On 2015 Jun 30, Jung Weon Lee commented:

Indeed, Na J, Ryu J, Kim HJ, and Nam SH should be recognized as the co-second authors.

2015.06.30 Jung Weon Lee

On 2015 Feb 18, Airton Stein commented:

This is quite an interesting article and reaching a conclusion in which it is mandatory to enhance prenatal care and newborn care at hospitals and basic healthcare units to prevent infant mortality.

On 2015 Jan 28, William Grant commented:

Vitamin D does reduce risk of upper respiratory infections!

The paper by Yawn and colleagues reported that the evidence for vitamin D for the treatment of respiratory diseases was weak since random controlled trials (RCTs) have not supported the observational studies finding inverse correlations between 25-hydroxyvitamin D [25(OH)D] concentrations and incidence of respiratory diseases [1]. However, the analysis of the RCTs and the literature search were not conducted well.

There have been two vitamin D RCTs that demonstrated a beneficial effect in reducing risk of influenza. The first was conducted on black post-menopausal women living on Long Island. Those taking 800 or 2000 IU/d vitamin D3 had significantly lower incidence of colds and influenza than those taking the placebo [2]. However, the same research group conducted a second vitamin D RCT on a similar group without finding a beneficial effect as noted in Ref. 1 [3]. The primary difference between the two trials was the baseline 25(OH)D concentration. As reported in Ref. 3, in the first one, it was 46.9±20.6 nmol/l (95% CI 43.9–50.39) while in the second one it was 64.3±25.4 nmol/L. The second was a vitamin D RCT conducted on school children in Japan. For those not taking vitamin D prior to entering the trial, there was a 64% reduction in incidence of type A influenza for those taking 1200 IU/d vitamin D3 compared to the placebo [4].

Another vitamin D RCT overlooked was conducted in Mongolia. In this study, school children with mean baseline 25(OH)D concentration of 7.0 (5.0–9.9) ng/mL (to convert ng/mL to nmol/L, divide by 2.5). were given 300 IU/d vitamin D3 which raised 25(OH)D concentration to 18.9 (15.5–22.9) ng/mL [5]. A significant reduction in acute respiratory infections was found.

Two negative studies were reviewed in Ref. 1. A study from New Zealand started with a baseline 25(OH)D concentration of 29 (SD, 9) ng/mL, achieved a 25(OH)D concentration of 48 ng/mL, and found no beneficial effect [6]. Another vitamin D RCT followed two groups, each with mean baseline 25(OH)D concentration near 25 ng/mL and achieved 25(OH)D concentration near 32 ng/mL [7]. Again, no significant effect of vitamin D supplementation was found.

Summarizing the findings from vitamin D trials, those trials that had baseline 25(OH)D concentrations below 50 nmol/L (20 ng/mL) found reduced risk of respiratory tract infections from taking vitamin D3 while those with higher baseline concentrations did not.

Robert Heaney recently outlined guidelines for optimizing designs of nutrients such as vitamin D [8]. The important points for vitamin D are to start with an understanding of the 25(OH)D concentration-health outcome relation, measure 25(OH)D concentrations, enroll only those with low 25(OH)D concentrations, supplement with enough vitamin D3 to raise concentrations high enough to see an effect, and remeasure 25(OH)D concentrations. Very few vitamin D trials satisfied these steps. Many 25(OH)D concentration-health outcome relations show rapid changes below 15 ng/mL with very little change after 30 ng/mL [9, 10]. Thus, vitamin D RCTs should seek to enroll people with baseline 25(OH)D concentrations below 20 ng/mL.

Additional evidence that vitamin D reduces risk of respiratory tract infections comes from an ecological study of influenza case-fatality rates in 12 communities in the United States from the 1918–1919 influenza pandemic. Case fatalities were generally due to pneumonia and occurred about 10 days after the influenza infection. In a comparison with solar UVB doses for summer or winter, rates were significantly lower in communities with higher solar UVB doses [11]. The mechanisms proposed were that vitamin D induces cathelicidin, which can kill bacteria, and reduction in the cytokine storm that often accompanies influenza. The cytokine storm can damage the lining of the lungs, thereby permitting the ever present bacteria to give rise to pneumonia. The influenza in that pandemic was type A H1N1.

