The results of this study support the validity of both structural empowerment and the authentic leadership theory. This contributes to knowledge leaders can utilize to make positive changes in nursing work environments and promote nurses’ health and retention (Laschinger et al., 2013). Health care work environments are extremely stressful - however, the results of this study show relationship-driven leadership strategies have the ability to transform current nursing environments positively and ensure all nurses are empowered to provide optimal patient-centered care. This study is important because it examines methods such as structural empowerment and authentic leadership and how these practices may lead to less emotional strain, turnover and burnout for the acute care nurse. The study also explores how a healthy work environment may render less cynicism amongst nurses - leading to happier patients, effective communication and better health outcomes.

Implications:

This article is important to nursing practice because it examines how healthy work environments are essential to effective and optimal patient-centered care. Nursing burnout and emotional strain is a very real dilemma the acute care setting faces and leads to job dissatisfaction, poorer health outcomes and turnover. In a field already facing vast nursing shortages, studies such as this one are pertinent to finding the best practice environment. This study provides evidence to implement across the healthcare setting in order to promote happier, healthier and safer work environments. As medicine and technology advance, leadership tactics and qualities must also in order to compete with a constantly evolving environment. This article resonated personally with group members who work and/or aspire to work in the acute care setting and have witnessed the reality of nurse burnout and cynicism. The nurse is already given the difficult task of treating and caring for the patient’s mind, body and spirit. Having to provide such care in a strained and/or unsupportive environment sets the nurse up for failure. As our class faces graduation, we want to feel confidently that all of our study and practice efforts will be rewarded in an environment that promotes support, empowerment and authenticity. As a result of reading and discussing this article, our group has learned preventing emotional exhaustion is key to preventing advanced burnout and can be accomplished through structural empowerment and authentic leadership practices (Laschinger et al., 2013). Moving forward as professional nurses, we will aim to act authentically, seek support when needed and ensure our work environments are those that promote empowerment in order to prevent emotional exhaustion and provide the best patient care possible.

Reference:

Laschinger, Heather K. Spence, Carol A. Wong, and Ashley L. Grau. Authentic leadership, empowerment and burnout: A comparison in new graduates and experienced nurses. Journal of Nursing Management 21, no. 3 (April 2013): 541–52 12p. doi:10.1111/j.1365-2834.2012.01375.x.

On 2014 Jan 31, George W Hinkal commented:

Lee GY The National Cancer Institute has been investing in the development of an online webportal of curated cancer nanotechnology data called caNanoLab. The numerical data and additional nanomaterial characterizations related to this publication have been added to the database and can be found at:

https://cananolab.nci.nih.gov/caNanoLab/characterization.do?dispatch=summaryView&sampleId=40566787&page=0&tab=ALL

https://cananolab.nci.nih.gov/caNanoLab/characterization.do?dispatch=summaryView&sampleId=40566788&page=0&tab=ALL

https://cananolab.nci.nih.gov/caNanoLab/characterization.do?dispatch=summaryView&sampleId=40566789&page=0&tab=ALL

https://cananolab.nci.nih.gov/caNanoLab/characterization.do?dispatch=summaryView&sampleId=41418754&page=0&tab=ALL

The left navigation links on these pages provide information about each sample (under Navigation Tree).

For general information on how to use caNanoLab, please visit https://cananolab.nci.nih.gov/caNanoLab/home.jsp

On 2014 Jun 27, David Keller commented:

H2-water therapy improved Parkinson disease in this pilot study - can it be replicated?

The results of this small but apparently well-designed pilot study were very encouraging, and seemed to demonstrate in humans with Parkinson disease [PD] significant benefits from drinking water saturated with dissolved hydrogen gas (also called "H2-water"). H2-water is hypothesized to be an effective antioxidant in vivo, and has demonstrated benefits in animal models of PD (1,2,3). More than a year has elapsed since the publication of the provocative findings of this pilot study. We need a large clinical trial to either confirm or refute the benefits of H2-water in humans with PD.

