On 2015 Sep 04, RAIMONDO D'AMBROSIO commented:

We find that the authors' conclusions are invalidated by poor experimental design, application of inappropriate epilepsy diagnosis criteria, and disregard of antecedent literature that is incompatible with their thesis. These issues are addressed briefly in a Letter to the Editor in the Journal of Neuroscience, and thoroughly discussed in a document found at http://arxiv.org/abs/1509.01206

First, there is no evidence that the authors managed to master the FPI technique to induce any epileptogenesis. They simply failed to induce any of the convulsive and non-convulsive epileptic seizures induced by classic FPI by many different independent laboratories. The discussion in ArXiv details why the nominal matching of the parameters of the pressure pulse used by the authors cannot be used to claim equivalency of injury.

Second, the authors' report of frequent bilateral SWDs in virtually all male Sprague Dawley rats by 3 months of age is unprecedented and in conflict with all other reports we know of, which originate from many different independent laboratories. Thus, their claim that young male Sprague Dawley rats commonly exhibit SWDs is unfounded.

Third, the authors do not use valid diagnostic criteria to distinguish focal epilepsy from absence epilepsy, but dwell instead on the morphology of the ECoG discharge which is not recognized as clinically valid to differentiate these seizure types. In the clinic, diagnosis of focal epilepsy is based on the identification of a pathological brain area from which seizures consistently precipitate (the epileptic focus) and the analysis of the consistent focality, lateralization and spread of the seizures. Using the clinically recognized criteria, it is apparent that the authors were dealing with absence epilepsy in both their control and FPI rats. The analysis in ArXiv details why the authors' diagnostic criteria are wrong, why they err in asserting that it is established that short duration and spectral power at 7-9Hz is uniquely characteristic of rodent absence epilepsy, and why the SWDs described here are clearly distinct from the focal seizures observed after effective FPI.

Fourth, the study systematically fails to acknowledge published data that are inconsistent with their conclusion, including: 1) focal seizures induced by FPI are not sensitive to drugs (valproate and carisbamate) that potently control genetic SWDs, 2) FPI-induced focal seizures can be potently prevented by mild focal cooling of the perilesional neocortical epileptic focus, which proves both their acquired nature and their origin from an epileptic focus, and 3) spectral power in the theta band is common in acquired epilepsy in both rats and humans.

As a result of these many flaws, this study simply amounts to a comparison of absence epilepsy in control rats with the same absence epilepsy in FPI rats. Contrary to the authors’ assertion, this study has no discernible implication for the translational relevance of the FPI model of PTE, or for any of the many other etiologically realistic acquired epilepsy models that are currently being used in a growing number of laboratories and that induce non-convulsive seizures with a dominant ECoG spectral power in the theta band.

On 2015 Jun 20, Christopher Southan commented:

Molecular identifiers for lead compound, some analogues and the target @ http://cdsouthan.blogspot.se/2014/06/getting-into-box-with-some-recent.html

On 2015 Oct 16, NephJC - Nephrology Journal Club commented:

This study was discussed on Aug 25th and 26th in the open online nephrology journal club, #NephJC, on twitter. Introductory comments from Joel Topf and Suzanne Norby are available at the NephJC website and blog. The discussion was quite detailed, with more than 40 participants, including nephrologists, hematologists, fellows and residents. The highlights of the tweetchat were:

• Dr Agarwal and his team, as well as the funding agency (NIDDK and the Indiana Institute for Medical Research) should be congratulated for designing and conducting this trial, though most participants were somewhat perplexed by the choice of outcome for this intervention.

• There was a very intriguing signal in worse outcomes with intravenous iron (compared to oral iron) which, despite some uncertainty due to small sample size and early stoppage of the trial, is coherent with other non-renal data, as also some data from the dialysis literature, resulting in broad agreement that oral iron could be used more often, if needed.

• Some bold questions were discussed: Is there any reason to treat anemia in chronic kidney disease unless hemoglobin was < 9 g/dL? Is there a role for regular monitoring of hemoglobin at all, given lack of data that iron or erythropoietin result in improvement of any meaningful outcome?

A transcript and a curated (i.e. Storified) version of the tweetchat are available from the NephJC website.

Interested individuals can track and join in the conversation by following @NephJC or #NephJC, or visit the webpage at NephJC.com.

On 2015 Oct 13, David M Patrick commented:

I thank Dr. Kindlon for his comment. As he indicates, this study was not designed to be a comprehensive evaluation of POTS in this context. We used a simple clinical screening test rather than a lengthy evaluation. We also had a largely ambulatory patient grouping which will not represent the full spectrum of ME/CFS.

On 2015 Oct 05, Tom Kindlon commented:

Is 1 minute of standing sufficient to test for orthostatic intolerance?

Perhaps not a major point regarding the main thrust of the paper, but I thought I would highlight how orthostatic intolerance was tested:

"Heart rate and blood pressure were measured lying and after 60 seconds of standing, with postural hypotension defined as a drop in systolic blood pressure by ≥20 mm Hg or in diastolic blood pressure by ≥10 mm Hg; postural tachycardia was defined as an increase from lying to standing heart rate of ≥30 beats per minute."

I'm not an expert on dysautonomia and the like but my impression is that testing using 1 minute of standing is non-standard.

On 2016 Jun 16, Lanfranco Troncone commented:

Adding to the discussion on the role of monoamines in violent aggression, this new data shed light on results we published many years ago on three models of aggression in REM sleep deprived rats. Aggression can be elicited in these animals by dopaminergic agonists like apomorphine, dopamine/noradrenaline precursor L-DOPA and noradrenaline synthesis inhibition by fusaric acid. We observed the clear differences in these three models and fusaric acid induced the more violent aggression while L-DOPA the most stereotyped mild boxing type. Our discussion proposed then that noradrenergic transmission was responsible for moderating the behavior while dopamine was triggering it. We offer the full paper at: Troncone LR, & Tufik, S. - Physiology & Behavior (1991), 50, 173-178 (PMID 1946713).

On 2015 Aug 31, Bill Cayley commented:

From the fore-going discussion, it is clear that there remain questions about this approach, but it seems that this is an area where, at least in some cases, "less" may be "more". See also: https://lessismoreebm.wordpress.com/?s=appendicitis

On 2015 Aug 20, Paulina Salminen commented:

In response to colleagues Bertele and Garattini, we agree that it is of great importance to evaluate the optimal treatment for uncomplicated acute appendicitis regarding all pros and cons for both surgery and antibiotic therapy. The aim of the APPAC trial was to test the hypothesis that uncomplicated acute appendicitis can be successfully treated with antibiotics by comparing antibiotic therapy with emergency open appendectomy. Even though more patient-centric outcomes are valuable, we aimed to identify a clear and concise definition of efficacy that would apply to both treatments in order to enable fair comparison – resolution of acute appendicitis. The non-inferiority approach was used as the goal was not to find an approach that is more effective than current standard treatment as appendectomy cures appendicitis. The non-inferiority approach allows us to evaluate other advantages and a novel treatment that is almost as effective as standard treatment might be preferred in practice or for some patients.(1)

Based on our APPAC study results, we now know that the majority (73%) of patients with uncomplicated acute appendicitis were successfully treated with antibiotics.(2) We agree with Bertele and Garattini that major complications in both treatment arms are of vital importance. In the APPAC trial none of the patients treated initially with antibiotics and later with appendectomy had major complications; thus antibiotics are a safe first-line treatment for CT-proven uncomplicated acute appendicitis.

The optimal use of antibiotic therapy regarding both spectrum and duration of the treatment in patients with uncomplicated acute appendicitis needs to be prospectively evaluated in large patient series including the important assessment of relevant patient-centric outcomes.

1.Kaji AH, Lewis RJ. Noninferiority Trials: Is a New Treatment Almost as Effective as Another? Jama. 2015 Jun 16;313(23):2371-2. 2.Salminen P, Paajanen H, Rautio T, et al. Antibiotic Therapy vs Appendectomy for Treatment of Uncomplicated Acute Appendicitis: The APPAC Randomized Clinical Trial. Jama. 2015 Jun 16;313(23):2340-8.

On 2015 Jul 29, Vittorio Bertele' commented:

The question of antibiotic therapy as an alternative to surgery for acute appendicitis keeps being addressed by a non-inferiority approach. We have previously challenged this attitude as it is not in the best interest of patients(1). The APPAC trial aimed to prove that fewer than one out of four patients with uncomplicated acute appendicitis would have required surgery within one year if they had been treated with antibiotics. The trial failed to prove this as the arbitrarily selected non-inferiority margin of antibiotics relative to surgery, although huge (24%), was not met. Still, the technically negative outcome of APPAC does not necessarily signify a failure(2). Avoiding surgery may be an advantage but patients also want to know what else could happen if they undergo, avoid or just defer appendicectomy. Unfortunately, the APPAC trial does not specify what patients can gain or lose with the two therapeutic strategies apart from the possibility of avoiding surgery in the antibiotic group. Besides –or even rather than– the greater than expected need for surgery in this group (27% instead of 24%), what matters most to patients is how many cases of peritonitis, other severe infections, complications of surgery, undue interventions, etc. there are in the two study arms. A trial addressing the superior overall benefit-risk profile of either the surgical or the antibiotic approach would have put these alternative strategies in a clearer perspective, enabling physicians and patients to make firmer decisions. A composite outcome measure including clinically relevant efficacy and safety endpoints –to address the sum of benefits and risks– would have answered the question whether failure to avoid surgery in spite of antibiotics still provides an actual advantage to patients, not just an acceptable disadvantage in exchange for unknown benefit. APPAC is one further demonstration that non-inferiority trials are often useless for patients(3): they assume an advantage (avoidance of surgery), allow too large a margin of failures –several patients needed surgery later in spite of the antibiotics– and, most important, do not address the possible overall benefit for patients (fewer complications?).

Vittorio Bertele’ and Silvio Garattini IRCCS-Istituto di Ricerche Farmacologiche Mario Negri email: vittorio.bertele@marionegri.it, silvio.garattini@marionegri.it

References 1. Banzi R, Torri V, Bertele V, Garattini S. Antibiotics versus surgery for appendicitis. Lancet 2011;378:1067-8. 2. Livingston E, Vons C. Treating Appendicitis Without Surgery. JAMA 2015;313:2327-8. 3. Garattini S, Bertele V. Non-inferiority trials are unethical because they disregard patients' interests. Lancet 2007;370:1875-7.

On 2015 Jun 25, Bill Cayley commented:

A good example of when less is more: https://lessismoreebm.wordpress.com/2015/06/17/arthroscopic-surgery-for-degenerative-knee-systematic-review-and-meta-analysis-of-benefits-and-harms/

On 2017 Jul 24, Richard E Goodman commented:

As one of the senior authors of the Siruguri et al., 2015 publication, with 20 years experience in evaluating the safety of Genetically Engineered (GE or GM) crops, I feel obligated to respond to the statements Dr. Sunil Verma is posting on PubMed COMMONS and now also in Science as an e-letter to the 2016 publication by Priyanka Pulls describing the development of this GE mustard. Dr. Verma's second comment posting here lists his letter in Science. Importantly, neither my comments, nor those of Verma are peer reviewed. We are giving our opinions (which differ markedly as does our experiences). I have written a response to Verma's e-letter in Science and it is available at http://science.sciencemag.org/content/352/6289/1043/tab-e-letters . It addresses the issues of the accepted hazard and risk evaluation of GE crops in India and internationally. Our 2015 publication here describes the assessment looking at the source of the genes, the sequences of proteins and the scientific rational is to evaluation potential risks for those who might be allergic to the protein (Barnase, Barstar or Bar), or to proteins that are highly identical, and could share IgE binding. Dr. Verma did not provide any data that demonstrates we are wrong, or that there are risks from this mustard. Instead in his supplemental information, he compared the sequence of Ani s 9, a minor allergen of a fish parasitic worm, to other sequences in the AllergenOnline.org database. And he implies that cross-reactivity might occur due to associated proteins like the SXP/RAL-2 proteins (Ani s 5 and Ani s 9). However, as noted by Garcia-Mayoral et al., 2014), similar proteins do not exist outside of worms (Nematodes). Ani s 9 has very little sequence similarity to Barnase, as described in our paper. Comparing Ani s 9 in AllergenOnline demonstrates that it is rather unique and unlikely to have cross-reactivity outside of the parasitic worm allergens. This mustard contains Barnase, not Ani s 9. Furthermore, he points to the six amino acid match of Barnase to Ani s 9. But as described in our paper and in my letter in Science, that six amino acid segment matches hundreds of proteins in the NCBI database, without any evidence of cross-reactivity or allergy. Furthermore, there is no evidence that a six amino acid match predicts cross-reactivity. The standard in CODEX is sequences matching >35% identity over 80 amino acids, and such matches are quite conservative (overpredict) both primary and confirmational epitopes (Goodman, 2006, Goodman et al., 2008). CODEX indicates you may do a short sequence match, but must justify the methods. If there are matches of >35% identity over 80, then serum IgE tests would be warranted using sera from at-risk (specifically allergic subjects (Goodman, 2008). In the future there will be improvements in the assessment (Goodman and Tetteh, 2011), however, Dr. Verma has not described any new method or any proof that he has an improvement. Instead, he has proposed hypothetical issues, a letter in Science and he has not posted the letter on facebook. If he thinks there can be improvements, he should do experiments and submit his results to a peer reviewed journal for scientific evaluation. The authors of the Siruguri et al 2015 paper stand by our results that this GM mustard is as safe as the non-GM mustards in use in India today.

References: Garcia-Mayoral MF, Trevino MA et al., (2014). Relationships between IgE/IgG4 epitopes, structure and function in Anisakis simplex Ani s 5, a member of the SXP/RAL-2 protein family. PLOS, Negl Tropical Dis 8(3):e2735. Goodman RE. (2006) Practical and predictive bioinforamtics methods for the identiifcation of potentially cross-reactive protein matches. Mol Nutr Food Res 50:655-660. Goodman RE, Vieths S et al, (2008). Allergenicity assessment of genetically modified crops--what makes sense? Goodman RE. (2008) Performing IgE serum testing due to bioinformatics matches in the allergenicity assessment of GM crops. Food Chem Toxicol 46(Suppl 10):S24-S34. Goodman RE, Tetteh AO. (2011). Suggested improvements for the allergenicity assessment of genetically modified plants used in foods. Curr Allergy Asthma Rep 11(4):317-324.

On 2017 Jul 21, Sunil Verma commented:

Reply to - Comment of Prof. Richard E. Goodman, and Vasanthi Siruguri 2017 Jul 19 2:50 p.m.

Sunil Kumar Verma, Principal Scientist CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.

Dear Authors,

I read your elaborated reply with great enthusiasm and with hope to find specific answers to my very specific queries on this specific paper (PMID: 26079618). Instead of replying to my scientific observations on the methodology used in this paper, your reply focus more on defending the release of GM mustard in India, which is not really the central theme of this specific paper in question. The scope of this specific paper was limited to assess the allergenic potential of the transgene Bar, Barnase, and Barstar expressed in Genetically Modified Indian Mustard for heterosis breeding, wherein, the technical methodology which was used in this paper was questionable as specifically explained in my previous comment (supported by 15 pages of supplementary information, please see attachment in previous comment) in detail.

Of course, GM Mustard release could be a consequence of this publication, which could be discussed in detail at appropriate forum when safety data and complete dossier which you have cited in your reply above is made available for public review by the concerned authorities (see ref. 1). Until that complete dossier is available for review, your justifications on GM mustard release would be one sided. And for that reason, I would not like to even touch upon the matter beyond the scope of this specific paper and my technical comments which are still unaddressed.

With reference to your first argument that I have misinterpreted recommendations of Indian Council of Medical Research (ICMR, 2008) guidelines on the safety of Genetically Engineered (GE) crops, I would like to highlight that even the ICMR guideline also emphasize on overall structural similarities to predict the allergenic potentials (see page 13-14 in ref. 2), however, in the light of my previous comment and provided data therein, we now know that primary amino acid sequence comparisons are not the best known indicators of structural similarities among proteins, therefore my technical objection remains valid.

My second technical comment was on Barnase-barstar complex and its possible differential immune response compared to individual proteins in free form; this aspect you have left unaddressed in your reply above.

In response to my third technical objection, you have written that AllergenOnline.org database is a peer reviewed database. I understand that it is a peer-reviewed database; however, it's peer-reviewed status does not change the newly revealed fact that this database fails to detect the known allergen 'Ani s 9' (GenBank: ABV55106.1) as allergen using the strategy as was followed to examine the GM Mustard transgenes; thus, the conclusions drawn in this analysis remain erroneous.

My commentary on your paper and flawed conclusion therein has been published in Science (3). Authors are welcome to take this debate further so that a consensus could be reached beyond a reasonable doubt on the conclusion of this specific paper in question and a suitable action could be taken to correct the conclusions of this paper by the way of erratum if appropriate, as an outcome of this post publication review.

References:

(1) Priyanka Pulla, India nears putting GM mustard on the table. Science 27 May 2016: Vol. 352, Issue 6289, pp. 1043. DOI: 10.1126/science.352.6289.1043

(2) Guidelines for the Safety Assessment of Foods Derived from Genetically Engineered Plants. Indian Council of Medical Research. New Delhi. 2008. https://goo.gl/HuAnGC Link accessed on 22/07/2017

(3) Sunil Kumar Verma, RE: Letter on In Depth "New India nears putting GM mustard on the table" Science 10 July 2017. http://science.sciencemag.org/content/352/6289/1043/tab-e-letters Link Accessed on 22/07/2017

On 2017 Jul 19, Richard E Goodman commented:

Sunil Verma, CSIR Hyderabad, India, provided hypothetical comments about our study (PMID 26079618). He suggests our evaluation methods and conclusions were wrong and suggests our conclusions should be changed. We reviewed our publication and Verma’s comments. We conclude that he is misinterpreting the rationale for our study, the recommendations of the CODEX Alimentarius guidelines (2003/2009) and Indian Council of Medical Research (ICMR, 2008) on the safety of Genetically Engineered (GE) crops and our study. The dossier including study was reviewed and approved by the RCGM and GEAC committees of the government of India. Dr. Verma did not present data from studies of the GE plant or the proteins.

Our response: 1) Indian mustard (Brassica juncea) is an important oil seed crop consumed as oil, seed and greens in India. However, production efficiency and plant diseases limits yield. Heterosis breeding often leads to production of agronomically superior hybrid plants.