Thus, there is plenty of evidence that vitamin D can prevent and treat upper respiratory tract infections.

References 1. Yawn J, Lawrence LA, Carroll WW, Mulligan JK. Vitamin D for the treatment of respiratory diseases: Is it the end or just the beginning? J Steroid Biochem Mol Biol. 2015 Jan 24. pii: S0960-0760(15)00029-1. doi: 10.1016/j.jsbmb.2015.01.017. [Epub ahead of print] 2. Aloia JF, Li-Ng M. Re: epidemic influenza and vitamin D. Epidemiol Infect. 2007;135(7):1095-6; author reply 1097-8. 3. Li-Ng M, Aloia JF, Pollack S, et al. A randomized controlled trial of vitamin D3 supplementation for the prevention of symptomatic upper respiratory tract infections. Epidemiol Infect. 2009;137(10):1396-404. 4. Urashima M, Segawa T, Okazaki M, Kurihara M, Wada Y, Ida H. Randomized trial of vitamin D supplementation to prevent seasonal influenza A in schoolchildren. Am J Clin Nutr. 2010;91(5):1255-60. 5. Camargo CA Jr, Ganmaa D, Frazier AL, et al. Randomized trial of vitamin D supplementation and risk of acute respiratory infection in Mongolia. Pediatrics. 2012;130(3):e561-7. 6. Murdoch DR, Slow S, Chambers ST, et al. Effect of vitamin D3 supplementation on upper respiratory tract infections in healthy adults: the VIDARIS randomized controlled trial. JAMA. 2012;308(13):1333-9. 7. Rees JR, Hendricks K, Barry EL, et al. Vitamin d3 supplementation and upper respiratory tract infections in a randomized, controlled trial. Clin Infect Dis. 2013;57(10):1384-92. 8. Heaney RP. Guidelines for optimizing design and analysis of clinical studies of nutrient effects. Nutr Rev. 2014;72(1):48-54. 9. Grant WB. Relation between prediagnostic serum 25-hydroxyvitamin D level and incidence of breast, colorectal, and other cancers. J Photochem Photobiol B, 2010;101(2):130–6. 10. Garland CF, Kim JJ, Mohr SB, et al. Meta-analysis of all-cause mortality according to serum 25-hydroxyvitamin D. Am J Pub Health. 2014;104(8):e43-50. 11. Grant WB, Giovannucci E. The possible roles of solar ultraviolet-B radiation and vitamin D in reducing case-fatality rates from the 1918–1919 influenza pandemic in the United States. Dermatoendocrinol. 2009;1(4): 215-9.

Disclosure I receive funding from Bio Tech Pharmacal (Fayetteville, AR), MediSun Technology (Highland Park, IL), and the Vitamin D Council (San Luis Obispo, CA).

On 2015 May 20, Bill Cayley commented:

This is a useful study, but it would have been helpful to know whether or not the non-everted wound closure was QUICKER.

On 2015 Feb 27, T Eugene Day commented:

This is an excellent use of simulation to tease out how complex factors influence ED crowding. In the real world, the (as the authors put it) "chaotic" nature of the ED can make identifying factors associated with crowding extremely difficult to understand. However, simulation allows us to make controlled experiments which reveal insight into system dynamics. This is an interesting and thoughtful approach to a difficult problem.

On 2016 Jan 03, Sargis Dallakyan commented:

Visit http://pyrx.sourceforge.net to learn about the new features in the latest PyRx releases.

On 2015 Feb 26, Anders von Heijne commented:

It would be of great interest if the thalamic volumes in this study were to be correlated with a simple linear measurement, such as Third Ventricular Witdh. If changes in TVW can be shown to correlate with cognitive impairment in multiple cohorts it would be much easier to implement in clinical practice than volumetric measures.