References

1) Fu Y, Ito M, Fujita Y, Ito M, Ichihara M, Masuda A, Suzuki Y, Maesawa S, Kajita Y, Hirayama M, Ohsawa I, Ohta S, Ohno K. Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of Parkinson's disease. Neurosci Lett. 2009 Apr 3;453(2):81-5. doi: 10.1016/j.neulet.2009.02.016. Epub 2009 Feb 12.PubMed [citation] PMID: 19356598

2) Fujita K, Seike T, Yutsudo N, Ohno M, Yamada H, Yamaguchi H, Sakumi K, Yamakawa Y, Kido MA, Takaki A, Katafuchi T, Tanaka Y, Nakabeppu Y, Noda M. Hydrogen in drinking water reduces dopaminergic neuronal loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. PLoS One. 2009 Sep 30;4(9):e7247. doi: 10.1371/journal.pone.0007247.PubMed [citation] PMID: 19789628, PMCID: PMC2747267

3) Matsumoto A, Yamafuji M, Tachibana T, Nakabeppu Y, Noda M, Nakaya H. Oral 'hydrogen water' induces neuroprotective ghrelin secretion in mice. Sci Rep. 2013 Nov 20;3:3273. doi: 10.1038/srep03273.PubMed [citation] PMID: 24253616, PMCID: PMC4070541

On 2013 Nov 22, Attila Csordas commented:

data available via ProteomeXchange with identifier PXD000596 http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD000596

On 2015 Oct 01, Egon Willighagen commented:

Dear Christopher, when you studied the indexing of IKs by Google, did you also look at the effect of SEO tricks? That is, Google suggests that writing a good page title and description will help them correctly index pages. For example, I can imagine you had looked at one of the lists, e.g. for atorvastatin, and determined if the IK was used in the page title and/or description. Would you expect that if chemical databases adopted this SEO technologies for the IK, it would further improve the situation? Or would the effect be minor? Could it help overcome the problem with partly matching IKs, as you noted here about a year ago?

On 2014 Sep 16, Christopher Southan commented:

Unfortunately Google has now become capricious in matching either part of the Key and thus producing false positives from just the second half. It is therfore more effective to try the inner 14-characters of the Key (the connectivity layer) first. The overall utility still stands and the better news is that, even though the full InChI strings are truncated to 32 caracters in a search, they can give useful partial matches.

In reply (Oct 2015) to Egons Q above. As we know, rankings move so its difficult to know what (legitimate) SEO steps are making the difference (exepting traffic per se). Yes, databases could do more, in particular PubChem has a backlog problem (i.e. new entries not indexed). It would also be geat if UniChem and SureChEMBL contrived to get fully crawled. Then we really could check the global 100+ million in a ~0.3 sec pop

On 2013 Dec 30, Gregory Francis commented:

In response to my critique of their original article, Elliot & Maier (E&M) raised three points. I respond in reverse order, because the first point is the most interesting.

3) E&M propose that the discussion about bias should be at the field level rather than at the level of individual articles. I strongly disagree. A discussion at the field level dilutes responsibility, so researchers can simultaneously believe that the field is biased and believe that their own effects are sound. One gets a hint of such an attitude in E&M, where they describe the problems of bias in the field, but insist that their findings related to the red-attractiveness effect are valid. Scientists fundamentally care about individual effects, so that is the appropriate place to consider the influence of bias.

2) E&M suggest that my article was incautious because it considered several different analyses of effects. Perhaps the text in Francis (2013) did not make clear that different bias tests were applied in order to see if there might be some way to interpret the experiments so that they did not indicate bias. This exploratory search was unsuccessful; every combination of effects and measures lead to a conclusion of bias for at least some of the effects.

E&M also suggest that my use of the test is inappropriate because the analytical approach was originally developed by Ioannidis & Trikalinos (2007) to detect bias in large sets of experiments. This claim is entirely without justification. If E&M want to argue that my analysis is flawed, then they should demonstrate an error in the calculations or reasoning.