2) Mustard is usually self-pollinating. Therefore, scientists at the University of Delhi South Campus developed a GE Indian mustard system producing a male sterile plant and a fertility restorer line that allows hybrid production. The male sterile line was created by insertion of a gene that encodes an RNase (Barnase) that is expressed only in the tapetum (outer cell layer of the anther or pollen sac in early flower buds). The DNA construct includes a herbicide tolerant gene (bar). Expression of Barnase is tightly regulated by the construct of DNA as explained in Jagannath et al., 2002, where a long spacer DNA was inserted between the bar gene and promoter and the Barnase gene to ensure it is only expressed in the anther as demonstrated from field trial plants. Barnase is not detectable in seeds or green tissue of the plant (Jagannath et al., 2001). The fertility restoring line was developed by inserting a gene encoding Barstar also using an anther (tapetum) restricted promoter. Barstar binds to Barnase in a very tight protein complex that inhibits the RNase activity of Barnase (Bisht et al., 2004). The restorer line also includes the bar herbicide tolerance gene. Data on the two GE plants and their hybrid offspring were well characterized and data, including a full safety evaluation, was reviewed by the RCGM and GEAC committees of the Indian government with recommendations the plants are as safe as non-GE mustard.

3) The safety evaluation followed the Indian Council of Medical Research guidelines (2008), which are consistent with the international guideline (CODEX Alimentarius, 2003 and 2009). One primary concern is whether the protein expressed by the novel genes encode allergens or proteins of moderate sequence identity (e.g. >35% identity over 80 amino acids) to an allergen so that allergenic cross-reactivity might be expected. The guidelines are referenced in the paper and are publically available. The methods and databases are cited. There is no evidence that proteins of lower sequence identity (including the FAO/WHO 2001 suggestion of 6 consecutive amino acid matches) are sufficient to demonstrate potential risks of allergy.

4) The assessment for potential risks of allergenicity of GE crops have been extensively addressed (e.g. Goodman et al., 2008). The risks of food allergy recognized by food regulators around the world are that foods must be labeled with the source of ingredients so that people who have allergies to allergenic sources (e.g. peanuts, specific tree nuts, milk, wheat, eggs), can avoid ingesting foods that cause their allergies. Governments have adopted specific food labeling laws for all foods and hold producers accountable. The standard is the same for GE crops. When Pioneer Hibred was developing a nutritionally enhanced soybean by adding a gene from Brazil nut, Dr. Taylor at the University of Nebraska conduct tests since the gene was from an allergenic tree nut. The results demonstrated that the protein was an allergen that would put Brazil nut allergic subjects at risk (Nordlee et al., 1996) That GE plant never entered food production. There are no published scientific reports of a GE protein in an approved crop causing allergic reactions in humans.

5) The methods suggested by Verma have not been demonstrates to predict allergenicity. A number of publications demonstrate that six-amino acid matches are common and not predictive (e.g., Hileman et al., 2002). Proteins with less than 50% overall identity are unlikely to be cross-reactive. Similar three-D shapes do not predict allergenicity. The AllergenOnline.org database is a peer reviewed database that includes Ani s 9, and there is no significant math. The database is updated every year and is accepted by international governments (Goodman et al., 2016).

6) A similar GE mustard (canola) was produced PGS in Europe, now owned by Bayer CropScience. The canola include the same proteins, expressed in similar amounts and patterns. Seven events have been approved for production and consumption in Canada, the US, and Australia. Food and feed products from those events are approved for import and consumption in Japan, China, South Africa, Korea and the European Union. The food and feed safety evaluation summaries and details are available on the CERA-GMC.org/GmCropDatabase Result website. First approvals were in 1995 and there is no evidence of harm even though 95% of canola in North America is GE.

7) The standard for food safety of GE crops in all countries that approve GE crops is that the GE plant does not pose any greater risk of allergy or toxicity than the similar non-GE crop. This GE Indian mustard developed at South Delhi campus meets those standards.

We stand by our conclusions that the male-sterile and restorer Indian mustard events and progeny are as safe as the non-GM counter-parts. There is no data that suggests otherwise. Statements by Dr. Verma are hypothetical, not based on facts. This mustard meets the same standards used to evaluate GE crops approved in the US, Canada, Australia-New Zealand, Japan and other countries.

Richard E. Goodman, and Vasanthi Siruguri

References: 1.Siruguri V, Bharatraj DK, Vankudavath RN, Rao Mendu VV, Gupta V, Goodman RE. 2015. Evaluation of Bar, Barnase and Barstar recombinant proteins expressed in genetically engineered Brassica juncea (Indian mustard). For potential risks of food allergy using bioinformatics and literature searches. Food Chem Toxicol 83:93-102. 2.Jagannath A, Bandyopadhyay P, et al. 2001. The use of a spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high-frequency generation of transgenic male sterile lines in Brassica juncea L. Mol Breeding. 8:11-23. 3.Jagannath A, Arumugam N, et al., 2002. Development of transgenic barstar lines and identification of a male sterile (barnase)/restorer (barstar) combination for heterosis breeding in Indian oilseed mustard (Brassica juncea).Curr Sci 82:46-52. 4.CODEX Alimentarius Guidelines 2003, 2009. 5.ICMR Guidelines. 2008. 6.Bisht NC, Jagannath A et al., 2004. A two gene-two promoter system for enhanced expression of a restorer gene (barstar) and development of improved fertility restorer lines for hybrid seed production in crop plants. Mol Breeding 14:129-144. 7.Hileman RE, Silvanovich A et al., 2002. Bioinformatic methods for allergenicity assessment using a comprehensive allergen database. Int Arch Allergy Immunol 128:280-291. 8.Goodman RE, Ebisawa M, et al. 2016. AllergenOnline: a peer-reviewed, curated allergen database to assess novel food proteins for potential cross-reactivity. Mol Nutr Food Res 60:1183-1198. 9.Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK. 1996. Idnetification of a Brazil-nut allergen in transgenic soybeans. N Engl J Med. 334:688-692. 10.Goodman RE, Vieths S, et al. 2008. Allergenicity assessment of genetically modified crops—what makes sense? Nat Biotechnol 26:73-81.

On 2017 Jul 09, Sunil Verma commented:

Comment on - Evaluation of Bar, Barnase, and Barstar recombinant proteins expressed in genetically engineered Brassica juncea (Indian mustard) for potential risks of food allergy using bioinformatics and literature searches

Sunil Kumar Verma, Principal Scientist CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.

In this study, the authors have tested the allergenic potential of the transgene Bar, Barnase, and Barstar expressed in Genetically Modified Indian Mustard for heterosis breeding. To this end, the authors have done the primary amino acid sequence comparisons of these proteins with the primary amino acid sequences of the known allergens listed in Allergenonline.org and NCBI Entrez protein database until January 2015 and 9 March 2015, respectively. Based on these bioinformatics comparisons authors concluded that the Bar, Barnase and Barstar proteins are unlikely to present any significant risk of food allergy to consumers. The authors also recommended not to perform any human serum IgE testing to further evaluate possible binding to the Bar, Barnase or Barstar proteins.

I hereby propose that the above conclusions drawn by the authors in this study are incorrect and require a major revision.

The main criteria used by the authors in these bioinformatics comparisons was the primary amino acid sequence homology searches of the proteins in question with that of the primary amino acid sequences of the potential allergen listed in above databases. All the hits with less than 50% primary amino acid sequence identities for full length proteins and less than 35% identity in the sliding window 80 amino acid segments of each proteins were ignored; the argument was that these matches could not have led to significant structural similarities among the proteins in question, therefore can be ignored.

Several independent studies have shown that in many cases, even though the primary amino acid sequence similarity between two proteins / domains are very less (<20%), but the tertiary structures of the proteins may be highly similar. One classical example of this is high structural similarity between N terminal half of the Krit-B41 domain with that of the RA domain of RalGDS (1RAX:A) with an r.m.s. deviation of 2.9A for 80 aligned positions; despite a very low homology in their primary amino acid sequences (sequence identity =8.7%). [1, S1] It is notable that both RalGDS and Krit-1 interact with Rap1A through the RA and B41 domains, respectively [2, 3], and so the talin [4]. Thus, the high primary amino acid sequence similarity between two proteins may though infer greater chances of structural homology between these proteins; however, low primary amino acid sequence similarity does not necessarily infer that proteins in question will necessarily have higher structural dissimilarities.

Since it is the conformationally determined structure of the proteins/epitopes which finally decide immunogenicity and allergenicity - and not just the primary amino acid sequences; the conclusion drawn in this study based on merely the primary amino acid sequence comparisons are scientifically inappropriate.

Secondly, in real scenario, both the Barnase and Barstar proteins are expressed simultaneously and these two proteins remain in a complex and not as individual proteins in plant [5, 6]. It is not unlikely that structure of a specific protein in complex may be different than that of the structure of the same individual protein in free form. Also, there may be the possibilities of formation/exposure of new epitope(s) surfaces, particularly as we know now that there are several antibodies known that recognize just the native proteins and some may indeed require complex assembly.

Thus, these conformationally determined epitopes that are recognized in the complex but not the free protein of interest may be reveled in differential screening between a protein and a complex form of the same protein. The conformationally determined epitopes could then be compared for structural homology with the epitopes in known allergens to determine the allergenic potential of two proteins in complex; such studies however, were not conducted in this paper; and the fact that Barnase and Barstar remain in complex and not in free form, was completely ignored throughout the study.

Finally, I found that the overall implication of the Allergenonline.org database itself on correctly predicting the allergenic potential of a new antigen was also questionable.<br> To test this, I assumed that 'Ani s 9' (which is a very well known allergen from SXP/RAL-2 protein family) [7] is a new putative allergen and that this group of proteins are not yet listed in the database; and asked whether or not one can predict if 'Ani s 9' is a potential food allergen using the strategy as was used in this study for Barnase, Barstar and Bar transgenic proteins. The full length primary amino acid sequence comparison of 'Ani s 9' (GenBank: ABV55106.1) using default parameter i.e 'E' value cut off = 1 identified 7 hits (excluding the hits with its own sequences) with 'tropomyosin' allergen from various organisms and 'AAEL002761-PC ' allergen from Aedes aegypti, respectively; however, none of the hits was with significant similarity cut off (>50%). Thus, this bioinformatics search criteria wrongly predicted that the 'Ani s 9' is not a potential food allergen. [S2]

The another criteria i.e. greater than 35% identity in the sliding window of 80 amino acid segment also did not produce any hit at all (other than self hits, which were excluded as explained above), indicating that this criteria also failed to identify 'Ani s 9' as potential food allergen. [S3] The third criteria i.e. 8 continuous amino acid segment search also did not identify any hit with any of the allergen in the database.[S4]

Thus, the bioinformatics search as used in this study following any of the criteria defined could not identify 'Ani s 9' as a potential food allergen. This confirms that the criteria used in this study by authors could easily give false negative results.

The only strategy that could have identified 'Ani s 9' as possible food allergen was a '6 continuous amino acid segment search, which could have identified its match with Allergen 'Lol p 5' for the 6-aa segment 'ANAPPA'. [S5]

This criteria however, was not used in current study to predict the allergenic potential of Bar, Barnase and Barstar. If this specific criteria was used, Barnase transgenic protein also could have given a potential hit with Allergen Ber e 2 and Ani s 9 for the 6 continuous amino acid patch LFSTAA, and WVASKG, respectively [S6]; hence, the conclusion of this paper could have been different.

In view of the above, I conclude that the criteria implemented in this study were not sufficient to exclude the possibility of the transgenic protein Bar, Barnase and Barstar being a possible allergen; therefore the conclusion drawn by authors that "the above transgenic proteins are unlikely to present any significant risk of food allergy to consumers" is not beyond a reasonable doubt, and hence need an appropriate correction by the way of erratum.

Further, as discussed above, the Barnase and Barstar proteins are expressed simultaneously in final plant and they remain in a tight complex (i.e. barnase-barstar complex) and not as free form. The current study has not even touched upon the barnase-barstar complex; therefore, until the systematic studies on this complex is conducted and concluded, it is not appropriate to give a 'safe' tag to these transgenic proteins. This is particularly important since the conclusion drawn from this study was one of the major evidence which was used by the Indian regulatory authorities to recently give a safety clearance to the genetically engineered Brassica juncea (Indian Mustard) for commercial cultivation in India. [8, 9]

Ref & Suppl Information

On 2015 Jul 23, Neil Davies commented:

Østergaard and colleagues report that fewer people with a high genetic risk of high blood pressure develop Alzheimer disease compared to those with a lower genetic risk of high blood pressure (1). This is consistent with evidence that use of anti-hypertensive medication is associated with incidence and progression of Alzheimer disease (2–4). We investigated these associations in an observational longitudinal cohort study using data from the Clinical Practice Research Datalink, a database of anonymous UK National Health Service primary and secondary health care data. We found that patients prescribed angiotensin-receptor blockers were less likely to be diagnosed with Alzheimer disease (OR 0.47, 95% CI: 0.37 to 0.58) than those prescribed other anti-hypertensives. Our results were based on observational data and may suffer from residual confounding, but they provide suggestive evidence that blood pressure or blood pressure medication is implicated in the aetiology of Alzheimer disease. A meta-analysis of randomised controlled trials has also suggested that treatment with blood pressure lowering medication may help slow cognitive decline (5).

However, there are alternative explanations for these associations. Although Mendelian randomization analyses may remove biases due to confounding and reverse causality, survival bias is still likely to be an issue. Individuals with a greater burden of high blood pressure SNPs may have higher mortality, which could cause them to die before developing Alzheimer’s disease and reduce the frequency of blood pressure increasing SNPs in cases compared to controls. Survival bias may also explain the apparent protective effect on Alzheimer’s disease risk of variants that are associated with heaviness of smoking. The smoking heaviness increasing allele located in the CHRNA5-A3-B4 gene cluster has been shown to be associated with increased mortality risk amongst ever smokers (6), which is likely to explain the reduced frequency of this allele amongst older populations of ever compared to never smokers.(7) This highlights the difficulties in using Mendelian randomisation for diseases of old age.

The authors discuss survival bias, and conclude it is unlikely to be a problem. However, in our opinion more research is needed to quantify the extent and impact of survival bias in Mendelian randomisation studies. It may be possible to assess the extent of survival bias by comparing risk allele frequencies to known allele frequencies in younger populations, or to track their change in a population as it ages. Survival bias is likely to only occur if there are fewer risk alleles in older populations. Simulations may allow us to estimate the size of survival bias and aid the interpretation of Mendelian randomisation studies.

Neil M. Davies, Amy E. Taylor, Marcus R. Munafò

[1] S. D. Østergaard et al., Associations between Potentially Modifiable Risk Factors and Alzheimer Disease: A Mendelian Randomization Study. PLOS Med. 12, e1001841 (2015).

[2] N. Li et al., Use of angiotensin receptor blockers and risk of dementia in a predominantly male population: prospective cohort analysis. Br. Med. J. 340, b5465 (2010).

[3] N. M. Davies, P. G. Kehoe, Y. Ben-Shlomo, R. M. Martin, Associations of anti-hypertensive treatments with Alzheimer’s disease, vascular dementia, and other dementias. J. Alzheimers Dis. JAD. 26, 699–708 (2011).

[4] P. G. Kehoe, N. M. Davies, R. M. Martin, Y. Ben-Shlomo, Associations of Angiotensin Targeting Antihypertensive Drugs with Mortality and Hospitalization in Primary Care Patients with Dementia. J. Alzheimers Dis. JAD. 33, 999–1008 (2013).

[5] N. Levi Marpillat, I. Macquin-Mavier, A.-I. Tropeano, A.-C. Bachoud-Levi, P. Maison, Antihypertensive classes, cognitive decline and incidence of dementia: a network meta-analysis. J. Hypertens. 31, 1073–1082 (2013).

[6] L. Rode, S. E. Bojesen, M. Weischer, B. G. Nordestgaard, High tobacco consumption is causally associated with increased all-cause mortality in a general population sample of 55,568 individuals, but not with short telomeres: a Mendelian randomization study. Int. J. Epidemiol. 43, 1473–1483 (2014).

[7] A. E. Taylor, M. R. Munafò, CARTA consortium, Commentary: Does mortality from smoking have implications for future Mendelian randomization studies? Int. J. Epidemiol. 43, 1483–1486 (2014).

On 2016 Aug 24, Lily Chu commented:

In response to public comments, Dr. Smith and her colleagues have conducted sensitivity analyses on the data, assessing the impact of CBT and GET on various outcomes when only subjects fitting Oxford criteria are considered versus when subjects fitting non-Oxford case definitions (i.e. 1994 Fukuda) are considered. They concluded in an Addendum to the original report that:

"Our sensitivity analysis would result in a downgrading of our strength of evidence on several outcomes which can be attributed to the decrease in power, dominance of one large trial, or lack of trials using criteria other than the Oxford (Sharpe, 1991) case definition for inclusion. Blatantly missing from this body of literature are trials evaluating effectiveness of interventions in the treatment of individuals meeting case definitions for ME or ME/CFS."

Almost all patients are diagnosed in the United States and most countries using the Fukuda criteria. The United Kingdom is the only region that uses the Oxford criteria on a regular basis. This means that clinicians need to be aware of low strength of evidence or the lack of evidence behind CBT and GET when considering this treatment for their ME/CFS patients.

The full revised report may be read at: https://effectivehealthcare.ahrq.gov/ehc/products/586/2004/chronic-fatigue-report-160728.pdf

On 2016 Feb 12, Ellen M Goudsmit commented:

Postscript 2016. The errors noted were rejected as inaccuracies by the editors and thus my comment (under Haney et al) was not published as a letter or correction. Example, they refused to accept that a trial discussed used two sets of criteria, not just the one listed. And if they had checked the reference given for the London criteria, they would have known that it does not give a list of authors. I was not named, so the citation is factually incorrect. I submit that this attitude to factual errors is inconsistent with good science.

On 2015 Jun 24, Ellen M Goudsmit commented:

I've already noted some of the factual errors and misleading comments in this review online: http://annals.org/article.aspx?articleid=2322800

Due to the word limit, I could not add that Haney et al named four individuals as authors of the London criteria for ME, despite the fact that their reference, the Westcare Report, did not [1]. The people listed in the review did not write the version published in the Westcare Report and this information has been in the public domain since 1994.

I was also unable to point out that that Haney et al refer to clinical criteria (e.g., p. 834), when most of the case definitions they discussed were formulated for research.