On 2015 May 25, Tove Alm commented:

Response to letter by Islam

Shahidul Islam refers to our research article in Science “Tissue-based map of the human proteome” (M. Uhlén et al 23 January, 2015, p. 394) and claims that “they have skipped the control experiments because that would slow down the productivity”. This is of course not true; a large portion of the resources for the project are invested in validation of the antibodies.

A considerable amount of the resources in the program have been used to perform control experiments for the antibodies displayed in the portal and a lot of effort has been put into the visualization of the primary data to allow the scientific community to explore the data behind the control experiments. At present, more than 60,000 antibodies have been validated using Western blots and high-density micro arrays and these efforts have been published in hundreds of articles from our group (www.proteinatlas.org/about/publications). The result in each application is also compared to RNA expression levels and what is known in literature. In addition, to further examine the antibodies, a pipeline for validation of antibody specificity using gene silencing has been established. The results are currently available for a subset of the antibodies in the Protein Atlas.

The open-source policy applied to all antibodies published on the Atlas allows transparency and users may themselves review the experiments supporting the specificity of a particular antibody through the “Antibody/antigen” page (see example: www.proteinatlas.org/ENSG00000141510-TP53/antibody).

The statement by Islam that the antibodies are available through Atlas Antibodies and therefore “reduces the reliability of the immunohistochemistry images” is also difficult to understand. The Human Protein Atlas includes antibodies from more than 40 commercial providers and all primary data supporting the respective antibody is shown as an open resource on the antibody/antigen page for each antibody (see example link above). In the Human Protein Atlas program, a requirement for including an antibody has from the start been that the corresponding antibody must be available to the scientific community through a commercial provider. This is important, since it allows for the use of the same antibody by all researchers interested in human biology and medicine.

As far as we know, there are no past or current efforts with a more comprehensive pipe-line of systematic antibody validations.

On 2015 Apr 19, Md. Shahidul Islam commented:

Immunohistochemistry images published in the “Human Protein Atlas” are not reliable

IN THE ARTICLE “Tissue-based map of the human proteome” (M. Uhlén et al 23 January, 2015, p. 394), the authors point out that they have produced more than 13 million tissue-based immunohistochemistry images by using 24,028 antibodies. These images are freely available from the “Human Protein Atlas” portal. However, when it comes to the interpretation of the images, the key issue is whether the antibodies detect only the proteins that they are intended to do, under the conditions of the immunohistochemistry experiments. What is not widely known is that the scientists behind the “Human Protein Atlas” publish the immunohistochemistry images without performing the proper control experiments. For interpreting immunohistochemistry images, it is essential to perform the appropriate control experiments (1, 2). They have skipped the control experiments because that would slow down the productivity. They have tested the specificity of the antibodies mainly in the protein microarray experiments, and assumed that the antibodies that turn out to be specific in the protein micro array experiments will be specific also in the immunohistochemistry experiments. This assumption is not correct. Only about 45% of these antibodies detect the intended protein in the Western Blot. Scientists cannot publish immunohistochemistry images in peer-reviewed journals, without appropriate control experiments (1, 2). “Human Protein Atlas” is an exception. Here, millions of immunohistochemistry images have been published without requiring any independent peer-review process. The antibodies used for generating the images are being sold by the “Atlas Antibodies”, founded by the researchers from the “Human Protein Atlas” project. This conflict of interest further reduces the reliability of the immunohistochemistry images.

Md. Shahidul Islam Department of Clinical Sciences, and Education, Södersjukhuset, Karolinska Institutet, SE-118 83, Stockholm, Sweden and Department of Internal Medicine, Uppsala University, Uppsala, Sweden. E-mail: shahidul.islam@ki.se

Reference List

1.  R. W. Burry, J. Histochem. Cytochem. 59, 6 (2011).
2.  A. Lorincz, Z. Nusser, J. Neurosci. 28, 9083 (2008).

On 2015 Apr 24, Björn Hallström commented:

We agree that it is not immediately obvious what the colors in Fig 1F refer to, and that this could have been better explained in the figure legend. However, the yellow bands refer to genes that have RNA evidence either from this study or from Uniprot. Since the categories ("Not detected" etc) are determined solely from our RNA data it is possible that Uniprot has RNA evidence for a gene that we could not detect. This is what the yellow band above "Not detected" in Fig 1F refers to. These are generally genes that are expressed in more specialized tissues that were not part of our panel of 32 tissues, such as retina, olfactory bulb and related to hair growth.