1) E&M claim that there is independent evidence that the red-attractiveness effect is real. They cite additional studies (including some from other labs) that report the presence of the effect. These studies may indeed demonstrate the validity of the effect, but their properties do nothing to remove the appearance of bias in the study by Elliot et al. In fact, if E&M intend to use these additional studies as evidence for their theoretical claim, then the success of these experiments only makes the bias problem worse. The probability of success for all the experiments in Elliot et al., low as it is, must be larger than the probability of success for those experiments and the additional studies.

E&M also reported a new experiment designed to check on the validity of the red-attractiveness effect. In the original study, this experiment produce a standardized effect size of 0.83 (n1=16, n2=17). The new study found a smaller effect size of 0.25 (n1=75, n2=69), which did not produce a significant effect (p=.13). E&M concluded that the red-attractiveness effect is in the small to medium range rather than the large range. Most research psychologists would go further and suggest that there is insufficient evidence to interpret the red-attractiveness effect as being different from zero (for many researchers the null hypothesis is the default position, until proven otherwise).

Given that Francis (2013) argued that the original experiments were too successful, one might wonder if the new (non-significant) experiment diffuses that critique. A new version of the analysis using the original five experiments in Elliot et al. and the new experiment produces a pooled effect size of 0.53. This value is substantially smaller than the pooled effect for the original studies (0.78) because the new experiment has a small effect size and carries substantial weight in the pooling due to its larger sample size.

With the new pooled effect size, the estimated power of each of the experiments (in order of publication) is 0.21, 0.30, 0.316, 0.49, 0.26, and 0.89. It is rather odd that the experiment with the largest power estimate is the only experiment to not reject the null hypothesis. Moreover, the probability of rejecting (any) five (or six) out of six experiments like these is 0.03, which is so low that it indicates bias.

Since it presumably is not biased, the new experiment arguably gives the best estimate of the red-attractiveness effect. The estimated power derived from an effect size of 0.25 for the original experiments is 0.08, 0.10, 0.11, 0.15, and 0.10. The probability that all five such experiments would reject the null is the product of the power values, which is 0.000013. So, if the new experiment provides a valid estimate of the effect, readers should be very skeptical about the validity of the original experiments in Elliot et al.

Finally, it is worth noting that E&M never explicitly discussed the presence of bias in their reported experiment set. They did not disclose whether there were unreported null findings or whether some experiments used sampling methods that inflate the Type I error rate. It is possible that bias occurred without their awareness, but the absence of a clear statement highlights that readers should be skeptical about the reported experiments.

Ultimately, the new experiment in E&M only strengthens the argument for some form of publication bias in Elliot et al. The other points deserve a frank and creative exchange of ideas, but they do nothing to alter the unbelievability of their original findings.

On 2015 Oct 23, Vojtech Huser commented:

This is a comment from the first author of the article:

Several months after publication, the journal changed publishers (from BMJ group to Oxfort University Press) and as a result, the appendix data for the article are not available via the current journal site.

To obtain the online supplemental data, use the following ResearchGate links:

• https://www.researchgate.net/publication/283089519_AppendixA_query_details_in_word_document
• https://www.researchgate.net/publication/283089520_AppendixB-v02_Counts_of_trials_in_ICMJE_approved_registries
• https://www.researchgate.net/publication/283089365_appendix_C_-_excel_file_-_journal-based-sample-trial-articles

On 2013 Oct 31, John Cannell commented:

I congratulate the authors on a fine study. I wonder if they are aware that Mostafa et al found that an anti neural antibody found in autism had a -.86 correlation coefficient with 25(OH)D levels? If not it goes to show that scientists become experts in their own niche but apparently do not keep up with relevant science in other fields.

Mostafa GA, Al-Ayadhi LY.Reduced serum concentrations of 25-hydroxy vitamin D in children with autism: relation to autoimmunity. J Neuroinflammation. 2012 Aug 17;9:201. Mostafa GA, 2012

Thus the vitamin D theory of ASD (vitamin D deficiency being the environmental risk factor for this highly heritable disorder) is consistent with the authors work. Three recent studies, using community controls, have found 25(OH)D levels are significantly lower in children with ASD. Two of the studies below (Mostafa et al and Gong et al) also found ASD severity, as rated on standard ASD rating scales, is inversely correlated with 25(OH)D levels. Mostafa et al found an R value of -.86 for the association of serum 25(OH)D with ASD severity.