The omission of the new research criteria for classic ME is baffling as they have already been cited in the literature, most recently by Jason et al [2]. If people are going to make decisions about ME, or ME/CFS, they need to know what ME is. I suggest readers look online or access the original paper from 2009 [3].

[1]. The UK Patient Organisations. “London Criteria”. In: Report from The National Task Force on Chronic Fatigue Syndrome (CFS), Post Viral Fatigue Syndrome (PVFS), Myalgic Encephalomyelitis (ME). Bristol: Westcare; 1994. Appendix B, Names, Definitions and Descriptions: p. 96-8. Available from: http://www.actionforme.org.uk/Resources/Action for ME/Documents/get-informed/national task force.pdf

[2]. Jason, LA., Sunnquist, M., Brown, A and Reed, J. Defining essential features of myalgic encephalomyelitis and chronic fatigue syndrome. Journal of Human Behavior in the Social Environment, 2015, 25, 6, 657-674. Online 6th May. doi:10.1080/10911359.2015.1011256

[3]. Goudsmit E, Shepherd C, Dancy CP, Howes S. ME: Chronic fatigue syndrome or a distinct clinical entity? Health Psychol Update. 2009;18(1):26-33. Available from: http://shop.bps.org.uk/publications/publications-by-subject/health/health-psychology-update-vol-18-no-1-2009.html

Alternatively see: http://www.foodsmatter.com/me_and_cfs/cfs_me_causes_general/articles/goudsmit-me-clinical entity-10-12.html

Revised article (2014): http://www.axfordsabode.org.uk/me/mecrit2014.htm

On 2015 Dec 02, Ellen M Goudsmit commented:

Sad to note today that the journal chose to publish letters, favouring opinion above the correction of factual errors. How can science progress if editors collude in the perpetuation of inaccuracies and myths? Who will know that there ARE research criteria for ME as described by Ramsay? That there are helpful alternatives to CBT and GET?

Postscript 2016. The editors decided not to publish the comment as a letter in the journal as they rejected the view that there were factual errors. Thus while I was not a co-author of the criteria cited in the article, and I'm not named in the reference given, listing me as a co-author is not a 'factual error' in their eyes. Nor is the claim that a trial I discussed selected patients using two sets of criteria, not just the one referred to in the article. In my view, this failure to correct errors and misleading information is inconsistent with good science.

On 2015 Jul 17, Ellen M Goudsmit commented:

As someone who has studied myalgic encephalomyelitis (ME) and chronic fatigue syndrome (CFS) since the 1980s, I appreciate the work completed by The National Institutes of Health Pathways to Prevention Workshops on what is now known as ME/CFS. Unfortunately, some of the information in the two reviews is inaccurate, incomplete and misleading (1,2). For example, in the report on diagnostic methods, the reviewers included the London criteria for ME but gave details in the table based on a version written by a layperson, rather than the four individuals cited in their reference (3). Moreover, they did not consider the updated criteria for ME (4), although one of the authors had emailed the panel on two separate occasions during the consultation phase to alert them to their existence.

The second review (2) encourages further research on subgroups and outcomes other than fatigue and function but did not identify one of the few controlled studies which had employed such a design (5). For instance, in the Appendix, Table 1 lists the programme evaluated by Goudsmit and colleagues under ‘counseling and behavioural therapies’, and describes the treatment as ‘counseling’. It also states that patients were selected using the Oxford criteria, that the duration of follow-up was six months and that the outcomes were function and fatigue.

In fact, the trial evaluated a physician-led multi-component programme comprising medical care, information on the illness, diet and relaxation, as well as advice on activity management and some counselling (5). It was conducted in the naturalistic setting of an NHS hospital clinic, patients were diagnosed using criteria for post-viral fatigue syndrome as well as the Oxford criteria, and data were available for a number of symptoms including cognitive impairment, as well as other variables. Fatigue improved as noted in the review but the latter did not convey that 82% of the patients rated themselves as ‘better’, that 23% were well enough to be discharged at six months and that the improvements were maintained at 1 year. Given the missing details, the study’s rating as ‘poor’ is understandable.

The reviewers concluded that “more definitive studies comparing participants meeting different case definitions, including ME... are needed to fill research gaps”. It was therefore disappointing that they did not recognise the positive aspects of a study that used a different case definition and assessed a range of symptoms, not just fatigue.

1. Haney E, Smith MEB, McDonagh M, Pappas M, Daeges M, Wasson N, et al. Diagnostic methods for myalgic encephalomyelitis/chronic fatigue syndrome: A systematic review for a National Institutes of Health Pathways to Prevention Workshop. Ann Intern Med. 2015;162: 834-40. [PMID: 26075754] doi:10.7326/M15-0443

2. Smith MEB, Haney E, McDonagh M, Pappas M, Daeges M, Wasson N, et al. Treatment of myalgic encephalomyelitis/chronic fatigue syndrome: A systematic review for a National Institutes of Health Pathways to Prevention Workshop. Ann Intern Med. 2015;162:841-

3. [PMID: 26075755] doi:10.7326/M15-0114

4. Dowsett E, Goudsmit E, Macintyre A, Shepherd C. London Criteria for myalgic encephalomyelitis. In: Report from The National Task Force on Chronic Fatigue Syndrome (CFS), Post Viral Fatigue Syndrome (PVFS), Myalgic Encephalomyelitis (ME). Westcare. 1994. 96-98. Available from: http://www.actionforme.org.uk/Resources/Action for ME/Documents/get-informed/national task force.pdf

5. Goudsmit E, Shepherd C, Dancy CP, Howes S. ME: Chronic fatigue syndrome or a distinct clinical entity? Health Psychol Update. 2009;18(1):26-33. Available from: http://shop.bps.org.uk/publications/publications-by-subject/health/health-psychology-update-vol-18-no-1-2009.html

6. Goudsmit EM, Ho-Yen DO, Dancey CP. Learning to cope with chronic illness. Efficacy of a multi-component treatment for people with chronic fatigue syndrome. Patient Educ Couns. 2009;77:231–6. [PMID: 19576714 ] doi:10.1016/j.pec.2009.05.015

Ellen M Goudsmit PhD FBPsS

Comment also on Annals of Internal Medicine website under Haney et al.

On 2015 Dec 15, Diana Frame commented:

Interesting article and, as a researcher engaged in systematic reviews, I really appreciate the empirical testing done by the authors. I wonder whether the true benefit of machine learning algorithms will be not so much in performing the screening (what is often called "Level I" screening, on titles and abstracts), but in helping to plan search strategies. I could see using this tool at the very beginning of a project to find other non-obvious terms that describe the concepts to be covered in the review. Might be especially helpful as more and more full-text articles become available to churn through. Looking for a needle in a haystack can be challenging for human reviewers, but computers don't get tired.

On 2015 Jun 22, Robin Andersson commented:

Preprint of accepted manuscript is available at BioRxiv, http://dx.doi.org/10.1101/019844

On 2017 Apr 09, Harri Hemila commented:

A manuscript version is available at HDL.

On 2016 Aug 24, 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 NCT001226810. We believe the correct ID, which we have found by hand searching, is NCT00122681.

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 2015 Nov 02, R Andrew Moore commented:

Peter has a number of problems, and it is difficult to cover them all in a short space. He has not reported his concerns through the Cochrane Feedback mechanism.

Our searches did identify a large number of unpublished trials. We deplore this, but are powerless to change things. In a previous review for topical NSAIDs we contacted all identified manufacturers for published or unpublished data. The yield was small (as others have found), but some unpublished studies were brought into the public domain. Waiting for all studies and clinical trial reports of all studies would take forever, and could probably never be achieved. We believe that many of these unpublished studies relate to drugs and/or formulations that have never been manufactured commercially. While these would be of interest in determining what does and doesn’t work, they would have little clinical relevance.

Peter’s main issue appears to be heterogeneity. Tests for heterogeneity are problematical anyway (Pain. 2000 85:415-24), and the I square of 92% was for all topical diclofenac formulations combined. We demonstrated in the review that the (at least five) different diclofenac formulations produced different results from one another using L’Abbé plots and in our detailed analyses showing large variations in efficacy between them. In the circumstance, a high I square for all combined (clinical heterogeneity) is to be expected. The bulk of the studies on diclofenac were published in the last five years, were of decent quality, and moderate to large size. There were older data for ketoprofen, but again major differences between formulations.

Trying to determine publication bias using funnel plots or other measures is something of a lost cause (Journal of Clinical Epidemiology 2000;53:207-16). It is especially so with small number of trials (Journal of Clinical Epidemiology 2000 53: 477-484), and making sense of funnel plots is anything but easy for most people (Journal of Clinical Epidemiology 2005 58: 894-901). A useless method seems an odd choice to make to criticise our review.

There are very good scientific reasons why drug and formulation may play a big part in the efficacy of topical NSAIDs. This is also the case for oral analgesics used in acute pain, where formulation improvements generating rapid absorption confers greater efficacy. Simple lumping strategies may have been permissible in past systematic review methodology, but a more forensic approach is needed now and in the future. This is what we have attempted to do in this latest review.

Comparisons with Peter’s 2010 review seem inappropriate since that review was based on other reviews that are now out of date.

On 2015 Oct 28, Peter Gøtzsche commented:

The authors found that the results were missing from 5900 patients. Furthermore, there was extreme heterogeneity in their meta-analyses, e.g. I square was 92% for the diclofenac trials, which were the most common ones, and there was extreme funnel plot asymmetry, with the largest trials showing the smallest effects (the authors didn’t show funnel plots but I constructed one for diclofenac). Moreover, the trials were industry funded, of relatively poor quality, and the authors analysed published data, not data from clinical study reports, and did not try to obtain all the missing trials and data from the manufacturers.

The authors concluded that topical NSAIDs are effective in providing pain relief but also cautioned that the large amounts of unpublished data “could influence results in updates of this review.” They certainly could. I believe it is plain wrong to perform meta-analyses on the authors’ data. When I most recently reviewed this area for the BMJ in 2010, I concluded that we don't know whether topical NSAIDs are beneficial (1).

1 Gøtzsche PC. NSAIDs. Clin Evid (Online). 2010 Jun 28.

On 2017 Jun 05, Michael Greicius commented:

Thanks for your interest in the paper. The discrepancy you note arises from the reannotation step described in the manuscript. We reannotated each probe sequence to a gene using the ReAnnotator tool [1]. The used probe-to-gene mapping is listed in supplementary data file S2 [2]. The complete reannotated file is available through sourceforge [3].

[1]http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139516 [2]http://science.sciencemag.org/highwire/filestream/631209/field_highwire_adjunct_files/2/Richiardi_Data_File_S2.xlsx [3]https://sourceforge.net/projects/reannotator/files/annotations/

On 2017 May 30, Xiang-Zhen Kong commented:

I found that there are 8 of 136 genes (listed below) not available from the Allen Brain website, http://human.brain-map.org/static/download. I used the "Complete normalized microarray datasets". Could you share how you downloaded the expression data with all the 136 genes? Thanks.

8 genes not available from the Allen Brain website: CDK1 PRSS35 SHISA9 SIX3-AS1 TINCR LINC00617 MS4A8 NUPR1L

On date unavailable, commented:

None

On 2017 May 02, Jonas Richiardi commented:

A detailed reply has been posted at https://doi.org/10.1101/132746, showing that our original results stand. We provide a short summary here for convenience.

To a first approximation, all connectivity, like all politics, is local and these two features -- nearness and connectivity -- are challenging to disentangle. Evidence abounds that the majority of connectivity is local, but this critical attribute of functional anatomy is perhaps most efficiently conveyed in a macaque tracer study by Markov et al (2012, see in particular Figure 7). Thus, in undertaking our analysis, we were well aware that spatial nearness is correlated with connectivity, which we addressed using a measure of "fine" tissue-tissue similarity derived from an ontological atlas provided by the Allen Institute. The peer-reviewers at Science were also aware of this confound and insisted on an additional level of tissue-tissue similarity correction which our analysis survives (note that Data File S1 of our paper also included information about "coarse" tissue classes (field coarsetissueclass)).

The commenters suggest that the tissue-tissue similarity correction we applied is inadequate and point out that in the brain samples we used there remains a significant linear correlation between Euclidean distance and correlated gene expression between brain regions. The r-value for this correlation is 0.1. When the coarse tissue-tissue correction is applied, the r-value for the correlation drops to 0.094. In terms of variance explained, this means that slightly less than 1% of the correlated gene expression measure used in our analyses can be explained by Euclidean distance.

We thank the commenters for providing an independent replication of our core results, using the same method and data as in our original paper, and our rebuttal can be summarized in the following 5 points:

• Our analysis survives correction for Euclidean distance (see details in full reply), applied on top of the tissue-tissue similarity correction we used, as well as distance-aware permutation tests. Here, we note that there is an intrinsic contradiction in the commenters' counter-argument that a linear regression of Euclidean distance is inadequate, despite the fact that their critique (see their Figure 1B) is founded on this very same linear correlation.
• The random clusters generated by the commenters, meant to show the non-specificity of our results, consist of nodes that are roughly twice as close to one another as the nodes in the actual functional networks (see Figure 1 in our full reply).
• We replicated results of our connectivity gene in both a mouse connectivity analysis and a resting-state fMRI connectivity analysis. The commenters did not generate gene lists for any of their random cluster analyses to try and replicate in these or other independent datasets.
• Euclidean distance correction will wrongly assign "nearness" to two "neurally distant" regions on the crowns of adjacent gyri (see Figure2 in our full reply). This is essentially the opposite problem of the limitation to tissue-tissue correction that the commenters rightly point out in their figure 1A.
• Correcting the connectivity of two regions for nearness, using any measure, is bound to dilute the measure of connectivity. That our gene list survives two levels of tissue-tissue similarity correction plus a correction for Euclidean distance and is then replicated in a mouse structural connectivity dataset and a human resting-state fMRI connectivity datasets strikes us as strong support for the conclusion that these genes are important to functional connectivity.

Detail of these and several other points are available in the full reply paper at https://doi.org/10.1101/132746.

On 2016 Oct 19, Spiro Pantazatos commented:

Mind the distance: spatial proximity confounds tissue-tissue gene expression correlations reported in this study.

This is a novel and very interesting study. However, the authors do not adequately control for spatial proximity, which, contrary to the authors’ claims in the original article, accounts entirely for high within-network strength fraction according to our recent replication/reanalysis of these same data. Furthermore, “null networks”, (i.e. contiguous clusters with center coordinates randomly placed throughout cortex), also have significantly high strength fractions, indicating that high within-network strength fraction is not related to resting-state networks identified by fMRI.

Here is a link to the full technical commentary and replication/reanalysis write-up with additional supplementary discussion: http://biorxiv.org/content/early/2016/10/04/079202

And here is a link to the replication/reanalysis code on Github: https://github.com/spiropan/ABA_functional_networks

The lead authors are aware of these findings and concerns (I notified them via personal email in March, 2016) and they have let me know they plan to respond. I have submitted the commentary for peer review to Frontiers in Neuroscience. If accepted, they have the option to publish a formal rebuttal/response letter there, and/or respond in the comments section here.

Commentary Abstract

A recent report claims that functional brain networks defined with resting-state functional magnetic resonance imaging (fMRI) can be recapitulated with correlated gene expression (i.e. high within-network tissue-tissue strength fraction, SF) (Richiardi et al., 2015). However, the authors do not adequately control for spatial proximity. We replicated their main analysis, performed a more effective adjustment for spatial proximity, and tested whether 'null networks' (i.e. clusters with center coordinates randomly placed throughout cortex) also exhibit high SF. Removing proximal tissue-tissue correlations by Euclidean distance, as opposed to removing correlations within arbitrary tissue labels as in (Richiardi et al., 2015), reduces within-network SF to no greater than null. Moreover, randomly placed clusters also have significantly high SF, indicating that high within-network SF is entirely attributable to proximity and is unrelated to functional brain networks defined by resting-state fMRI. We discuss why additional validations in the original article are invalid and/or misleading and suggest future directions.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

On 2015 Jun 20, Arnaud Chiolero MD PhD commented:

Reinvention of cohort studies as platforms to test interventions is probably a great idea. It could give a new life to epidemiology, which seems to be lost in the data-deluge of observational studies.

On 2016 Apr 06, Jakob Suckale commented:

This is a good article on how to improve our undergraduate teaching. Below is a verified list of the resources mentioned in the text ordered by usefulness - a few links no longer work as of early 2016 others lead to locked or difficult to get resources, many are very useful.

Most useful

• openly accessible repository of science problems at U Delaware categorized by discipline
• list of teaching tips at Wieman Science Education Initiative
• online course: Introduction to Evidence-based Undergraduate STEM Teaching last session Nov 2015 but still available
• accessible Repository of case studies at U Buffalo but solutions and teaching notes only available after paid registration & background check
• teaching journal focusing on evidence: CBE Life Science Education is available online and best searched via PubMed
• applied learning by U Georgia, requires making a free login and only contains 5 projects
• guided inquiry POGIL only links to difficult to get paper books; some links to publishers already broken, e.g. biochem. - correct publisher link; little in biology, most detailed in chemistry
• course-based research experience from U Texas - just a list of research courses without detailed content
• TopHat an internet-based feedback software requires a paid student subscription, dramatically reducing voluntary participation; free academic alternatives exist, e.g. ARSnova
• CPR, a student writing and peer reviewing tool, costs several thousand $ to license; the very first version CPR1 is freely usable as an online tool
• The National Academies Summer Institutes link is already broken

Least useful

On 2016 Feb 21, Joe Newton commented:

This revealing study, in the absence of established biological markers, and gene sequencing for epistasis, highlights the diagnostic dilemma of uncertainty of animal control systems. For example, "The diagnosis of schizophrenia was made with great rigor by obtaining a consensus between clinical diagnosis of an expert clinician and a research diagnosis generated by MINI-Plus interview. Moreover, diagnostic stability over 1–3 years was examined by reviewing the follow-up notes; ten subjects were excluded from the final sample following this exercise." In addition, the cohort sample size (45 each) is likely too small.

Statistical method, although following established procedures, is also uncertain for a many degree-of-freedom simultaneous non-linear system. For example, the null HYPOTHESIS, based on smooth Gaussian curves, applied to such a system, is questionable.

Congratulations to the authors. Joe Ray Newton

On 2015 Aug 24, Martine Crasnier-Mednansky commented:

Relevant published data were not taken into consideration by the authors, as indicated below.

In 2007, Vibrio fischeri was reclassified as Aliivibrio fischeri Urbanczyk H, 2007.