On 2015 Mar 05, University of Kentucky Systems Biology and Omics Integration Journal Club commented:

Figure 1F appears to have a significant discrepancy. The definition for the "Not Detected" category in the first column section of Figure 1F is defined as "FPKM < 1 for all tissues" in Table 1. The definition would indicate that there is no RNA evidence for putative protein coding genes in this category; however, there is a yellow band indicating significant number of these genes have RNA evidence.

On 2015 Jul 12, Guo-Liang Jiang commented:

Proposed revision of CT-based cervical and thoracic lymph node levels for esophageal cancer in UICC 7th version.Radiother Oncol. 2014 Nov;113(2):175-81. We correct the first author Liu M's affiliation as the following: 1. Department of Radiation Oncology, Fudan University Shanghai Cancer Center, Shanghai 200032, China; 2.Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China; 3.Department of Radiation Oncology, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, China

On 2016 Jun 03, Ewen Gallagher commented:

A detailed review on MEKK1 (encoded by Map3k1) and MAPK signaling, and looking at the roles of Map3k1 in cell death, survival and differentiation. Recent research relating to the genetic analysis of the MEKK1 PHD motif is included in the review (1).

Related work.(1) Charlaftis N, Suddason T, Wu X, Anwar S, Karin M, Gallagher E. The MEKK1 PHD ubiquitinates TAB1 to activate MAPKs in response to cytokines. The EMBO journal 2014; 33:2581-96.

On 2017 Jul 13, Randi Pechacek commented:

Katherine Dahlhausen described this paper after traveling to South America. She wrote of its important findings on microBEnet.

On 2017 May 04, Jeremy Horst commented:

Can the actual results of this study be extended to infer a role of human genes in the disease? The influence of sex on the relationship to and influence of the environment is known to be strong for this disease. No assessment of any genotype besides assumed XX vs XY was performed.

Also, a role for horizontal transmission of dental plaque bacteria from parents to children has been established. This obfuscates simple heritability estimates.

Are there any loci that have shown an association with disease or disease severity in different studies? Perhaps lactoferrin. Clearly there is a role when tooth-development genes are mutated, but these are rare and do not extend to the general population.

The conclusions here go to far in purporting genes as the cause of the difference in disease outcomes by sex.

On 2015 Feb 13, Amanda Capes-Davis commented:

The two cell lines used here are both HeLa. KB is not an oral cancer cell line. It was cross-contaminated by HeLa - most likely during establishment of the cell line in 1955 - and no authentic stocks are known.

A number of studies have used KB for testing of therapeutic activity and concluded that an effect will be relevant for oral cancer. Unfortunately that is not the case.

Cell lines can be easily tested for cross-contamination and an increasing number of journals require authentication testing before publication. In my view, authentication testing would address the widespread use of KB as a model for oral cancer.

Short tandem repeat (STR) profiling is the current consensus for authentication testing. Online databases can be used to compare a laboratory's result to HeLa and other commonly occurring contaminants.

For more information on STR profiling see: http://iclac.org/resources/human-cell-line-authentication/.

For a list of known cross-contaminated cell lines see: http://iclac.org/databases/cross-contaminations/.

On 2015 Nov 12, RUBEN ABAGYAN commented:

Additional disclosure. One of the authors, R.A., has an equity interest in Molsoft, LLC. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies.