Gong ZL, Luo CM, Wang L, Shen L, Wei F, Tong RJ, Liu Y. Serum 25-hydroxyvitamin D levels in Chinese children with autism spectrum disorders. Neuroreport. 2013 Oct 1. Gong ZL, 2014

Meguid NA, Hashish AF, Anwar M, Sidhom G. Reduced serum levels of 25-hydroxy and 1,25-dihydroxy vitamin D in Egyptian children with autism. J Altern Complement Med. 2010 Jun;16(6):641-5. Meguid NA, 2010

Mostafa GA, Al-Ayadhi LY.Reduced serum concentrations of 25-hydroxy vitamin D in children with autism: relation to autoimmunity. J Neuroinflammation. 2012 Aug 17;9:201. Mostafa GA, 2012

There is a plethora of basic science explaining why low gestational or early childhood 25(OH)D levels would adversely effect brain development.

DeLuca GC, Kimball SM, Kolasinski J, Ramagopalan SV, Ebers GC. Review: the role of vitamin D in nervous system health and disease. Neuropathol Appl Neurobiol. 2013 Aug;39(5):458-84. doi: 10.1111/nan.12020. DeLuca GC, 2013

Eyles DW, Feron F, Cui X, Kesby JP, Harms LH, Ko P, McGrath JJ, Burne TH. Developmental vitamin D deficiency causes abnormal brain development. Psychoneuroendocrinology. 2009 Dec;34 Suppl 1:S247-57. doi: 10.1016/j.psyneuen.2009.04.015. Epub . Review. Eyles DW, 2009

Furthermore, the vitamin D theory of autism explains most of the epidemiological facts of ASD.

Cannell JJ. On the aetiology of autism. Acta Paediatr. 2010 Aug;99(8):1128-30. Cannell JJ, 2010

Cannell JJ. Autism and vitamin D. Med Hypotheses. 2008;70(4):750-9. Cannell JJ, 2008

70% of American toddlers do not take the American Pediatric Association's recommended vitamin D supplement of 400 IU/day. As milk consumption has fallen, so have toddler’s vitamin D levels.

Cannell JJ. Autism, will vitamin D treat core symptoms? Medical Hypotheses. 2013 Aug;81(2):195-8. Cannell JJ, 2013

Some autism researchers seem cognizant of the entire body of autism research. For example, a group of well-known European autism researchers, including Professor Christopher Gillberg of the Gillberg Neuropsychiatric Institute, have recently called for the need for “urgent research” into the vitamin D deficiency theory of ASD.

Kočovská E, Fernell E, Billstedt E, Minnis H, Gillberg C. Vitamin D and autism: clinical review. Res Dev Disabil. 2012 Sep-Oct;33(5):1541-50. doi: 10.1016/j.ridd.2012.02.015. Epub 2012 Apr 21.Kočovská E, 2012

However, these scientists appear to be in the minority. Until all autism researchers become cognizant of the wider body of autism research, we will continue to wonder how to prevent - and perhaps even treat - this modern day plague.

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2015 Feb 14, Satyanarayan Hegde commented:

Interesting observation. Authors do not mention the patient's FEV1 at other time points prior to starting HFCWO which they probably did not have access to. How do we know the modest increase in FEV1 (~400ml) after starting HFCWO is not because of resolution of an exacerbation? If the patient were to have neutrophillic inflammation, he would not respond to systemic steroids. Patient reported symptoms as a measure of disease severity can be notoriously unreliable. Authors should have attempted in obtaining more objective data such as frequency of systemic steroid use, unscheduled physician office visits, ED/hospitalization rates, missed work days and medication refill history. Nevertheless, they describe another mode of potential therapy for a select group of patients.