Transcription of the cpdA gene, encoding the cellular cAMP phosphodiesterase CpdA, is positively regulated by the CRP-cAMP complex, as reported for the closely related Vibrio vulnificus cpdA gene Kim HS, 2009. By itself, this finding infers homeostatic control of the cAMP cellular concentration by CpdA in A. fischeri. This is reminiscent of Pseudomonas aeruginosa CpdA whose gene transcription is also under cAMP positive control Fuchs EL, 2010.

As regards the periplasmic cAMP phosphodiesterase CpdP, Dunlap PV, 1993 reported its specific activity was 'exceptionally' high, and allowed growth on cAMP as the sole source of carbon and energy. Therefore the role of CpdP in altering the periplasmic cAMP concentration in A. fischeri is important and, as shown by data in figure 4A, likely regulates indirectly the cytoplasmic cAMP concentration by controlling excretion of cAMP from the cell.

On 2016 May 25, Jonathan Rakar commented:

This paper is part of two dissertations (2016), here: http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-125925 and here: http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-123313

On 2016 Jan 24, Venkata Kodali commented:

Should be read as VRR Kodali

On 2015 Jun 25, David Keller commented:

Does Deep Brain Stimulation slow the progression of neurodegeneration in early Parkinson disease?

The authors report that "Subjects receiving an earlier STN-DBS showed a sustained improvement in the activities of daily living and motor complications, never reaching the severe levels of disability reported by Late-Stimulated patients at the time of surgical selection." Did this sustained level of symptom improvement require ongoing increases in DBS stimulation parameters (such as total daily stimulation current)? The need for increasing DBS stimulation to maintain a constant level of symptom improvement implies continued progression of the patient's underlying neurodegeneration. On the other hand, if sustained symptom improvement was achieved while requiring constant or decreasing amounts of electrical brain stimulation, this might reflect slowing or reversal of the neurodegenerative process.

It would be informative to assess the neurological status of the subjects who received early DBS, with their stimulators turned off, and track these UPDRS scores over time. If the rate of worsening of their stimulator-off UPDRS scores is slower than the rate of worsening for comparable patients who have not received DBS, that would imply possible disease-altering effects for DBS in early PD. Was the crucial stimulator-off UPDRS data collected in this study, and, if so, what do they tell us about how DBS affects the rate of neurodegeneration?

On 2015 Jul 10, Miguel Lopez-Lazaro commented:

The authors show a pharmacological strategy that might improve the therapy of a variety of cancer types. However, they tested the anticancer activity of their drug combinations using a preclinical validation approach that, in my opinion, does not reliably predict drug efficacy in cancer patients. Their approach was based on evaluating cytotoxic potency against cancer cells and tumor regression in mice. Although this experimental approach is used by many researchers (including myself in the recent past), it may be inadequate to detect the type of drugs that cancer patients need. I recently proposed an alternative way to assess preclinical anticancer activity: the new drug or drug combination should improve the selectivity (in vitro) and survival rate (in vivo) of the standard treatments used in cancer patients (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381701/pdf/oncoscience-02-0091.pdf). This patient-oriented approach may help reveal whether or not a combination of kinase and BET inhibitors can improve the efficacy of the existing therapies. I wrote the following letter for Cancer Cell, but the Editors did not find it adequate for their journal. Perhaps someone finds it useful.

Combination of Kinase and BET Inhibitors to Overcome Resistance to Therapy: Preclinical Validation Required

Miguel López-Lázaro Department of Pharmacology, Faculty of Pharmacy, University of Seville, Spain

Kinase inhibitors are the standard of care for a variety of cancer types. Patients treated with kinase inhibitors often develop resistance to therapy, which has been associated with upregulation of tyrosine kinase receptors (RTKs) and subsequent activation of the PI3K pathway. In an article recently published in Cancer Cell, Stratikopoulos et al. showed that BRD4 is important for the feedback activation of several RTKs, and that combined PI3K and BET inhibition blocks PI3K reactivation in a variety of cancer cells. Then, the authors carried out experiments to validate this combination in preclinical in vitro and in vivo models. They observed in several cancer cell types that the cytotoxicity of the combination of a PI3K inhibitor and a BET inhibitor at a fixed concentration was higher than that observed when the inhibitors were tested alone at the same concentrations. They also found a better antitumor activity when mice with breast tumors were treated with kinase and BET inhibitors together than when they were treated with each inhibitor alone at the same doses. The authors discussed that these findings provide the rationale for combining kinase and BET inhibitors to improve therapy in several human cancers (Stratikopoulos et al., 2015).

Patients with unresectable or metastatic cancers are difficult to cure and require the development of better pharmacological therapies. The first step to developing better therapies is to find good drug candidates or pharmacological strategies for testing. Stratikopoulos et al. find that combined PI3K and BET inhibition sustains PI3K inhibition in a variety of cancer cells, thereby providing the rationale for testing the anticancer potential of this combination. The next step to developing better treatments is to evaluate the new drugs using robust preclinical models; these models should predict whether the anticancer potential of the new drugs is high enough to deserve clinical evaluation. The experimental approach used by Stratikopoulos et al. does not reveal, however, whether or not the combination of PI3K and BET inhibitors deserves clinical testing. The main reason is that their approach was based on evaluating cytotoxic potency in cancer cells and tumor shrinkage in animal models, and these parameters are poor predictors of clinical efficacy. Despite their widespread use, these parameters do not reliably detect the type of drugs that cancer patients need.

Cancer patients do not need drugs or drug combinations that target their cancer cells at low concentrations if they also target their normal cells at similar concentrations. Cancer patients will probably not benefit from drugs that reduce tumor volumes in mice if they do not extend their lives. Cancer patients need drugs that improve the efficacy of the existing treatments. Establishing the best parameters to measure efficacy in vitro and in vivo is essential, and evidence suggests that selectivity (in vitro) and survival rate (in vivo) are the most reliable parameters to predict drug efficacy in cancer patients (Lopez-Lazaro, 2015c). In vitro, the new treatment should improve the selectivity of the existing drugs when tested in cancer cells versus nonmalignant cells from a variety of healthy tissues (Lopez-Lazaro, 2015a; Lopez-Lazaro, 2015b; Lopez-Lazaro, 2015c). The efficacy of a drug combination should be assessed by testing in cancer cells versus nonmalignant cells if it improves the selectivity of the standard treatment, and not by testing in cancer cells if its cytotoxicity is enhanced in relation to the cytotoxicity induced by each drug alone. It is important to realize that a drug combination that induces a strong cytotoxic synergism in cancer cells will not be clinically effective if it induces a stronger synergism in nonmalignant cells, or that a drug combination that induces synergism in cancer cells and antagonism in nonmalignant cells will not be clinically useful if its selectivity towards cancer cells is lower than that of the standard treatment (Lopez-Lazaro, 2015c). In vivo, the new drug or drug combination should improve the survival rate of the existing therapy when tested under equivalent experimental conditions (e.g., equitoxic doses) in animal models representative of the patients who would eventually receive the new treatment (Lopez-Lazaro, 2015c). Unfortunately, many drugs and drug combinations are tested in cancer patients after showing cytotoxic potency against cancer cells and tumor regression in mice. This preclinical validation approach may contribute to explain why our ability to translate preclinical cancer research to clinical success is remarkably low (Hutchinson and Kirk, 2011). A preclinical validation model based on assessing whether the drug candidates improve the selectivity and survival rate of the standard therapies would facilitate the development of better treatments, and would also prevent many cancer patients from receiving ineffective drugs (Lopez-Lazaro, 2015c).

In conclusion, Stratikopoulos et al. have discovered a pharmacological strategy that might improve the therapy of a variety of cancer types. However, they assessed the anticancer potential of their strategy using a preclinical validation approach that does not reliably predict drug efficacy in cancer patients. Testing if BET inhibitors improve the selectivity and survival rate of the kinase inhibitors used in oncology would help reveal whether or not kinase and BET inhibitors together can overcome resistance to therapy.

REFERENCES

Hutchinson, L. and Kirk, R. (2011). High drug attrition rates--where are we going wrong? Nat. Rev. Clin. Oncol. 8, 189-190.

Lopez-Lazaro, M. (2015a). A Simple and Reliable Approach for Assessing Anticancer Activity In Vitro. Curr. Med. Chem. 22, 1324-1334.

Lopez-Lazaro, M. (2015b). How many times should we screen a chemical library to discover an anticancer drug? Drug Discov. Today. 20, 167-169.

Lopez-Lazaro, M. (2015c). Two preclinical tests to evaluate anticancer activity and to help validate drug candidates for clinical trials. Oncoscience 2, 91-98.

Stratikopoulos, E.E., Dendy, M., Szabolcs, M., Khaykin, A.J., Lefebvre, C., Zhou, M.M., and Parsons, R. (2015). Kinase and BET Inhibitors Together Clamp Inhibition of PI3K Signaling and Overcome Resistance to Therapy. Cancer Cell. 27, 837-851.

On 2015 Jul 03, Martine Crasnier-Mednansky commented:

Gene identification number VC1822 and VCA1045 relate to PTS proteins with A, B, and C domain (not just an A domain as inferred from the text). In accordance with the 'Proposed Uniform Nomenclature' for the PTS proteins Saier MH Jr, 1992, gene VC1822 encodes an Enzyme IIACB^N/D and VCA1045 an Enzyme IICBA^Mtl . It would be a great benefit for PTS researchers if the nomenclature was used beyond for example Escherichia coli.

Based on figure 6A, the authors report the CRP-cAMP complex negatively regulates the expression of tcpA in the absence of the PTS. However, because figure 6A shows there is no significant difference in expression between the cya and the cya ptsH mutant strain or the crp and the crp ptsI mutant strain, and no significant difference in expression between the wild type and the crp or cya mutant strain, it could be concluded from figure 6A that the CRP-cAMP complex does not play a major role in the transcription of tcpA in the TCP-producing AKI medium used by the authors. This conclusion is not supported by previous data indicating CRP-cAMP indirectly regulates negatively the expression of tcpA by inhibiting the transcription of tcpPH (Kovacikova G, 2001). However the cAMP level varies in response to changes in carbon and energy sources. Thus, if the level of cAMP is relatively low in the AKI medium as compared to LB medium, production of TcpA occurs. This provides an explanation for the present observation indicating deletion of crp does not significantly affect production of TcpA (figure 6A). It also provides an explanation why incorporation of a crp mutation in the El Tor strain C6706 allows production of TCP in LB medium (Skorupski K, 1997).

Figure 6B does not indicate that "…in the EI or Hpr mutants, intracellular cAMP concentrations were significantly higher than that in wt cells" - as stated. In fact, intracellular cAMP concentrations are significantly higher in the cpdA mutant strains, possibly indicating a role for cAMP phosphodiesterase in regulating the cAMP levels.

On 2015 Jun 26, Donald Forsdyke commented:

The review covers a field that has occupied geneticist Jianzhi Zhang and colleagues for many years. Their publications are in journals that have usually not permitted direct commenting. The present new PubMed facility allows the release of past comments on their work that previously had only limited circulation (see Yang JR, 2012, Lin F, 2012, Park C, 2013). There are also comments on a paper in PLOS Biology that are accessible at http://www.plosbiology.org/annotation/listThread.action?root=81241.

The review considers the scope of negative selection, which usually associates with low evolutionary rates and, following Hurst and Smith (1999), only briefly alludes to positive selection, which usually associates with high evolutionary rates. In so doing, the review seems to exclude evidence from studies of positive selection that might reflect on its thesis (see comment on Park C, 2013). Indeed, biochemists have long known that some proteins that are deemed “important” for their host organism evolve slowly under negative selection. Other “important” proteins evolve rapidly under positive selection. Within this broad negative-to-positive range are scattered many other “important” or “essential” or “non-dispensable” proteins. Thus, the review correctly concludes that “the functional importance of a protein only has a weak impact on its evolutionary rate,” and “the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance.”

However, the latter statement can be interpreted as implying that, above a certain minimum, expression level and function are not connected. This misinterpretation could be compounded by (i) the narrow range of papers considered the “foundations in the field,” and (ii) frequent allusions to the functional importance (note singular) of a protein, and (iii) focusing too closely on recently acquired genomic datasets (important as they are).

There is extensive literature showing that collective functions of proteins, which are dependent on expression level, can underlie biological phenomena (e.g. the Donnan equilibrium; Donnan FG, 1927). Thus, a protein can have both specific (e.g. enzymic) and general functions (1). The discovery of X chromosome dosage compensation (reviewed by Muller in 1948; see http://post.queensu.ca/~forsdyke/xchromos.htm) gave an early indication of the importance of the general role.

Since many proteins contribute to collective functions, the loss of an individual protein type is more likely to affect its specific function than its contribution to collective functions. Depending on the collective function, some proteins have properties (e.g. size) that would better support that function than other proteins. Thus, there can be degrees of specificity.

And long ago (McConkey EH, 1982) attention was drawn to the importance of functional constraints due to “quinary” interactions between proteins in the crowded intracellular environment. This referred to “macromolecular interactions that are transient in vivo” which should “constitute an important source of constraints on changes in primary structure” (see also Monteith WB, 2015). The E-R anticorrelation, and selection to avoid protein misinteractions, are further considered in a recent review. A cell mutation that may be deemed as imposing a “gain-in-toxicity,” may function to alert an organism’s cytotoxic T cells that the mutant cell should be destroyed before becoming cancerous. We neglect the immunological concomitants of mutation at our peril (Forsdyke DR, 2015).

(1)Forsdyke DR (2012) Functional constraint and molecular evolution. In: Encyclopedia of Life Sciences. Chichester: John Wiley.

On 2015 Jul 21, Weijun Liu commented:

None

On 2015 Jul 16, Weijun Liu commented:

Same flow cytometry figures for cell cycle analysis in Figure 3C and Figure 5A but with different experiment conditions.

On 2015 Oct 21, Gerard Bruder commented:

Jesulola et al. (2015) reviewed the literature of EEG alpha asymmetry in relation to behavioral withdrawal in depression. One of the confusing issues in this literature is whether one is referring to hemispheric asymmetry of EEG alpha (α) or brain activity, which is thought to be inversely related to alpha. To their credit, the authors attempted to deal with this by using separate abbreviations for the direction of alpha asymmetry in depression (Lα > Rα) and asymmetry of brain activity (Ra > La). The problem is that these were not consistently used in describing findings in literature. Specifically, on page 60 (3.2.1.4) in referring to the findings of Lopez-Duran et al. (2012), they state that “greater relative left frontal activation (i.e., the opposite of the La>Ra finding for depressed individuals)” but the typical finding for depression is greater right than left frontal activity (Ra>La). Immediately below, in referring to the findings of Bruder et al. (2007), they state “that children whose parents and grandparents were depressed demonstrated greater Lα > Rα EEG asymmetry in the parietal…”, but it should be La>Ra, meaning that activity was less over right than left parietal sites. In section 3.2.2.1 on this page, they state “other studies have examined EEG asymmetry in participants with comorbid anxiety and depression”…and “In general, depressed patients who also had an anxiety disorder had the opposite direction EEG asymmetry (i.e., La>Ra) than depressed patients who did not have comorbid anxiety, although most significant findings have been reported in the parietal sites.” The Bruder et al. (1997) study referred to, which measured EEG alpha in depressed patients with versus without a comorbid anxiety disorder, found greater activity over right than left parietal sites in those with a comorbid anxiety disorder and so it should read “Ra>La”. The other EEG studies referred to were in non-clinical samples or did not include resting alpha measures. They also state, “This contrary finding is unpredicted as it might be expected that co-morbid anxiety and depression might combine to give a much larger Ra>La frontal asymmetry...” The Bruder et al. (1997) study actually found the co-morbid group had Ra>La at frontal sites, whereas depressed patients without an anxiety disorder did not, and so the having both an anxiety and depressive disorder was associated with Ra>La. It was only at parietal sites where the alpha asymmetry in the co-morbid group (Ra>La) was opposite to depressed patients without an anxiety disorder (La>Ra). The favoring of right over left parietal activity in the co-morbid patients could reflect hyperactivation of right parietal regions due to anxious arousal (Heller et al., 1997), hypoactiation of left hemisphere regions or some combination of both (Bruder et al. 1999).

The authors also discuss several key research issues that require addressing for this field to move forward. The issue of where EEG asymmetry occurs in depression is important and can be dealt with by including frontal and more posterior electrode sites. However, this review does not mention more recent methodological developments, in particular application of current source density (CSD), that can refine the quantification and spatial resolution of EEG measures, improving neuroanatomical and spectral resolution (Tenke et al., 2005; Tenke & Kayser, 2015; Tenke et al., 2015; Kayser & Tenke, 2015). The use of CSD measures also deals with the EEG “reference” problem, which no doubt contributed to differences in alpha asymmetry findings across studies, and can lead to greater consistency of EEG findings between studies using “resting” and task-based EEG measures (Stewart et al., 2014). References Bruder, G.E., Fong, R., Tenke, C.E., Leite, P., Towey, J.P., Stewart, J.E., McGrath, P.J., Quitkin, F.M (1997). Regional brain asymmetries in major depression with or without an anxiety disorder: a quantitative electroencephalographic study. Biological Psychiatry, 41(9), 939-948. Bruder, G.E., Tenke, C.E., Warner, V., Weissman, M.M. (2007). Grandchildren at high and low risk for depression differ in EEG measures of regional brain asymmetry. Biological Psychiatry, 62(11), 1317-1323. Bruder, G.E., Wexler, B.E., Stewart, J.W., Price, L.H. (1999). Perceptual asymmetry differences between major depression with or without a comorbid anxiety disorder: A dichotic listening study. Journal of Abnormal Psychology, 108 (2), 233-239. Heller, W., Nitschke, J.B., Etienne, M.A., Miller, B.A. (1997). Patterns of regional brain activity differentiate types of anxiety. Journal of abnormal Psychology, 106, 376-385. Kayser, J., Tenke, C.E. (2015). Issues and considerations for using the scalp surface Laplacian in EEG/ERP research: A tutorial review. International Journal of Psychophysiology, 97(3), 189-209. Lopez-Duran, N.I., Nusslock, R., George, C., Kovacs, M. (2012). Frontal EEG asymmetry moderates the effects of stressful life events on internalizing symptoms in children at familial risk for depression. Psychophysiology, 49 (4), 510-521. Tenke, C.E., Kayser, J. (2015). Surface Laplacians (SL) and phase properties of EEG rhythms: simulated generators in a volume-conduction model. International Journal of Psychophysiology, 97(3), 285-298. Tenke, C.E., Kayser, J., Abraham, K., Alvarenga, J.E., Bruder, G.E. (2015). Posterior EEG alpha at rest and during task performance: comparison of current source density and field potential measures. International Journal of Psychophysiology, 97(3), 299-309. Stewart, J.L., Coan, J.A., Towers, D.N., Allen, J.J.B. (2014). Resting and task-related prefrontal EEG asymmetry in depression: Support for the capability model. Psychophysiology 51, 446-455.