On 2015 Sep 20, Ralph Brinks commented:

Most guidelines in health economic modeling stress the importance of the quality of the input data for the results of an economic model [1]. The input data of the transition probabilities between the BMI groups in the model of this work is based on empirical data from Germany and a method, which is stated to be described in [3]. Essentially, the transition probabilities are estimated from age-specific prevalence data, which is a well-known problem in epidemiology [4]. The method used in [2], however, is inappropriate, which I will demonstrate by showing the magnitude of the error. Terminology here is similar to [2], the BMI groups are numbered: normal (1), overweight (2), obese (3). The transition probability from state x to state y is denoted by tpxy

Given the data of 53 year old males tp12 = 0.02159, tp23 = 0.01035, [2], and assumed that the probability of dying is 0.006 for all BMI groups [5], then at age 53 the prevalences in the respective BMI groups are about P(53) = (0.45, 0.3, 0.25), see Figure 2 of [2]. After simulating one cycle of the Markov model, we obtain the prevalence at age 54 as P(54) = (0.4402, 0.3067, 0.2531). If we now use Equation (1) in [2] to estimate tp12, we obtain (0.3067-0.3)/0.45 = 0.01489. Compared to tp12 = 0.02159, this is an relative error of -31%, a magnitude which exceeds the relative errors in the sensitivity analysis (+/-20%) by far. This is not acceptable.

Thus, I query whether the results of [2] are valid. Instead of using the inappropriate Equations (1) and (2) in [2] I suggest to use studies surveying the transition probabilities between BMI groups directly.

[1] Penaloza Ramos MC, Barton P, Jowett S, Sutton AJ. A systematic review of research guidelines in decision-analytic modeling. Value Health 2015; 18: 512-29.

[2] Sonntag D, Ali S, Lehnert T, Konnopka A, Riedel-Heller S, König HH. Estimating the lifetime cost of childhood obesity in Germany: Results of a Markov Model. Pediatr Obes 2015 [Epub ahead of print]

[3] Miller DK, Homan SM. Determining transition probabilities: confusions and suggestions. Med Dec Making 1994; 14: 52-58.

[4] Keiding N. Age-specific incidence and prevalence: a statistical perspective. J R Statist Soc A 1991; 154: 371-412

[5] Statistisches Bundesamt. Generationensterbetafeln Deutschland: Wiesbaden 2011.

On 2015 Nov 11, Luis Quadros commented:

It's impressive to reach such a conclusion with only 36 patients.

On 2016 Oct 13, Lydia Maniatis commented:

In addition to the circularity of the noise revelation, the claim in the abstract regarding "spatial pooling" is also misleading:

" Our results suggest that chickens use spatial pooling of cone outputs to mitigate photon-shot noise."

The results show nothing of the sort, since "spatial pooling" was introduced as a free parameter to aid in data-fitting. If we were to label this parameter "the tooth fairy" then the data would "suggest" that the tooth fairy mediates color discrimination.

Again, we are talking about cosmetic applications of concepts to data-fitting techniques and arguments consisting of multiple casual assumptions that license no such interpretation.

On 2016 Oct 05, Lydia Maniatis commented:

Thanks for your reply, Peter. I've copied it below and respond to the various points in the order they came up.

You: There are measurements of noise in the visual system of animals which we have cited in the paper, so I can not agree with you that there is no evidence for noise in the visual systems of animals.

Me: I think that all of the cited papers assume they are measuring "noise", without actually testing that assumption.

You: In this paper we are describing the smallest colour difference that chickens were able to discriminate to figure out the equivalent Weber fraction which describe these limits. Whether that is actually caused by noise or not we can not say from our experiment, but it is adressed in some of the cited literature.

Me: The title of your paper says "behavioural thresholds reveal receptor noise" and the abstract states that your "experiments allowed us to compare behavioural results wtih model expectations and determine how different noise types limit colour discrimination." So it sounds like a claim that your experiments did, in fact, corroborate the "noise" assumptions. In fact, the noise argument was never in any danger of falsification, because it was not tested, only assumed to be indirectly measurable. The confusion about this issue has seeped into other publications; the reason I was looking at yours was that Scholtyssek, Osorio and Baddeley (2016) claim that your paper "validated experimentally" the "receptor-noise limited model of Vorobyev and Osorio (1998)." This didn't seem likely, so I wanted to check.

You: We find, similar to other experiments, that there this Weber fraction is the same in relatively brighter light (thresholds are very similar) but that the Weber fraction we need to assume to describe the thresholds needs to be higher in dim light, consistent with addition of photon-shot noise.