On 2014 Jan 15, Satoko Hattori commented:

"ImageLD" and "ImageEP", image analysis application softwares used in this article, are now freely available from http://www.mouse-phenotype.org/software.html.

On 2014 Jun 16, Dave Hayes commented:

Erratum: Note that the final three columns of Table 3 should be titled "Regional BP x vmPFC % signal change" and NOT "Regional vmPFC...", i.e. the GABA(A) receptor binding potentials of each region from the first column were correlated with BOLD signal changes in the vmPFC.

On 2017 Jan 20, Claudiu Bandea commented:

To be productive translational research must be driven by scientifically sound working hypotheses

(This is an abbreviated version of a comment originally posted in Nature in 2013: http://www.nature.com/news/translational-research-medicine-man-1.12380)

Many effective therapies and drugs have been discovered fortuitously or with little science backup. This door for discovery is still open. However, for some diseases, the development of prevention and therapeutic approaches requires deep scientific understanding of their etiology and pathogenic mechanisms. Take, for example, neurodegenerative diseases. Despite decades of research, continuous effort by thousands of scientists and clinicians, and huge investments in the order of billions of US dollars per year, there are no successful preventive or therapeutic approaches.

Incidentally, it was the dramatic experience of ‘Losing a life’ to amyotrophic lateral sclerosis (ALS) that drove Christopher Austin, the director of the National Center for Advancing Translational Sciences (NCATS), to biomedical research and his quest for drug discovery almost three decades ago (1). Since then, millions of people have dies of ALS, Alzheimer’s (AD), Parkinson’s (PD), Huntington’s (HD), Creutzfeldt-Jacob disease (JCD) and other neurodegenerative diseases.

In light of the extraordinary public health and economic burden associated with neurodegenerative diseases, including tens of millions of affected patients and their families and economic losses estimated at over two hundred billion dollars per year in the Unites States alone, it is imperative to ask: (i) why there are no successful prevention and therapeutic approaches, and (ii) how can this unfortunate situation be resolved in a matter of years, not decades?

Unfortunately, these uncomfortable questions are often marginalized or obscured by empty optimism or deceiving ambiguity. Articulating these questions, though, would not only establish a high sense of urgency and concern for people’s life, but might also help with the science. By emphasizing that despite decades of research the etiology of these diseases and the physiological function of the primary proteins implicated in these diseases are not known, these questions point to the possibility that the current working hypotheses might be wrong.

Indeed, I recently proposed (2) that the protein misfolding theory and the prion hypothesis, which have directed most of the thinking and research on neurodegenerative diseases, are scientifically flawed, and that, by misdirecting the research and strategies for developing prevention and therapeutic approaches, these working hypotheses have been responsible for the lack of progress. According to the protein misfolding theory, AD, PD, HD, ALS and CJD are caused by the misfolding of the primary proteins implicated in these diseases: amyloid-beta and tau in AD, alpha-synuclein in PD, huntingtin in HD, TDP-43 in ALS, and prion protein in CJD (3). And, the prion hypothesis posits that protein misfolding is caused by prions (i.e. self-replicating proteins) or prion-like pathogens (4). In summary, according to these two working hypotheses, the isomeric conformation changes of amyloid-beta, tau, alpha-synuclein, huntingtin, TDP-43 and prion protein and their assembly into various protein complexes, including oligomers, plaques and tangles, are protein misfolding or prion replication activities.

In a radical departure from the current paradigms, I proposed that:

(i) A-beta/APP, tau, alpha-synuclein, huntingtin, TDP-43, and prion protein are members of the innate immune system;

(ii) The isomeric conformational changes of these proteins and their assembly into various oligomers, plaques, and tangles are not protein misfolding events or prion-replication activities, but part of their normal, evolutionarily selected innate immune activities;

(iii) The immune reactions associated with the function of these proteins in innate immunity lead to AD, PD, ALS, HD, CJD and other neurodegenerative diseases, which are innate immunity disorders.