On 2015 Jun 12, Pavel Baranov commented:

Would it be useful to refer to the signal obtained with this technique as heelprinting instead of footprinting?

On 2015 Jun 06, Christopher Southan commented:

This post resolves the featured ML probe numbers to PubChem CIDs and chemical structures http://cdsouthan.blogspot.se/2015/06/resolving-small-molecule-probes.html

On 2015 Nov 15, NephJC - Nephrology Journal Club commented:

This paper was discussed on Sep 8th in the open online nephrology journal club, #NephJC, on twitter. Introductory comments, prepared by Hector Madariaga, are available at the NephJC [website](www.nephjc.com/obesity-and-transplant/). This controversial topic was discussed in detailed with more than 50 participants, including the authors of the paper (Jennifer McCaughan and Chris Hill) as well as nephrologists, transplant specialists, transplant surgeons, fellows and residents. The highlights of the tweetchat were:

• The authors did a commendable job in compiling the data for this review together, trying to answer this vexing question about the suitability of kidney transplantation in the obese individual with kidney failure, and provided useful quantitative data on clinical outcomes.

• The participants acknowledged the difficulties of obese patients losing weight and accessing bariatric surgery pre-transplant. The additional specific concern, which could not be assessed in this systematic review, is that these patients may be challenging for kidney transplantation surgery due to technical aspects (length of stay, wound complications, infections). Balancing equity and utility are crucial from a societal perspective.

• The consensus was that BMI is an imperfect criterion for kidney transplantation and that each case should be assessed individually. There was still a transatlantic difference, with the European discussants still being wary above a BMI of 40 kg/m2. A transcript and a curated (i.e. Storified) version of the tweetchat are available from the [NephJC website](www.nephjc.com/obesity-and-transplant/). Interested individuals can track and join in the conversation by following @NephJC or #NephJC, or visit the webpage at [NephJC.com](www.nephjc.com).

On 2015 Dec 11, Mark Johnston commented:

An interactive protocol from this article is freely available at protocols.io.

On 2015 Jun 19, Christopher Southan commented:

Please provide (figshare?) a full list of the 647 candidate genes and the 1,497 for the seven complex diseases from PMID 25077696 so that others can extend these results. The journal should have mandated this.

On 2015 Jul 13, CREBP Journal Club commented:

The authors concluded that the “use of written information leaflets in general practice consultations are effective in reducing antibiotic prescribing, and actual antibiotic use by patients and their intention to reconsult for future episodes of illness. It is unclear whether information leaflets also actually affect reconsultation rates.”

The group generally agreed that this was a well conducted review and liked the arrangement of the data in Table 3. We discussed a number of areas:

In this review, two reviewers screened 200 titles and abstracts (with no disagreements) and a single author screened the rest with double checking by a second. We noted that individual reviewers miss around 3% of articles; thus, this approach may have led to a number of missed articles (Doust et al 2005).

The authors stated they would exclude multifaceted interventions but included a study by Francis et al. 2009. It included both information and training in delivering of the intervention (and some members of the journal club believed it could be classed as a shared decision-making intervention). However, the authors did acknowledge this training and stated that it may have affected the results of this study.

The authors assessed the risk of bias as high for blinding of participants and personnel for each study. GRADE processes suggest that the risk of bias for blinding should be assessed for each outcome (rather than study) so that the objectivity or subjectivity of the specific outcome measure can be taken into account when deciding on whether bias was likely.

Overall, we believed this was a well conducted review. Clinicians in the journal club had a lively debate about whether they would use information for their patients with common infections. We concluded that it was one tool that you could have in your toolkit to help minimise antibiotic use and the risk of antibiotic resistance.

References:

Doust et al. Identifying studies for systematic reviews of diagnostic tests was difficult due to the poor sensitivity and precision of methodologic filters and the lack of information in the abstract. Journal of Clinical Epidemiology 2005; 58 (5): 444-449

Francis et al. Effect of using an interactive booklet about childhood respiratory tract infections in primary care consultations on reconsulting and antibiotic prescribing; a cluster randomised controlled trial. BMJ 2009; 339:b2885

O’Sullivan et al. Written information for patients on the use of antibiotics in acute upper respiratory infections in primary care. Cochrane Database of Systematic Reviews 2014; Issue 10: CD011360.

Full journal club report: CREBP Journal Club

On 2016 Jul 24, JAVIER MATA commented:

Sorry not to have written before. We know that selection of an ES of 0.75 in the study establishes a stricter requirement to prove the efficacy of acupuncture than is used in the evaluation of NSAIDs. We used a higher effect size in order to improve the statistical power. A study without statistical significance does not necessarily mean that a relevant association does not exist between the factor under the study and the response. The statistical power of a study is determined by: the variability of the variables within the study, the size of the sample, the level of statistical significance and the effect size. The size of the effect on pain compared to sham acupuncture is 0.4 which is considered ‘moderate’ but the 95% CI around this estimate are wide (0.1, 0.6), this effect size was calculated with manual and electroacupuncture (EA). Use of EA was shown to be superior to manual stimulation alone for knee pain in 2010, but there is not effect size calculation with EA alone and from our experience it is higher.

On 2015 Jun 15, Arthur Yin Fan commented:

Why use Effect size 0.75? This is much higher than NIH recommended 0.50 for testing pain medication and higher than hinman's acupuncture trial for chronic knee pain (ES= 0.60).

On 2015 Jun 15, Prashant Sharma, MD, DM commented:

This letter to the Editor is available free full text at http://archive.nmji.in/archives/Volume-27/Issue-5/CORRESPONDENCE.pdf

• Author

On 2015 Jun 23, Nazzarena Labo commented:

Video abstract: https://vimeo.com/126172348

On 2017 Jul 10, Randi Pechacek commented:

Mitja Remus-Emsermann, one of the authors of this paper, wrote a blog post on microBEnet to reveal the "story behind the paper."

On 2017 Mar 14, Andrea Messori commented:

Promoting the use of Markov simulation models to study outcomes of total hip arthroplasty

Andrea Messori

HTA Section, ESTAR Toscana, Regional Health Service, Firenze, Italy

Correspondence to: Dr. Andrea Messori, PharmD, HTA Unit, ESTAR Toscana, Regional Health Service, Via San Salvi 12, 50100 Firenze, Italy. andrea.messori.it@gmail.com Fax: +39-05-74701319

In patients receiving total hip arthroplasty (THA), simulation studies employing Markov models are increasingly being used [1-4]. The aim of these studies is to determine the “typical” clinical outcomes expected on the long term and to generate estimates of cost/effectiveness. If we consider these modelling tools, most of the simulation software published thus far shares the following characteristics:

a) Health states. The model implements, with minimal variations across different models, the health-states shown in Figure 1 along with the corresponding transitions from one health state to another. The probabilities of individual transitions are shown in Figure 1; these probabilities can be adjusted depending on the specific intervention under examination;

b) Clinical outcomes after THA. The following outcomes can occur after the first surgery: i) successful outcome; ii) complications; iii) death; the same outcomes can occur also after a repeat surgery for arthroplasty revision.

c) Life expectancy. The life-expectancy attributed to the simulated patients is determined by considering: a) the age-related and gender-related mortality of a healthy population [5]; b) the mortality attributable to arthroplasty surgery. These two factors are separately managed in different sections of the Markov model (see Figure 1).

d) Utilities and estimation of QALYs. Utility of patients is assumed to be around 0.72 [6] after surgery. Over the pre-specified time horizon (e.g. 20 years), QALYs are computed on the basis of the health states of the model, their utilities, and the corresponding transition probabilities.

e) Discounting. The annual discount rate (e.g. 3.5%) is incorporated in the calculation of QALYs according to standard discounting techniques [6].

As regards the practical use of these computer programs, the simulation models published in the past years are essentially based on two software tools: on the one hand, some researchers have used a general-purpose spreadsheet (namely: Excel by Microsoft) to develop these Markovian programs; on the other, other researchers [3] have used a specific, commercial program (in most cases: TreeagePro by Treeage Software Inc., Williamstown, Massachusetts, USA). The Markovian subroutines written under Excel, as well as the tools developed under Treeage, share a negative characteristic because they are not freely available. Even NICE does not provide these tools when a Technology Appraisal is released. The unavailability of these programming tools is a serious hurdle that limits the scientific advancement of cost-effectiveness research on THA. Hence, in the present report we have tried to facilitate the application of Markov models in the setting of THA by developing a simulation software which is an improved version of the tools previously employed for specific research projects [3]. Our simulation model, that can be downloaded from the following link http://www.osservatorioinnovazione.net/papers/total_hip_arthroplasty.trex, is designed to be run under TreeagePro version 2011 (or subsequent versions). The input variables for the model are shown in the legend to Figure 1. The output of the program is represented by the estimate of total QALYs per patient accrued over the pre-specified time horizon. The software manages only the clinical part of these simulations; however, cost data can be added quite easily by introducing new sections of programming.

References

[1] Losina E, Walensky RP, Kessler CL, Emrani PS, Reichmann WM, Wright[ EA, Holt HL, Solomon DH, Yelin E, Paltiel AD, Katz JN. Cost-effectiveness of total knee arthroplasty in the United States: patient risk and hospital volume. Arch Intern Med. 2009 Jun 22;169(12):1113-21.

[2] Bedair H, Cha TD, Hansen VJ. Economic benefit to society at large of total knee arthroplasty in younger patients: a Markov analysis. J Bone Joint Surg Am. 2014 Jan 15;96(2):119-26.

[3] Pennington MW, Grieve R, van der Meulen JH. Lifetime cost effectiveness of different brands of prosthesis used for total hip arthroplasty: a study using the NJR dataset. Bone Joint J. 2015 Jun;97-B(6):762-70.

[4] Mari K, Dégieux P, Mistretta F, Guillemin F, Richette P. Cost utility modeling of early vs late total knee replacement in osteoarthritis patients. Osteoarthritis Cartilage. 2016 Dec;24(12):2069-2076.

[5] ISTAT. Tavole di mortalità della popolazione italiana—Ripartizione: Italia—Maschi—Anno: 2005, Report of 2010. http://demo.istat.it/unitav2012/index.html?lingua=ita (last accessed 7 May 2014).

[6] Mason J, Drummond M, Torrance G. Some guidelines on the use of cost effectiveness league tables. BMJ. 1993 Feb 27;306(6877):570-2.

[7] Jørgensen CC, Kehlet H; Lundbeck Foundation Centre for Fast-track Hip and Knee Replacement Collaborative group.. Time course and reasons for 90-day mortality in fast-track hip and knee arthroplasty. Acta Anaesthesiol Scand. 2017 Apr;61(4):436-444.

Figure 1. States of the Markov model and transition probabilities.

The starting point of the simulation model is a Markov node (circled M) from which six branches originate. The explanation for these six branches is the following: 1) surgery for THA; 2) follow-up after first surgery (and also the occurrence of revision surgery): 3) follow-up after revision surgery; 4) follow-up after first surgery with complications; 5) follow-up after revision surgery with complications: 6) death. Second-level branches regard events defined according to the accompanying labels. The symbols adopted in this scheme reflect the syntax required by the Treeage software: Ο, probabilistic node;◄, terminal node.

Abbreviations: RWD, reward (which in this model represents the incremental increase in quality- adjusted survival).

The graph of Figure 1 can be downloaded from the following link: http://www.osservatorioinnovazione.net/tenders/tha.gif

On 2015 Jun 30, Frank Twisk commented:

Commentary on Jason et al. (2015):

Towards separate empirical Case Definitions of Myalgic Encephalomyelitis and chronic fatigue syndrome.

Health Psychol Behav Med. 2015; 3(1): 109-110. doi: 10.1080/21642850.2015.1027705.

Twisk FNM.

http://www.tandfonline.com/doi/pdf/10.1080/21642850.2015.1027705

On 2015 Jun 05, Weiwei Chen commented:

Weiwei Chen and Xiaonan Yang contributed equally to this work. Xiaonan Yang is an equal contributor.

On 2015 Sep 30, Peter Gøtzsche commented:

Augmentation therapy with alpha-1 antitrypsin cannot be recommended

Chapman et al. published the third trial of α1 proteinase inhibitor (A1PI) augmentation treatment for α1 antitrypsin deficiency.1 The new trial adds very little, if anything, to the results of the two previous trials, which we have summarised in our Cochrane review.2 In the Cochrane review, the decline in lung density measured by CT scan was slightly less on drug than on placebo, difference 1.14 g/l (95% confidence interval 0.14 to 2.14). This minute effect is about the same when the new trial is added to the analysis, difference 0.86 (0.31 to 1.42). The effect of drug on FEV1 was the opposite as that on lung density. In the Cochrane review, FEV1 declined slightly faster on drug than on placebo, difference 20 ml per year (-41 to 1; p = 0.06). Adding the new trial doesn’t change this estimate much, standardised mean difference -0.19 (-0.42 to 0.05). In the accompanying editorial,3 Crystal writes that there is now “evidence of clinical efficacy” and suggests that augmentation treatment should be used earlier than at present, despite the fact that the treatment costs far more than most people earn in a year before taxes. An effect on a surrogate marker is not “evidence of clinical efficacy” and we therefore don’t reimburse or use this treatment in Denmark. We conclude in our Cochrane review: “Augmentation therapy with alpha-1 antitrypsin cannot be recommended, in view of the lack of evidence of clinical benefit and the cost of treatment.”2

1 Chapman KR, Burdon JG, Piitulainen E, et al. ntravenous augmentation treatment and lung density in severe α1 antitrypsin deficiency (RAPID): a randomised, double-blind, placebo-controlled trial. Lancet 2015;386:360-8.

2 Gøtzsche PC, Johansen HK. Intravenous alpha-1 antitrypsin augmentation therapy for treating patients with alpha-1 antitrypsin deficiency and lung disease. Cochrane Database Syst Rev 2010;(7):CD007851.

3 Crystal RG. Augmentation treatment for α1 antitrypsin defi ciency. Lancet 2015;386:319-20.

On 2015 Dec 22, Allison Bischoff commented:

It recently came to our attention that an incorrect grant number for the NICHD was cited on this paper. The correct funding information is as follows:

Grant title: “Tracking neurodegeneration in early Wolfram Syndrome” Hershey, PI NICHD HD070855

On 2016 May 26, Lydia Maniatis commented:

Short version: Bottom-up, feed-forward, “feature detection” models that describe perception as a summation process and neurons as “detectors” are inconsistent with logic and the facts, and poking around in the brain will not make them any more credible.

On 2016 May 26, Lydia Maniatis commented:

Part 2: An understanding of the role of “higher levels” of processing to CREATE and to ADD structural interpretations to the raw point information registered in “lower levels” - an understanding that led Kanizsa to coin the phrase “going beyond the information given,” is obviously absent when the authors say things like: “These anatomical observations indicate that the visual system is progressively REDUCING [my caps] the amount of information encoded at higher levels of the form vision hierarchy.” If anatomical observations are supposedly contradicting perceptual facts, then there's obviously something wrong with the theory/methods that have led to these conclusions.

The authors themselves are at least aware that there is a problem with their description of the visual process as feedforward: “One major element missing from this ventral pathway scenario is any role for the ubiquitous feedback connections among areas (VanEssen et al., 1992).” This statement speaks for itself; a story has been created that ignores a major, relevant fact. If this fact couldn't be accommodated, then the story was woefully premature. The brief, vague, unintelligible speculation that follows this admission is not worth discussing.

The authors are also aware of the ad hoc nature of their conclusions. They hope this will not be a problem for relevance, concluding their paper by saying: “We believe that the observations and models for curved shapes presented here will inform and illuminate these future studies [involving angles and curves].” The authors belief-system aside, the probability that uninformed, ad hoc accounts will end up being corroborated or even useful is really very small. The authors are claiming that they have discovered correlations between shape concepts – curvature – and V4 activity. However, it is a sure thing that, given a particular pattern of activity in any particular V4 neuron, the authors would be in no position to infer anything at all about the stimulus - not even what it isn't. No experiments are needed to make this assertion.

The term “dimensionality” is thrown around a lot but its meaning and relationship to principles of perception is not at all clear; it obviously involves many unstated and likely unviable assumptions.

On 2016 May 26, Lydia Maniatis commented:

Part 1: This article is nonsense, a quality that unfortunately doesn't differentiate it from much (likely most) of the vision science literature today. It's particularly disconcerting that the authors are employing animals, and in particular, monkeys.

The authors are making a fundamental error in describing the visual process (see below). The error predates the period of German research known as Gestalt psychology. The latter generated cogent logical arguments and empirical evidence against this erroneous, myopic description. The modern proponents of this view are perpetually spinning their wheels, using arbitrary experimental situations to generate inconclusive data fed into ad hoc “models” built on arbitrary assumptions, perpetually adjusting these models, and treating failures and contradictions as temporary bumps in the road. This literature has become an impenetrable swamp of detail unguided by careful observations and lacking organizing concepts.

Since it involves correlations, there is obviously no point in trying to analyze perception at a neural level unless we have correctly described what it is that we are trying to correlate neural activity with. The authors are naive to the basic challenges that face the visual system as are their descriptions of “stimuli.” They describe the “stimulus” as a face, a shoe, a chair,”containing curves, straight lines, etc. Particular neurons or neural populations are supposed to be particularly sensitive to – i.e to “detect” particular features thus naively described. We're told, for example, that“Many V4 neurons” are “selectively sensitive to curvature extrema” located at “a particular position relative to the center of a closed, curved object.” Leaving aside that “a particular location relative to the center a closed, curve object” conveys no information whatsoever, there are serious theoretical and practical problems with this discussion.

First, it is a serious mistake to treat perception as a detection and information-reduction problem. The authors frame the problem as: here is a “face;” how does the visual system “detect” it, and how much of the information contained in the “face” is thrown away?

The problem is that the stimulus with which the visual system is confronted, and must use to create a percept, is never a “face,” a “shoe,” a “chair.” It is a disconnected set of points, which, moreover, are constantly changing location on the constantly-moving retinal surface. Being a sum of disconnected points (the stimulating photons), the stimulus does not intrinsically contain any type of shape or shape feature, such as lines or curves, and it certainly doesn't contain closed, curved objects. If it did, then the problem of computer vision would not be so difficult – we would just tell the computer to pick out – to match - the relevant shapes features. The problem is the computer hasn't been made that can infer/construct the absent forms in the first place. That's why reading the numbers or letters in captchas, a trivial problem for us, is so difficult for robots.

Any shapes eventually perceived are the result of what are, in effect, inferential processes. Whether and the degree to which the percept will resemble the physical object from which the photon pattern originated (which, again, the authors naively equate with the stimulus) depends on many factors. The percept might resemble it, and it might not. If an actual face, for example, is painted the same color as its background and everything is equally illuminated from all directions, then we will see a single homogeneous color instead of a face. Conversely, a flat surface may appear to possess 3D physical structure.

These simple examples are enough to show the problem, which is that photons reflected from objects don't possess the properties of these objects, shape or otherwise. Obviously, variations in intensity and wavelength of the photons are required in order for there to be a chance that the percept will resemble, in experienced structure and properties, the physical object. But this is not enough. Even luminance boundaries can't be said to intrinsically possess shape properties; we need to solve the figure-ground problem first, and the illumination/reflectance problem. And as the Gestaltists showed conclusively, non-local points influence what forms will be seen locally, whether or not these forms correspond to actual intensity changes. How these forms are constructed is a non-trivial problem that must be solved before we can refer to “a curve” or “a face.” Wilson and Wilkinson don't see these problems because they describe stimuli in terms of what they see – in terms of the problems already solved by their respective visual systems. (Again, if there were a simple correlation between what they see and the photon information, even in a simple geometrical or mathematical sense, then computers would be better at seeing by now.)

Therefore, to say that a neuron in V4 simply “detects” a particular feature that lies within its particular receptive field, and feeds this information to higher levels for combination (a summary process) with information from other such neural detectors, flies in the face of perceptual facts (and exhaustive arguments, available in the Gestalt literature, of why these claims can't hold in principle). Even more so given that we know that the activity of visual neurons even below V4 - neurons which “detect,” for example, illusory contours which are not physically present - is determined on the basis of entire visual field (and on the principles by which this field is organized).

Furthermore, as Teller (1984) pointed out, the logic behind the assumption that a higher level of firing in a particular neuron indicates a special role in “encoding” the “feature” that is supposedly being responded to, is faulty. Perceptual outcomes depend on the relative activity of neurons, not on peak activity of individual neurons. For an example she refers to cone responses and their role in coding color perception. What matters are relative, not maximum, firing rates.

On 2015 Sep 15, NephJC - Nephrology Journal Club commented:

This study was discussed on Aug 11th and 12th in the open online nephrology journal club, #NephJC, on twitter. Introductory comments are available at the NephJC website .

The discussion was quite detailed, with more than 50 participants, including nephrologists, critical care physicians, fellows and residents with additional input from one of the authors, John Kellum. This activity was also supported by the editors and staff at the journal JAMA, which made the full text of the article available for free and provided prizes for best tweet comments.

A transcript and a curated (i.e. Storified) version of the tweetchat are available from the NephJC website.

The highlights of the tweetchat were:

• The authors were to commended for designing and conducting this trial, using an inexpensive and low-risk intervention, and choosing an ideal target population to test it on.

• The use of biomarkers as damage and alarm markers was quite elegant and thought provoking, but didn’t necessarily convince everyone. Biomarkers, and not just creatinine, do seem to be crucial in future acute kidney injury research.

• The consensus was that though this trial was quite promising, given other negative data (ERRIKA and RIPHEART) the overall body of evidence is not conclusive yet to drive change in practice. There were concerns about the time required and impact on busy/tight operating schedules, and there was a hope that a mortality powered trial would be done.

Interested individuals can track and join in the conversation by following @NephJC or #NephJC, or visit the webpage at NephJC.com.

On 2015 Sep 30, Lydia Maniatis commented:

This study has very serious problems in terms of both its purpose and its methods.

Purpose: The authors' guiding question is “how color constancy supports object selection,” an issue they claim is “not understood.” What, exactly, is not understood? If color constancy is achieved, if the percept is clear and the task unambiguous, then what do we hope to gain by having subjects choose a matching surface (out of a limited number of pre-selected options) rather thanfreely adjusting a patch, and then comparing it to a target in order to judge the correspondence between the two? Unless the means of gauging the subjects' perceptual experience is expected to affect the experience itself, then why not choose the most direct, precise, and easy-to-analyze means, rather than a cruder, ambiguous, difficult-to-analyze method deemed to be more “naturalistic”? (The authors themselves acknowledge the crudeness of their method – basically a forced-choice method - in relation to the free-choice “asymmetric matching” method).

Methods: 1) The authors' use the dimensions “naturalistic” vs “simple” as their independent variables, without explaining what they mean by the terms. The study purports to compare color comparison/selection in a “naturalistic” stimulus vs a “simple” stimulus. The problem with this may be appreciated if we describe the “naturalistic” stimulus, which was actually a series of “simulated naturalistic scenes:” “The target objects were embedded in a multifaceted cube suspended in midair in a room in which the illumination, coming from multiple light sources, varied spatially.” Each facet of the cube contained 49 differently-colored, semi-randomly assigned checks. The illumination on one side was blue or yellow, on the other “standard.” Targets were placed on top of these colored checkerboards. The “simple” stimuli were “flat patches embedded in a textured color background across which simulated illumination varied.” It is not clear why the backgrounds in the “simple” stimulus were not checkerboards matched to those of the “natural” stimuli, or, alternatively, a solid background. Even though the authors state, at one point, that they are controlling for “low-level” features of the stimuli, methodological choices are never clarified beyond the “simple/natural” dichotomy.

The authors themselves acknowledge the theoretical gap at the heart of this dichotomy: “A systematic characterization of how color constancy varies with the degree of stimulus naturalness is challenging, because a definition of naturalness remains elusive.” So, “Rather than attempting to define dimensions along which naturalness varies, we chose to study two configurations that we judged differed considerably in how natural they appeared.” Despite this vagueness in their chosen dimensions, they qualify the undefined value, “stimulus naturalness,” as a “key factor” in their results (at another point they refer to their stimulus as “nearly naturalistic” and the task as “fairly natural”). The problems here are beyond methodological, they are epistemological – failure to specify conditions in an experimental study means it does not meet the basic demands of the scientific method. Scientists go from nature to the lab in order to control and test potential causal variables; these authors are arguing that going "natural" is a merit - is somehow informative - even if they can't define what they mean by the term.

2) The authors conclude that “a reasonable degree of color constancy operates effectively in support of object selection.” (Again, why wouldn't it?). The assertion turns the scientific method on its head. This is because the surfaces and illumination were “simulated.” Thus, judgment of whether observers' choices are 'color-constant' or not hinged on assumptions about what the true reflectance of a patch should be judged to be, based on the “simulated” illumination: “To find the reflectance match for each target under a test illuminant, we [derived] a surface reflectance function...using a three-dimensional linear model for surface reflectance...derived from analysis of the spectra of Munsell papers, using the tabulated spectral data reported by Nickerson (1957)...”

Here's the problem: Let's say I take a black surface, place it on a larger grey surface, and brightly illuminate the smaller surface such that its boundary and the boundary of the illumination precisely coincide. Then, based on my knowledge of the illumination, I declare that a well-functioning lightness constancy mechanism should label the smaller surface “black.” Of course, my subjects would all fail this test. But it will not have been fair test, because observers are required to use the luminance structure of the stimulus to guess at both the reflectance and the illumination of the surfaces; they don't have my inside information about the illumination. If the stimulus itself doesn't support the inference of differential illumination, then the best inference is that illumination is uniform. Similarly, observers (their visual systems) of Radonjic et al's stimuli are called on to make guesses about both the reflectance and the illumination of target surfaces based on stimulus structure (geometric/chromatic). In order for the authors' presumed “correct” answers to constitute a valid test of observers' choices, they would have to provide a specific theory of why these answers constitute the best-guess based on stimulus structure. They would need to explicitly state their assumptions, rather than “cheating” by simply stripping away the “simulated illuminant.” And if they had made and shared any such theoretical assumptions, it would probably be more appropriate to consider observer choices a test of the theory, rather than making the predictions of the theory a test of observer choices. Because if we're studying perception, then the typical observer is never wrong.

On 2015 Nov 22, Lydia Maniatis commented:

Another issue that deserves consideration is the distinction between perceptual salience and "detection." The visual system "sees" the data but the percept is a selective description of this data. The so-called laws of perceptual organisation are an attempt to determine how the data are exploited in the attempt to construct a (for practical purposes) veridical representation of the world.

"Detection" is not the right paradigm for describing the contents of perception, any more than "discovery" is the right description of the scientific process (with respect to discovering (I can't help it) the laws of nature. The phenomena of subjective contours and amodal completions should, on the basis of a detection paradigm, be errors, failure to detect the absence of edges/surfaces.

Conversely, as the Gestaltists have amply shown, the mere presence of a contour in the stimulus does not mean it will be perceived. But this does not mean it was not "detected."

We can prove this on the basis that a change in a distant part of the stimulus may make a previously unseen "contour" salient. A very simple example is if we arrange four dots so that they mark the four corners of a square, then we will tend to perceive a square shape, i.e. we will mentally connect the dots to form a square. We will not tend to mentally connect the dots diagonally. But if we remove one of the "corners," then one of these diagonal connections will become salient, as we will typically perceive a triangle shape.

It's the same with science. Natural laws aren't discovered, they're created with the goal of making them match the facts as closely as possible. Just as with the example above, a chance discovery can cause an entire theoretical structure to rearrange.

On 2015 Nov 03, Lydia Maniatis commented:

As is the case with “Bayesians” in general, the authors here perform a sleight-of-hand: They make highly predictable predictions, to which they have attached some type of “Bayesian” rationale; the prediction is borne out; they claim (invalidly) that their “Bayesian framework” has been validated.

The authors' proposition is that increasing complexity of a "contour" (via increasing bending) decreases its detectability. We are repeatedly told that this is a well-known fact:

“A number of recent studies have demonstrated that...detection performance declines with larger turning angles” (p. 1)

“Of course...it is well-known that contour curvature decreases detectability...” (p. 5).

“Contour curvature is well known to influence human detection of contours” (p. 12).

Lo and behold, the authors' results show that “ subjects are sensitive to the complexity of the target contour's shape, as expected under the Bayesian framework and the assumed generative model. This finding gives strong prima facie support for the Bayesian approach...” (p. 8). Thus, our predictable prediction comes out as expected, and is treated as corroboration of our fundamentally post-hoc model. This train of thought is analogous to my claiming that invisible angels push the sun up every morning, and consider the coming of the dawn as prima facie evidence for my theological framework. But in fact, my angel hypothesis can only be corroborated by testing its unique components - the existence of invisible angels. It happens to be vague and untestable. Similarly, the results of this study can only validate the “Bayesian framework” by clarifying and testing its uniquely “Bayesian” assumptions (whatever these may be), not by straightforwardly modeling "well-known" facts.

In addition, the authors frame their question in a way that doesn't reflect a real perceptual problem, but is amenable to their “model.” They state that “The detection of coherent objects among noisy backgrounds is an essential function of perceptual organization, allowing the visual system to distinguish discrete, whole forms from random clutter” (p. 1). As I look around me, all I see are discrete, whole forms. None is set against a background of noise. Nor are their long, skinny objects (“open contours”). The problem is framed this way because it corresponds nicely to the authors' “hammer.” The task is an awkward, not a natural one, akin to exploring motor patterns by tying subjects legs together. It is not an obvious candidate for modeling natural perceptual processes; we are told that conditions were set so that, in a pilot study, subjects performed near 75% “correct.” Results were, predictably, mixed.

The claims made are easy to falsify. Specifically, the authors are suggesting that subjects performance in detecting the “contours” presented is due to “noisy” encoding of turning angles, thus “depressing their performance relative to an ideal observer in possession of perfect image data. “ (p. 14). The definition of contours as chains of points of identical luminance in a “noisy” background, as well as the reference to “perfect image data” reveals a certain naivete on the part of the authors vis a vis basic principles of visual perception. Their model would obviously fail to predict performance of subjects shown images that produce illusory contours, or amodal completions, for example. It would not even predict grouping of points with gaps between them – for example, a straight contour composed of identical black pixels alternated with white ones.

Finally, one of the authors, (Feldman) has repeatedly and strenuously argued against the “frequentist” definition of probability (e.g. Feldman, 2015), contrasting it with the belief-based “Bayesian” definition. Here, he inscrutably cross-breeds “frequentism” and “Bayesianism,” referring, for example, to a “Bernoulli Bayesian decision problem ” (p. 13)). Thus, it is not clear where, for Bayesians, “frequentism” ends and belief begins.

References

Feldman, Jacob. "Bayesian models of perception: a tutorial introduction." Wagemans, J eds (2014).

On 2017 Jul 10, Randi Pechacek commented:

Rachel Adams, first author of this paper, wrote a blog post on microBEnet to provide some background.

On 2016 Mar 13, Geir Bjørklund commented:

CORRECTION: My family name is Bjørklund, not BjΦrklund.

On 2015 Sep 27, Raechel Damarell commented:

The Dementia Search Filter is available on the Flinders Filters webpage at http://www.flinders.edu.au/clinical-change/research/flinders-filters/search-filters/. Other validated topic filters available include: heart failure, bereavement, residential aged care, glaucoma, contraception, and lung cancer.

On 2015 Jun 20, Matthias Kern commented:

The editorial title listed here in Medline is incorrect and contradicts the intention of the editorial. The correct title reads:

Editorial: On the scientific evidence that the sterilisation of customised implant abutments is required

The correct title of the editorial and the corresponding pdf-file can be found at: http://ejoi.quintessenz.de/index.php?doc=toc&year=2015&issue=2

On 2015 Jun 19, Christophe Dessimoz commented:

This paper is also available on Wikipedia: https://en.wikipedia.org/wiki/Inferring_horizontal_gene_transfer

Furthermore, I have written a blog post providing some background on the "Topic Page" format adopted in this article.

On 2015 Aug 20, Yoonsoo Hahn commented:

The second Comamonas bacterium (strain E6) containing the HSR1 sequence has been published (see the sequence at http://www.ncbi.nlm.nih.gov/nuccore/821590236 and the paper at http://www.ncbi.nlm.nih.gov/pubmed/26089421). Now there are two Comamonas bacteria with the HSR1 sequence. The first one JL14 was isolated in a mine in China and the second one in Niigata, Japan.

On 2015 Sep 09, Bill Cayley commented:

A good example of when less is more in cardiac care - a collection of related examples is at: https://lessismoreebm.wordpress.com/tag/cardiovascular/

On 2015 Jun 11, thomas samaras commented:

My research conflicts with the mainstream findings that short height is related to greater coronary heart disease. My conclusions are related to human studies based on worldwide research and various ethnic groups.

I have studied the relationship between height, chronic diseases, and longevity for almost 40 years. One of my papers in the Indian Heart Journal (2013) summarized worldwide findings showing shorter people have inherently lower heart disease. In 2014, I had a paper published in the Journal of Scientific Research & Reports that summarized key findings showing shorter people live longer. If tall people had inherently better hearts, then why do today's taller Americans have more coronary heart disease (CHD) compared to the early 1900s when we were a few inches shorter? Also women are shorter than men and have less CHD.

Studies from the 20th C found that many populations were free of CHD and stroke. Papua New Guineans, Kitavans, Kalahari Bushmen, Yanomami, and the Congo pygmies. Obviously, there was nothing wrong with their genes at the time they were studied. I know of no tall, western population that is free of CHD. Of course, the Japanese have had one of the lowest death rates from CHD in the developed world. When Japanese are compared in Japan, Hawaii and California, they see an increase in CHD mortality along with an increase in height.

In 2007, The World Cancer Research Fund reported that until recently, CHD was rare, even among the elderly. However, in parallel with industrial development, we have seen increases in height, weight and chronic disease (which includes CHD).

Many within population studies show that shorter people have lower CHD. These include findings from Hawaii, Sweden, Italy, UK and the US. For example, shorter elderly males were found to have lower CHD compared to taller males based on an over 40-year study of 8000 elderly Hawaiian Japanese males (He et al.). A Harvard study found that the tallest athletes had the highest rates of mortality from CHD compared to shorter athletes and non-athletes (the lowest CHD). Davenport reported that taller WWI military recruits had more heart problems than shorter ones based on 1 million men. In addition, US Asians have the lowest CHD and are the shortest ethnic group. Latinos and Native Americans are taller and have higher CHD. Whites and Blacks have the highest CHD and are the tallest.

Unfortunately, when it comes to human health and mortality, conflicting studies abound. However, confidence in a study's findings should be based on support from different types of studies (e.g., ecological, longitudinal, cross-sectional) that provide consistent results and include populations from different parts of the world and different ethnic groups.

Studies have shown that low income is a risk factor for CHD independent of other risk factors. Poor people tend to be shorter and fatter than higher income groups. A source of error in many studies is failure to compare short and tall people with the same degrees of overweight. Studies have found that shorter people tend to be more overweight than taller people. Thus, a substantial error in findings can be related to failure to compare tall and short people with the same body builds—this requires comparing taller people with higher BMIs vs. shorter people. A 10% taller person should have a 10% taller BMI for the same build type. The 1979 Build Study is the only study that I found which compared similarly overweight short and tall people. They found that the shorter cohort had a slightly lower mortality.

In 2014, a study also found that shorter people live longer (He, et al.) A 2012 study also found shorter men lived longer (Salaris, et al.)

I have reported scores of examples showing that non-Western shorter people have less CHD than taller Westerners. See www.humanbodysize.com for a listing of over 45 papers, book chapters and books expanding on what is discussed here. Some papers and a book related to height and CHD are listed below.

In conclusion, it worth noting Silventoinen's observation: CVD has increased in parallel with the Western diet and increased height.

References:

Samaras TT. Shorter height is related to lower cardiovascular disease risk—A narrative review. Indian Heart Journal 2013; 65: 66-71.

Samaras, TT. Is short height really a risk factor for coronary heart disease and stroke mortality? A review. Med Sci Monit 2004; 10(4): RA63-76.