If correct as the current evidence indicate, these new paradigms will be eventually embraced, as science always corrects itself. However, this is a slow process that usually takes many years or even decades, and for the millions of people affected by these diseases time is everything. Therefore, when it comes to scientific issues that affect people’s life, the life of tens of millions of people in this case, the conventional approach of letting science takes its (usually lengthy) course is not acceptable.

The solution is rather straightforward: all new data, observations, ideas and hypotheses need to be immediately, openly and systematically evaluated. This is where NCATS and other agencies, institutions and associations could have the opportunity to make a life-or-death difference for millions of people. How can this be accomplished in a field, such as neurodegenerative diseases, in which the existing primary working hypotheses, the protein misfolding theory and prion hypothesis, have directed the work and the careers of an entire generation of researchers?

It is unrealistic to expect researchers in the field to systematically and specifically search for data and observations that challenge their current and previous work. It is reasonable, however, for the funding agencies to establish an open and comprehensive Peer-Review Platform (PRP) for critical and timely evaluation of all the research and publications. The PRP would be supported by a small percentage (e.g. 5%) of the biomedical funds, which would be awarded as ‘peer-review grants’ to qualified scientists (surprisingly, the funding agencies and the philanthropists invest billions of dollars in scientific projects but very little in the systematic evaluation of the results and publications, which undermines the entire process). Additionally, the scientists receiving research grants would be encouraged to contribute to PRP by providing critical evaluations of the research projects and scientific publications in their field of expertise. The PRP would be open to all scientists, including the authors of the research and publications under review. Generating data and observations is only half of the scientific process, the other half is their evaluation, interpretation, and integration into useful knowledge and productive working hypotheses.

References

1.Wadman M. 2013. Translational research: Medicine man. Nature. 494:24-6; Wadman M, 2013

2.Bandea CI. 2013. Aβ, tau, α-synuclein, huntingtin, TDP-43, PrP and AA are members of the innate immune system: a unifying hypothesis on the etiology of AD, PD, HD, ALS, CJD and RSA as innate immunity disorders. bioRxiv. doi: 10.1101/000604; http://biorxiv.org/content/biorxiv/early/2013/11/18/000604.full.pdf

3.Dobson, C. M. 2003. Protein folding and misfolding. Nature 426:884-90. Dobson CM, 2003

4.Prusiner SB. 2012. A unifying role for prions in neurodegenerative diseases. Science. 336:1511-3; Prusiner SB, 2012

On 2013 Oct 23, Stephen Turner commented:

This study set out to compare shotgun metagenomic sequencing to 16S rRNA amplicon sequencing to determine the taxonomic and abundance profiles of mixed community metagenomic samples. Thus far, benchmarking metagenomic methodology has been difficult due to the lack of datasets where the underlying ground truth is known. In this study, the researchers constructed synthetic metagenomic communities consisting of 64 laboratory mixed genome DNAs of known sequence and polymerase chain reaction (PCR)-validated abundance. The researchers then compared metagenomic and 16S amplicon sequencing, using both 454 and Illumina technology, and found that metagenomic sequencing outperformed 16S sequencing in quantifying community composition. The synthetic metagenomes constructed here are publicly available (Gene Expression Omnibus [GEO] accession numbers are given in the manuscript), which represent a great asset to other researchers developing methods for amplicon-based or metagenomic approaches to sequence classification, diversity analysis, and abundance estimation.

On 2016 Aug 23, Ben Goldacre commented:

This trial has the wrong trial registry ID associated with it on PubMed: both in the XML on PubMed, and in the body of the text of the article. The ID given is NCT0151529. We believe the correct ID, which we have found by hand searching, is NCT01515293.

This comment is being posted as part of the OpenTrials.net project^[1] , an open database threading together all publicly accessible documents and data on each trial, globally. In the course of creating the database, and matching documents and data sources about trials from different locations, we have identified various anomalies in datasets such as PubMed, and in published papers. Alongside documenting the prevalence of problems, we are also attempting to correct these errors and anomalies wherever possible, by feeding back to the originators. We have corrected this data in the OpenTrials.net database; we hope that this trial’s text and metadata can also be corrected at source, in PubMed and in the accompanying paper.