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 date unavailable, commented:

None

On 2015 Sep 23, Angelo Gaitas commented:

The entire response appears in the PLOS1 comment section under response: http://www.plosone.org/article/comments/info:doi/10.1371/journal.pone.0127219

In two recent articles [1, 2] two techniques for removing or inactivating blood borne pathogens were introduced. The initial experiments were performed in vitro under simplified conditions. First, the primary achievement of the PDT work deserves clarification [1]. PDT is a powerful therapeutic modality, but its clinical application has been hampered by the inability of light to penetrate deep layers of the tissue, which is mainly due to hemoglobins in the blood readily absorbing photons. Utilizing a millimeter- diameter transparent tube for extracorporeal blood circulation allows PDT to function well despite the presence of hemoglobins in blood. Another point that deserves clarification is that the tube capturing device is not a microfluidic device [2]. This technique can be adapted using existing medical tubing without the need for complicated microfluidics and micro-fabrication. The device is a medical tube that has been chemically modified using simple steps to adapt the internal surface for cell capturing. 

We would like to take this opportunity to respond to concerns brought up in [3]. We start off by addressing concern (1), which speculates about the possibility of overheating during the use of near IR light. Our control data (Fig.3 and Fig.4 of [3]), confirmed that controls illuminated without photosensitizer-antibody conjugates did not undergo cell death, whereas those with photosensitizer-antibody conjugates underwent significant cell death under identical conditions. Thus it is clear from our data that temperature did not affect the outcome. It has been shown that 660 nm irradiation is safe and effective [4-6]. 

Moving on to concern (2) part (a) that brings up the problem of using the CD-44 antigen as a target. Limitations of antibody specificity are common knowledge and not unique to CD-44, but to all antibodies. To our knowledge, a targeting method that exclusively binds only to cancer cells does not yet exist, making the use of such a compound an unreasonable standard for publication. We used CD-44 antibody to demonstrate feasibility. As targeting methodologies advance and better selectivity to target cells becomes available, this technique will have improved selectivity. Our experiments were designed to avoid non-specific damage to other cells by pre-staining pure cancer cells with the photosensitizer-antibody conjugates and subsequently removing extra free conjugates before spiking into blood (described in detail in [1]). This elimination of the possibility of side effects due to undesired binding to other blood cells and excess free photosensitizer-antibody conjugates precluded the need for a toxicity study, particularly because we were at the proof-of-principle stage.

Part (b) of concern (2) suggests that we may have caused non-specific damage to non-cancerous cells by ROS' convection in the blood stream. We believe that this is highly unlikely. One of the authors has been conducting research focusing on ROS and PDT for years, in collaboration with other researchers [7-15]. This research demonstrated that PDT is extremely selective to targeted cells [13]. 

 Part (c) of concern (2) states that we should have used additional cytotoxicity assays, such as Annexin V, TUNEL, and MTT. However, because none of these techniques are cell-type specific, they would be useless for the particular objective they were suggested. Once our line of investigation reaches a more mature stage, we plan to undertake more useful studies, such as applying separate fluorescent tags, or radio labels, in addition to a cell viability assay and analyzing cell death with a cell sorting technology, such as FACS, MACS, density gradient centrifugation, etc.  

Concern (3) is that the capturing work [2] lacked purity confirmation concerning non-specific capturing of blood cells. Though purity confirmation is critical in diagnostic testing, our work was strictly limited to in vitro conditions, using spiked pure PC-3 cells as a model. To visualize and quantify PC-3 cells in the presence of whole blood, PC-3 cells were pre-labeled using a fluorescence tag (Calcein AM) and the extra free dye was subsequently removed before spiking PC-3 cells into blood. Because only PC-3 cells can have fluorescence in the blood mixture, and because quantification was based on fluorescing cells, false-positive results from other blood cells can be reasonably excluded. Furthermore, if other blood cells were captured but not identified by our detection method our data would then indicate that the simple tube captured cancer cells despite being blocked by other blood cells. If our technique were applied to CTC diagnosis, independent isolation procedures could be used to ensure the purity of captured cells. In contrast, if used for therapy, the purity of captured cells would not be as critical, provided that CTCs are effectively removed. If, by chance, capturing is hampered by accumulation of non-specific binding in filtering the entire blood volume, this issue can be addressed with strategies such as scaling up the tube and carefully determining the tube dimensions, flow rate, frequency of tube replacements, etc. 

Finally, concern (4), points out that the experimental conditions were not translatable to clinical applications. Part (a) regards scaling up the system to show high throughput. The concept of extracorporeal cleansing of the entire blood volume has been used for years in cases such as hemodialysis. We already are working on optimizing the technique for larger blood volume processing. Part (b) of concern (4) discusses the static no-flow condition as being unrealistic. This issue was brought up during the review process, and we provided with our results showing data under constant flow conditions by peristaltic pump (to be published in future publication). The reviewers agreed that the use of a no-flow condition as a conservative approach during a proof-of-concept stage was appropriate.

Despite its preliminary nature, we believe that our work communicates novel ideas, an important objective of research and publication. Given the number of research articles dealing with diagnostics and microfluidics, perhaps a further point of confusion came about by thinking of our work in those terms. We want to clarify that diagnostics were not the primary objective in our work. Furthermore, as it becomes evident by this response our experimental design was carefully devised to minimized unnecessary interferences. We hope that this response mitigates any confusion and addresses the concerns raised. 

1. Kim G, Gaitas A. PloS One. 2014;10(5):e0127219-e.
2. Gaitas A, Kim G. PLoS One. 2015;10(7):e0133194. doi: 10.1371/journal.pone.0133194.
3. Marshall JR, King MR. DOI: 101007/s12195-015-0418-3. 2015;First online.
4. Ferraresi C, et al. Photonics and Lasers in Medicine. 2012;1(4):267-86.
5. Avci P, et al. Seminars in cutaneous medicine and surgery; 2013.
6. Jalian HR, Sakamoto FH. Lasers and Light Source Treatment for the Skin. 2014:43.
7. Ross B, et al. Biomedical Optics, 2004
8. Kim G, et al. Journal of biomedical optics. 2007;12(4):044020--8.
9. Kim G, et al Analytical chemistry. 2010;82(6):2165-9.
10. Hah HJ, et al. Macromolecular bioscience. 2011;11(1):90-9.
11. Qin M, et al. Photochemical & Photobiological Sciences. 2011;10(5):832-41.
12. Wang S, et al. et al. Lasers in surgery and medicine. 2011;43(7):686-95.
13. Avula UMR, et al.Heart Rhythm. 2012;9(9):1504-9.
14. Kim G, et al. R. Oxidative Stress and Nanotechnology, 2013. p. 101-14.
15. Lou X, et al. E. Lab on a Chip. 2014;14(5):892-901.
16. https://www.roswellpark.org/patients/treatment-services/innovative-treatments/photodynamic-therapy.
17. Yin H, et al. Artificial organs. 2014;38(6):510-5.
18. Yin H, et al. Journal of Photochemistry and Photobiology B: Biology. 2015.

On 2015 Aug 29, Michael King commented:

Our recent commentary discusses this paper:

http://link.springer.com/article/10.1007/s12195-015-0418-3

In cancer research, the discovery and study of circulating tumor cells (CTCs) have seemed to open a world of possibilities. We now have the potential to gain cellular and molecular understanding of individual cases of metastatic cancer without invasive procedures. This area of research is, however, not without some basic pitfalls. In this commentary, we address some of these pitfalls by considering two recent examples in the published literature and discuss ways to overcome their limitations with the hope of informing those who may be entering the growing field of CTC research. Careful research design should always be followed to prevent incomplete or misleading studies from entering the literature, and thereby avoid setting back this burgeoning field.

On 2015 Sep 10, Bill Cayley commented:

More evidence that for warfarin cessation, "less" is "more" - it seems better NOT to "bridge": https://lessismoreebm.wordpress.com/?s=bridg

On 2017 Mar 04, University of Kentucky Systems Biology and Omics Integration Journal Club commented:

The authors have tried to contact the journal at least a half-dozen times over the last few months to post an erratum for this article. At first the editor suggested waiting to fix the erratum until two other manuscripts submitted by the authors were fully reviewed. After both manuscripts were accepted, the authors started to inquire again about posting an erratum, but have not receiving any guidance on how to submit an erratum nor when it would be posted. Therefore, the authors of this manuscript are posting an impromptu erratum via the journal club onto PubMed Commons. We have a new version of Figure 6 that fixes a mistake in two of the images that have the wrong chirality due to the image being flipped. The corrected image can be found on a FigShare repo for the paper: https://figshare.com/articles/Zn_metalloprotein_paper/4229333

On 2015 May 27, Timothy Shakespeare commented:

Despite not being an expert in the field, I read this paper with interest. It seems that the definition of dyslexia is a difficult subject, and I wondered what the justification was for the definition of severe reading impairment including <level 4 KS2 in Science or English. Am I correct in thinking that this would put someone with very poor reading, but good science and English in the normally developing group? Is this the case, and if so, how many people are in that category and does that fit with other definitions of dyslexia? Also, in the online version there are typos in the inclusion criteria "Children who achieved scores <2 SD below the mean" should be >2SD below the mean (as it is in the abstract).

Finally, the discussion draws conclusions about the implications of this study for use of visual aids. This has been taken by some to mean this study provides evidence that coloured overlays don't work for people with dyslexia, but I am interested in whether 1) it leaves open the possibility that a subsection of people with dyslexia (e.g. those who describe words moving on page) could benefit whilst others don't and 2) coloured overlays could possibly have an effect on higher order visual processing that was not tested or shown to be normal in this study. There doesn't seem to be a strong evidence base for these interventions, but it's not clear whether that's absence of evidence or evidence of absence.

On 2016 Mar 04, Melissa Rethlefsen commented:

We attempted to replicate the authors’ Embase search strategy, as reported in the Appendix (pages 13-14). The authors, according to the PRISMA flow chart also located in the Appendix (page 37), retrieved 1,371 results for their Embase search strategy, prior to deduplication of records. The authors specifically mention that there was no language limit imposed on this search in the "search strategy and selection criteria" panel in the primary manuscript, but there are no other limits mentioned.

In case the phrase “for randomised controlled trials” in the "search strategy and selection criteria" panel indicated a limit to ‘randomized controlled trials’ as a study type (using the EMTREE term), we restricted to this term. There was also no indication of the dates of coverage of the version of Embase (via the Ovid platform) used. There are multiple possibilities of date ranges (1947-1979; 1980-1987; 1988-1995; 1947 to present; 1974 to present; 1980 to present; 1988 to present; 1989 to present; and 1996 to present). We attempted all of the different date ranges, until an entry date of February 1, 2014 to most closely mimic the published search. There was no exact search date mentioned, but the authors did note that the search went "up to January 2014"; we used a February 1, 2014 entry date to allow for a late January 2014 search, though it was not clear if the authors searched in January 2014, or limited to publications published prior to January 2014. Even with the assumed ‘randomized controlled trials’ EMTREE term applied, we could not replicate the search count for any year range available by default in any Embase via Ovid database option.

The search as presented by the authors retrieves over 50,000 records in Embase (up to an entry date of 2/1/14). With the ‘randomized controlled trials’ EMTREE term applied as a limit, it retrieves over 3,100 articles. Mimicking the smallest Embase database coverage at the time of the authors’ search (1996-present, limiting to an entry date before February 1, 2014), we still see nearly 3,000 articles. This is a 1,600 record difference between the authors’ published results and what their published search strategy retrieves, even with these assumptions applied.

This study highlights the need for more accurate and comprehensive reporting needed for search strategies in systematic reviews and other literature search-based research syntheses, and the need for better peer review of search strategies by information specialists/medical librarians. Though the searches in the Appendix are on face value replicable and high quality, on closer inspection, they do not in fact meet the reporting standards as outlined by PRISMA Statement items #7 and #8: “Describe all information sources in the search (e.g., databases with dates of coverage, contact with study authors to identify additional studies) and date last searched” and “Present the full electronic search strategy for at least one major database, including any limits used, such that it could be repeated.”

On 2015 May 29, Karl Herrup commented:

Shakespeare makes several good points. The tandem article was meant from the beginning as a strong 'counterpoint' to my own article. I find that it is a scholarly and well written piece and I encourage people to read both (especially students).

As for setting the age of onset where sporadic and familial AD diverge, I would have two responses. The first is to thank Dr. Shakespeare for raising the very real point that there is no bright line that separates the two. It is only a question of odds: AD that starts before age 65 is most likely fAD; AD after age 65 is most likely sAD. But "likely" is different from "is". The second response is to confess that I smiled when I saw the comment about how this was an 'unattributed' remark. I too complain about authors who assert that "it is commonly known that...." even when there is little or no primary source offered to back up the point. I have to admit that I was hoisted on my own petard here. I have glanced at the cited reference and am pleased to incorporate its findings in future writings...and to choose my words a bit more precisely.

Concerning my contention that there can be Alzheimer's without plaques. Here Shakespeare actually makes the exact point I was trying to make and I wish I had been clearer. Plaques may well contribute to the disease process and then disappear, which I assert is the message from the vaccine trials. The result is that mechanistically the plaques could have started the disease but then vanish. But given our current approaches, I assert that this is a serious problem. If this individual were to die and his brain come to autopsy, a trained neuropathologist would assure us that the individual's dementia could not possibly have been Alzheimer's, even though it was. I was trying to argue that the implications of this is that we may well distort our findings if we require the presence of plaques to reach a diagnosis of AD. My suggestion is that we should relax our dependence on them, detected through PET scans or neuropathologically, in our diagnostic regimens.

On 2015 May 27, Timothy Shakespeare commented:

This is an interesting article that I'm sure will stimulate much discussion. Readers may wish to see an alternative perspective published in tandem here: http://www.nature.com/neuro/journal/v18/n6/full/nn.4018.html

Working in the area of sporadic Alzheimer's disease with age at onset <65, I would disagree with the sentence "Sporadic AD first appears clinically after the age of 65." This sentence is uncited and incorrect. There is occasionally confusion between early onset or young onset Alzheimer's disease, and Dominantly Inherited Alzheimer's Disease (also known as Autosomal Dominant Alzheimer's Disease, familial Alzheimer's disease). An onset before 65 (early or young onset) should not be confused with a dominantly inherited genetic form of Alzheimer's disease. In fact sporadic AD is more common that familial AD even in people with an onset <61 http://dx.doi.org/10.1086/302553.

I would also make a point regarding the interpretation of vaccine trials that have not had great success in providing cognitive benefits. In this paper the interpretation is that this shows you can have Alzheimer's disease without plaques "The implication is that just as there can be plaques without AD, there can also be AD without plaques." The alternative interpretation, that the amyloid is being removed too late and has already had its effect, should be given consideration. I.e. you need to have plaques at some point, but perhaps only earlier in the disease process. Perhaps this would fit into the idea of rejecting the simple linear pathway tracing disease progression from Aβ to AD.

On 2015 Jun 10, Arthur Yin Fan commented:

The methodology flaws in Hinman's acupuncture clinical trial, part I: design and results interpretation. Fan AY. J Integr Med. 2015 Mar;13(2):65-8. doi: 10.1016/S2095-4964(15)60170-4. No abstract available. PMID: 25797635 Similar articles

On 2015 May 26, Wayne Butler commented:

There must be an error either by PubMed or the Brazilian journal in matching the proper abstract with the title and journal page numbers. The abstract above, pubmed/26005980, has nothing to do with the racial classification article by FR Romero, et al. in Int Braz J Urol, 2015;41(2):360-366. The abstract above is from the next article in the journal by A Billis et al. 2015;41(2):367-372, pubmed/26005981. 27 May 2015 – PubMed has corrected the error. What a fast response!

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 NCT00562293. We believe the correct ID, which we have found by hand searching, is NCT00562263.

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 2015 Sep 06, Martine Crasnier-Mednansky commented:

This review should have discussed transcription-coupled DNA supercoiling. Ma J, 2013 demonstrated that RNAP was fully capable of melting DNA at random, and indicated that RNAP-generated (-) supercoiling may facilitate initiation of transcription at adjacent promoters and binding of regulatory proteins. This agrees with the proposal CRP-cAMP has a preferential affinity for negatively supercoiled promoters. The same authors also indicated in vivo transcription-generated supercoiling may potentially dissociate DNA-bound proteins even at a distance. Thus current understanding suggests RNAP itself may have evolved to be a master regulator of gene expression. Simply said, transcriptional regulators do not recruit RNAP, they are recruited by RNAP.

On 2015 May 26, Christopher Southan commented:

Selected leads and targets resolved to molecular identities at http://cdsouthan.blogspot.se/2015/05/entity-resolution-for-antimalarial.html

On 2016 Apr 30, Robert Eibl commented:

The authors review many aspects of CD44 in the nervous system; they even discuss some findings in brain tumors, but lack to include the first paper on CD44v expression and functional assays of hyaluronan binding in primary astrocytomas and glioblastomas (GBM), as well as related tumor cell lines: PMID:8750182

J Neurooncol. 1995 Dec;26(3):165-70.

Expression of variant CD44 epitopes in human astrocytic brain tumors.

Eibl RH, Pietsch T, Moll J, Skroch-Angel P, Heider KH, von Ammon K, Wiestler OD, Ponta H, Kleihues P, Herrlich P.

On 2015 Dec 08, Patrick Schloss commented:

We have published a manuscript that is in part a response to this paper by He et al. https://peerj.com/articles/1487/

On 2015 Jun 10, David Keller commented:

Depression caused by early PD should respond to dopaminergic medication, not an SSRI or psychotherapy

Gustafsson and colleagues demonstrate an association between depression and a later diagnosis of Parkinson disease (PD). If a patient's depression is due to reduced brain dopamine levels caused by alpha-synucleinopathy involving the substantia nigra, then that patient's depression is probably a pre-motor symptom of their PD, rather than a psychological illness. Furthermore, any form of SSRI or psychotherapy should be futile to alleviate the depression caused by early PD; however, a therapeutic trial of levodopa or a dopamine agonist might be remarkably effective at relieving depression caused by low brain levels of dopamine. Dopaminergic therapy might also relieve a depressed patient's "psycho-motor retardation" if it is actually early Parkinsonian bradykinesia.

It might be difficult to reliably determine by clinical examination alone which depressed patients are actually exhibiting pre-motor PD, and a therapeutic trial of SSRI therapy should be avoided for these patients (since SSRI's can lower brain dopamine levels). Similarly, a therapeutic trial of dopaminergic medication in depressed patients who do not have early PD would be a waste of time if their psychiatric depression would respond better to SSRI therapy and / or psychotherapy.