Many thanks,

Jessica Fleminger, Ben Goldacre*

[1] Goldacre, B., Gray, J., 2016. OpenTrials: towards a collaborative open database of all available information on all clinical trials. Trials 17. doi:10.1186/s13063-016-1290-8

* Dr Ben Goldacre BA MA MSc MBBS MRCPsych<br> Senior Clinical Research Fellow<br> ben.goldacre@phc.ox.ac.uk<br> www.ebmDataLab.net<br> Centre for Evidence Based Medicine<br> Department of Primary Care Health Sciences<br> University of Oxford<br> Radcliffe Observatory Quarter<br> Woodstock Road<br> Oxford OX2 6GG

On 2013 Nov 24, Allison Stelling commented:

In chemistry and physics journals, there's something called "table of contents graphics"- one picture that "sums up" the findings of the whole paper, which are displayed with the just the title. (Here's mine from one of the papers I published for my PhD work Stelling AL, 2007, see the journal for the graphic: http://pubs.acs.org/doi/abs/10.1021/ja074074n.) I've published in both physical chemistry journals and biomedical journals, and it was interesting to note that this particular graphic was not required in biomedicine.

Searchable graphics and flowcharts for clinical trials would make life a lot easier, I think, esp. for practicing MDs who are quite busy and unfortunately have little time to "keep up with the news". (Esp. considering how fast the science "news" comes out these days- and the volume!)

Keyword searches are already most scientists' choice for discovering new literature- perhaps we should let the idea of bound journals with issues and word counts and constrained subjects fade away. (Don't worry, publishers will have still have very important tasks: organizing pre-publication checks on Ethics and study design etc. from experts; & digital data curation, organization, and efficient distribution.)

On 2013 Nov 24, John Sotos commented:

JAMA’s laudable effort to upgrade medical abstracts [1] represents only a syntactic improvement in communicating quantitative results. It was proposed by editors faced with reviewing multitudes of abstracts submitted to research meetings.

Of greater use to JAMA’s general medical readership, and especially to the innumerable members of the public who read JAMA abstracts online via the Pubmed system, would be a graphical flow chart describing each study’s design. Instead of syntactic sugar, this would provide at-a-glance understanding of what is often the most innovative part of a study.

Such charts are now familiar to readers, having been part of JAMA’s instructions to authors since at least 1998 [2]. However, because they are often laden with details [3], they are themselves candidates for abstraction.

Although Pubmed already supports graphics in its abstract pages, authors would be better specifying these proposed abstract-flow-charts declaratively, e.g. with an XML data description language. Pubmed could then, someday, support searches based on details of study design, thereby fulfilling the hope expressed during JAMA’s introduction of structured abstracts in 1991: to “allow more precise computerized literature searches” [4].

[1] Bauchner H, Henry R, Golub RM. The restructuring of structured abstracts: adding a table in the results section. JAMA. 2013; 309: 491-492.

[2] Anonymous. JAMA instructions for authors. JAMA. 1998; 279: 69-72.

[3] Paradise JL, Bluestone CD, Colborn DK, Bernard BS, Smith CG, Rockette HE, Kurs-Lasky M. Adenoidectomy and adenotonsillectomy for recurrent acute otitis media: parallel randomized clinical trials in children not previously treated with tympanostomy tubes. JAMA. 1999; 282: 945-53.

[4] Anonymous. Structuring abstracts to make them more informative. JAMA. 1991; 266: 116-117.

On 2015 Jun 29, Bill Cayley commented:

A good example of when "less" is "more" - https://lessismoreebm.wordpress.com/2015/06/29/effect-of-corticosteroid-injection-physiotherapy-or-both-on-clinical-outcomes-in-patients-with-unilateral-lateral-epicondylalgia-a-randomized-controlled-trial/

On 2015 Sep 15, Ivan Oransky commented:

This paper, along with a related article, has been retracted: http://retractionwatch.com/2015/09/14/fabricated-results-retract-jama-clinical-trial-plus-a-sub-analysis-of-the-data/