What this all suggests is that scintigraphic dopamine transporter imaging of the brain (the "DaT scan") may be helpful when a depressed patient fails to respond to SSRI therapy and does not exhibit psychological depression. A DaT scan which diagnoses early pre-motor PD in such a patient could be useful if it discloses that the proper therapy for that patient's depression would be dopaminergic medication, rather than an SSRI and / or psychotherapy. If SPECT scanning lacks the spatial resolution to be helpful in identifying depression due to early PD, then perhaps PET imaging could be employed.

On 2016 Oct 24, Sean Ekins commented:

attached are some slides on this topic from an ACS in 2015 http://www.slideshare.net/ekinssean/mining-big-datasets-to-create-and-validate-machine-learning-models

On 2016 Oct 24, Sean Ekins commented:

Attached are some slides on this paper from an ACS given in 2015 http://www.slideshare.net/ekinssean/development-and-sharing-of-admetox-and-drug-discovery-machine-learning-models

On 2015 May 21, DOMINIQUE BERGMANN commented:

There is an error in the assignment of two Arabidopsis gene names in Fig S6. SPCH should be AtbHLH098 and FAMA should be AtbHLH097.

On 2015 Jun 16, Robert G Sawyer commented:

I hope it continues a trend to shorten routine antimicrobial courses.

On 2015 Jun 10, Bill Cayley commented:

A great example of when "Less" is "more": https://lessismoreebm.wordpress.com/about/less-is-more-database/

On 2016 Apr 11, Gustav van Niekerk commented:

We (van Niekerk G, 2016) have recently argued that sickness associated anorexia (SAA), evolutionary conserved across both vertebrates and invertebrates, my represent a strategy for upregulating autophagic flux systemically. As an example, patients with sepsis typically decrease a number of amino acids (AA), including branched chain AAs (see table 2 in Su L, 2015). Similar, bile acids returning into circulation (bile acid reabsorption in the gut is very efficient) inhibit hepatic autophagy by binding to FRX (Lee JM, 2014). Thus, SAA likely represents an evolutionary conserved strategy to maintain elevated levels of autophagic flux during an infection.

An upregulation of autophagy during an infection may be critical for a number of reasons:

• Serum and AA starvation induces autophagy in macrophages and protects against TB infection (Gutierrez MG, 2004).

• We speculate that hepatic autophagy may play a critical role in clearing LPS and bacteria from circulation.

• Pathogens entering a cell must quickly subvert host processes to prevent being degraded by autophagy. In this regard, upregulation of autophagic flux would confront pathogens with a narrower window of opportunity to modulate the host machinery. Thus, autophagy enhances cell autonomous defence.

• Autophagy processes ribosomal components into antimicrobial peptides (Ponpuak M, 2010). Note that all nucleated cells have ribosomes and are capable of autophagy, thus suggesting that autophagy may again enhance cell-autonomous defence.

• Autophagy is also involved in the non-canonical expression of epitopes on MHC II by non-professional antigen presenting cells such as adipocytes, muscle and endothelium cells.

Autophagy may also be important in cell survival. As an example, in tissue ischemia, the release of biocidal agents from immune cells as well as the increase in misfolded proteins resulting from a febrile response may lead to the generation of toxic protein aggregates. Here, autophagy may promote cell survival by processing ‘overflow’ of damaged protein aggregates when the proteasome pathway is overwhelmed.

Collectively, these observations suggest that autophagy probably plays an important role in both pathogen clearance and in promoting host survival. It also provides a rational basis for permissive underfeeding as a form of nutritional support. We suggest that the reason why studies present mixed results on the effect of nutritional support results from the heterogeneous context and timing. As an example, a patient in recovery might benefit from aggressive nutritional support after the body switches from a catabolic towards an anabolic state. Similarly, the kind of pathogen challenge would also impact on the efficacy of nutritional support, as derailing autophagy may render cells more susceptible towards infection by certain pathogens.

On 2017 Oct 28, thomas samaras commented:

The findings of this study are supported by about 70 studies showing that shorter people live longer. Some key facts from a few of these studies include:

1.A 2017 study (Lemez) of basketball players showed that the shorter players lived longer

2.A 2016 study (Chmielewski) of 800,000 deceased Polish men and women showed the shorter lived longer

3.A 2009 study (Mueller and Mazur) of about 8000 West Point graduates who retired showed shorter lived longer after 60 years of age.

4.Studies of centenarians generally show that they tend to be very short and lean

5.Animal studies show that smaller individuals within a species live longer

6.A recent study (2017) identified 36 biological factors that favor greater longevity in smaller individuals

A collection of articles, book chapters and books showing shorter or smaller individuals live longer are given in website: www.humanbodysize.com

On 2015 May 21, Peter Hajek commented:

A great study. Two important results are not in the abstract. Sustained quit rate at 6 months was 5% and 3%, so neither treatment led to smoking cessation; but at six months, 13% still used the gum and 27% continued to use snus (p=0.0006). It is likely that dual use of either product reduces smoke intake and thus reduces harm and it may also facilitate quitting in future. This also suggests that snus is more attractive to smokers despite the initial rating, and so even if the two products were to have the same effect (and the same cost and availability), snus would have better population impact.

On 2015 May 31, Robert Withers commented:

It is indeed difficult to tread the fine line between the need to insure client confidentiality and the need to present accurate clinical material. This is why I have restricted myself to describing and reflecting on a real critical incident,without disclosing any information that could identify 'John'.

On 2015 May 23, Andrew Kewley commented:

In the manuscript, the author states: "While Chris has kindly granted me permission to use his material in this article, I am not in a position to request similar permission from John. So I will create a composite case, within which to frame a real ‘critical incident’ to which I wish to give particular attention. In doing this, I will aim to remain as true as I can to the dynamics of the original case without compromising client confidentiality."

I'm not convinced that this is ethical. Basically, it means that either privacy/confidentially is violated, or that the case is basically fictional and thus has no place being published.

On 2015 May 29, Michal Kicinski commented:

I thank Dr. Hilda Bastian for her interest in our recent study (Kicinski M, 2015). I strongly believe that post-publication comments very often raise important issues and help the readers to better understand the merits of a study and its limitations. However, I was disappointed to see that the comments of dr. Hilda Bastian do not correspond with the content of our study. For this reason, I feel obliged to clarify a number of issues.

The study of Ioannidis JP, 2007 points out one of the limitations of a large part of publication bias methods based on the asymmetry of the funnel plot, namely that they do not take between-study heterogeneity into account. This is indeed an important limitation of these methods, as also discussed by other researchers (Song F, 2010). However, please note that we did not rely on the asymmetry of the funnel plot in our analysis. Additionally, please note that our model is just an extension of the standard random effects meta-analysis model, which is a valid approach when between-study variability is present. In fact, the study of Ioannidis JP, 2007 is one of the contributions that motivates our approach to model publication bias since our model takes heterogeneity into account.

Dr. Hilda Bastian correctly points out that our study is not the first study on publication bias. There are many valuable studies on this topic and we discussed those most relevant to our research questions in our article. The contribution of our study is that we analyzed a very large number of meta-analyses using a model with strong theoretical foundations. Our study is the largest study on publication bias in meta-analyses to date. Please note that previous studies, e.g., Ioannidis JP, 2007, which Dr. Hilda Bastian mentioned, considered small study effects, a phenomenon that may have many different causes, including publication bias (Song F, 2010, Sterne JA, 2011). Another merit of our study is that we estimated the association between the size of publication bias and the publication year of the studies included in the meta-analyses.

I completely agree that the best solution to the problem of publication bias is the complete reporting of study results. In fact, our findings showing that publication bias is smaller in the meta-analyses of more recent studies support the effectiveness of the measures used to reduce publication bias in clinical trials. I strongly advocate the introduction of new policies aimed to completely eliminate reporting biases from clinical trials and, as written in our article, the implementation of measures to reduce publication bias in research domains other than clinical trials, such as observational studies and preclinical research.

Although we did not investigate the use of publication bias methods in the meta-analyses from the Cochrane Library, it is clear from previous research that the potential presence of publication bias is often ignored by researchers performing meta-analyses and that the methods accounting for publication bias based on the statistical significance are hardly ever used (Song F, 2010, Onishi A, 2014). When publication bias is present in a meta-analysis, ignoring the problem leads to biased estimates of the effect size (Normand SL, 1999). Therefore, similar to others (Sterne JA, 2011), we argue that researchers should investigate the presence of publication bias and perform sensitivity analyses taking publication bias into account. One difficulty with the use of publication bias methods is that they require researchers to make certain assumptions about the nature of publication bias. For example, the trim and fill method defines publication bias as suppression of a certain number of most extreme negative studies (Duval S, 2000). The use of the Egger’s test (Egger M, 1997) as a publication bias detection tool requires researchers to make the assumption that publication bias leads to a negative association between effect size and precision. The performance of a certain publication bias method depends on whether or not the method’s assumptions are met. For example, it has been demonstrated that publication bias detection tests based on the funnel are characterized by a very low power when publication bias based on the statistical significance is present and the mean effect size equals zero (Kicinski M, 2014). Publication bias based on the statistical significance is the best-documented form of publication bias (Song F, 2009, Dwan K, 2013), The results of our study add to this body of evidence. Therefore, we argue that publication bias tools designed to handle publication bias based on the statistical significance should be used by researchers.

In the tweet with the link to her comment on PubMed, Dr. Hilda Bastian wrote on the 25th of May: ‘27% of cochranecollab reviews over-estimate effects cos of publication bias? Hmm.’ Please note that our study did not investigate the proportion of meta-analyses that overestimate effects. In fact, the objectives of our study were completely different. We estimated the ratio of the probability of including statistically significant outcomes favoring treatment to the probability of including other outcomes in the meta-analyses of efficacy and the ratio of the probability of including results showing no evidence of adverse effects to the probability of including results demonstrating the presence of adverse effects in the meta-analyses of safety.

On 2015 May 25, Hilda Bastian commented:

This is an interesting study. But it's a rather enthusiastic self-assessment of a method not validated by other researchers, and some perspective is useful in thinking about the conclusions.

Kicinski M, 2015 is neither the first, nor the largest study, of publication bias (PB) in meta-analyses, and the presence of publication bias in them is well-known. These authors used a scraper they have made available on Github to extract meta-analyses from Cochrane reviews. They looked at reviews with placebo or "no treatment" control groups and 10 or more included studies. Whether or not these results are applicable to interventions with active or usual care control groups is unknown.

For perspective here: Ioannidis JP, 2007 considered PB in 1,669 Cochrane reviews, ultimately analyzing 6,873 meta-analyses. A half of the meta-analyses had no statistically significant results in them, so the problem identified here could not have applied to them. Ioannidis JP, 2007 concluded that only 5% of the full set of Cochrane reviews would qualify for the use of asymmetry tests, and only 12% of those with a larger number of events and participants. They found very little concordance between different asymmetry tests - only around 3-4%. A more important problem according to Ioannidis JP, 2007 was the misapplication and misinterpretation of statistical tests, not under use. False-positives are a problem with tests for PB when there is clinical heterogeneity. Ioannidis JP, 2007 conclude that the only viable solution to the problem of PB is full reporting of results.

Kicinski M, 2015 conclude that statistical tools for PB are under-utilized, but the extent to which PB is assessed was not part of their study. Although PB itself may be decreasing over time, assessment of PB is increasing, even if the methods for exploring it are still problematic:

• Palma S, 2005 found that PB was assessed in 11% of trials between 1990 and 2002, increasing from 3% in 1998 to 19% in 2002 (less frequently in Cochrane reviews than others).
• Moher D, 2007 found that about 23% of systematic reviews in 2004 assessed PB (32% in Cochrane reviews, 18% in others).
• Riley RD, 2011 found that only 9% of reviews from one Cochrane group assessed PB.
• van Enst WA, 2014 found that most systematic reviews of diagnostic test accuracy in 2011/2012 mentioned the issue, with 41% measuring PB.

In assessing only the meta-analyses themselves, and not the reviews that included them, it's not possible to know, as the authors point out, to what extent other studies were included, but without data that could be pooled. An issue not raised by Kicinski M, 2015 are trials reported only in conference abstracts, and thus with minimal data. Cochrane reviews often include studies reported in conference abstracts only, and those are apparently more likely to have non-statistically significant results (Scherer RW, 2007) - as well as relatively little data for the multiple meta-analyses in a review.

It's important to consider the review, and not just the effect summaries within meta-analyses, because the conclusions of the systematic review should reflect the body of the evidence, not only the meta-analyses. Over-favorable results in a meta-analysis shouldn't be equated with over-favorable conclusions about effectiveness in a review (although unfortunately it often will). We shouldn't jump to conclusions about effect sizes from meta-analyses alone. They can be skewed by clinical heterogeneity and small study size as well as (or instead of) publication bias, and the devil may be more in the interpretation than the calculations.

Disclosure: I work on projects related to systematic reviews at the NCBI (National Center for Biotechnology Information, U.S. National Library of Medicine).

On 2015 May 26, Yanju Ma commented:

I am very interested in your study. In this study, you found no correlation between the numbers of CTC-clusters and that of single CTCs (Supplementary Fig. 9). But we could see that three patients with melanoma have higher CTC clusters. So is there any correlation between higher CTC clusters with poor prognosis? I will be glad to get your reply. Thank you.

On 2015 Jun 14, Preben Berthelsen commented:

In this editorial, Prof Matthay contents that the paper by Blodgett: The continuous inhalation of oxygen in cases of pneumonia otherwise fatal, and in other diseases. (Boston Med Surg J 1890;123:481-) is the first report on oxygen therapy. Blodgett’s case story is indeed interesting and heart-breaking but it is not a first – and Blodgett certainly does not claim priority in his paper.

Fifty years earlier, in “On Asphyxia, and on the Resuscitation of Stillborn Children”(a paper read at the Westminster Medical Society, London, October 16, 1841 and printed in Boston Med Surg J 1842;25:409-) John Snow states on page 414 “Oxygen gas is sometimes mixed with air to be thrown into the lungs of asphyxiated persons” And, “if it is deemed advisable, oxygen gas can be generated in great purity, in a few minutes, from chlorate of potash by means of a spirit-lamp and a small retort” Furthermore, “No harm can arise from thus using oxygen.”

I do not know if John Snow – the first academic anaesthetist in the world – was also the first to advocate oxygen treatment.

P.G.Berthelsen, MD. Charlottenlund, Denmark

On 2015 May 22, thomas samaras commented:

Researchers should be aware that two earlier reviews were published showing that shorter people are more likely to have lower coronary heart disease (CHD) (1,2). This apparent conflict with the Nuesch, et al. paper is no doubt due to various confounders and failure to recognize the biological mechanisms that underlie the inherently lower risk of CHD for shorter, lighter people. In addition, ecological studies showing short people often have little or no CHD are often ignored because of the belief that ethnic, racial, climatic and geographical factors make the results unreliable. However, Shaper (1) stated that these factors are completely irrelevant to the incidence of CHD unless they are related to social class and economic status.

In my opinion, the facts covered in the two papers cited below (1,2) provide overwhelming evidence that shorter people have the potential for very low CHD. (Unfortunately, Dr. Elrick and Professor Storms have passed away and cannot co-write this response.) A summary of some of our findings follows.

Before the early 1900s, Europeans and Americans were shorter but CHD was rare. As we got taller and heavier, CHD increased due to the Western diet.

Based on 1 million WWI military recruits in the US, Davenport and Love found that taller men had a much higher incidence of heart problems.

In the developed world, the Japanese have ranked within the top three countries in terms of low CHD for a number of decades. Japanese males average a little over 5’7”.

A number of populations that had very low or no CHD during the 20th C now have much higher rates along with their increased height. For example, S. Korea has seen a large jump in heart disease in recent years along with a substantial increase in height.

Western Sicilian centenarians are short and free of CHD risk factors.

Compared to men, shorter women have lower lifetime death rates from CHD.

A US study found that in a low-income cohort, taller people have almost a 40% higher risk of heart attacks. US mortality statistics based on ages ranging from youth to over 85 years of age show that shorter Asians have an almost 80% lower risk of CHD compared to taller Blacks and Whites. Latinos and Native Americans are taller than Asians but shorter than Whites and Blacks and fall between the shortest and tallest ethnic groups in mortality. (Findings based on a 15-year period and about 8 million deaths.)

A study of 350,000 dogs found that smaller dogs have much lower risk of heart failure compared to bigger dogs. For example, the Great Dane has 70 times the risk of heart failure compared to the miniature Dachshund. And the standard Dachshund has 3 times the rate of heart failure as the smaller miniature Dachshund.

I have also identified over 15 biological factors that would explain the advantages of smaller body size and low CHD. These include longer telomeres, smaller left ventricular mass, lower blood pressure, lower HDL, lower homocysteine, higher sex hormone binding globulin, lower C-reactive protein, higher adiponectin and FOXO3, and lower pulse wave velocity. These factors are based on the assumption that we are comparing shorter and taller people who have the same body type or proportions. (Otherwise, comparing a tall, thin person to a short stocky one would change these parameters.)

Many studies have also found that shorter people live longer. These include studies from the Hawaii, Ohio, San Diego, Spain, Sardinia, Okinawa, Bama (China), and Cuba (3). Many animal studies and experimental research support these human findings (4).

Recently, a 40-year longitudinal study based on over 8000 elderly Hawaiian Japanese males found that shorter men live longer (5). It is unlikely that they had more heart problems than the taller men who died younger.

Unfortunately, the findings discussed above have not received much attention because of the widespread belief that taller people are healthier and longer lived. Over 45 papers, books and book chapters on the ramifications of increasing body size are listed in www.humanbodysize.com

References

1 Samaras TT, Elrick H, Storms LH. Is short height really a risk factor for coronary heart disease and stroke mortality? A review. Med Sci Monit 2004; 10(4): RA63-76.

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On 2015 Jun 22, Darko Lavrencic commented:

Your conclusions indicate that spinal extrathecal CSF collections are not the cause but direct consequence of intracranial hypotension/CSF hypovolemia, regardless of CSF leakage. For the most probable cause of intracranial hypotension/CSF hypovolemia see http://www.med-lavrencic.si/research/correspondence/

On 2015 May 29, Kim D Pruitt commented:

lncRNA-NR024118 is represented by NCBI Gene ID: 25602 and RefSeq accession NR_024118.1

On date unavailable, commented:

None