Me: "consistent with" doesn't mean causally connected to. If the claim is about causality, this needs to be tested, not assumed. The adjustments and assumptions in general seem ad hoc.

You: Which has been shown to limit visual sensitivity in many experiments.

I think this applies in very, very low light conditions. But your experiment does not constitute a test that this is relevant to your results. Again, it is simply assumed, even though the idea that raw responses of neurons early in the hierarchy are transmitted directly to visual experience is not credible.

You: We are using the Receptor Noise Limited model to describe the data, it is a tool that can be used to make predictions in other scenarios as well.

Me: Why do you consider it a useful tool? Have its "noise" related assumptions ever been tested and corroborated? How? When Vorobyev and Osorio (1998) say that the model "predicted" some psychophysical data, they just mean that they were able to find some datasets that fit more or less, and some that didn't, not that they predicted beforehand which datasets would match. And again, general consistency alone does not justify uncritical acceptance of whatever qualitative assumptions are tacked onto the math.

You: Regarding Percepts: We are not talking about how the animals perceive the colours, but rather how they are able to discriminate them. Absolutely, there is much more in the visual system going on that we yet do not understand. But the photoreceptor cells are the input of the system and the limits of the photoreceptors are going to be important.

Me: First, discrimination implies perception, at least to the extent that two surfaces are perceived as same or different. Second, the limits of photoreceptors are not discernible in ordinary percepts. Correct me if I'm wrong, but you don't even seem to be acknowledging the complexity of the neural code for color, which involves combining cone activity to produce color experiences that can't be qualitatively analyzed on the basis of the individual cone activity, for example the fact that "red" plus "green" plus "blue" cone activity (or, e.g. "red plus green") produces the experience of grey or white. With four cones, the code will be even more complicated.

On 2016 Oct 05, Peter Olsson commented:

Dear Lydia Thank you for your interest and comments.

There are measurements of noise in the visual system of animals which we have cited in the paper, so I can not agree with you that there is no evidence for noise in the visual systems of animals.

In this paper we are describing the smallest colour difference that chickens were able to discriminate to figure out the equivalent Weber fraction which describe these limits. Whether that is actually caused by noise or not we can not say from our experiment, but it is adressed in some of the cited literature.

We find, similar to other experiments, that there this Weber fraction is the same in relatively brighter light (thresholds are very similar) but that the Weber fraction we need to assume to describe the thresholds needs to be higher in dim light, consistent with addition of photon-shot noise. Which has been shown to limit visual sensitivity in many experiments. We are using the Receptor Noise Limited model to describe the data, it is a tool that can be used to make predictions in other scenarios as well.

Regarding Percepts: We are not talking about how the animals perceive the colours, but rather how they are able to discriminate them. Absolutely, there is much more in the visual system going on that we yet do not understand. But the photoreceptor cells are the input of the system and the limits of the photoreceptors are going to be important.

On 2016 Oct 01, Lydia Maniatis commented:

From the article (I've broken up a single continuous paragraph for clarity (caps mine):

"We ASSUME that discrimination thresholds of visual systems are set by noise (Vorobyev and Osorio, 1998; Lind and Kelber, 2009b) and that noise can arise from different sources in different light conditions. Over a wide range of relatively high light intensities...

...we EXPECT that Weber's law holds, so that sensitivity changes proportionally to light intensity and a constant Weber fraction (थscribes the signal-to-noise ratio that sets discrimination thresholds (Donner et al., 1990; Osorio et al, 2004; Lind et al., 2014). Under these conditions...

...the main source of noise is PROBABLY transducer noise (Lillywhite and Laughlin, 1979; Howard and Snyder, 1983) originating at the later stages of signal transduction in the photoreceptors, possibly by fluctuations in cGMP levels (Angueyra and Rieke, 2013).

There are no electrophysiological measurements of noise in bird photoreceptors, but rough estimates of noise have been deduced from the results of behavioural experiments on spectral sensitivity (Maier, 1992; Vorobyev et al., 1998; Lind et al., 2014)."

There is no empirical evidence that there is "noise" in the visual system and that this noise affects perception. The assumption is, furthermore, so vague that it could not be tested; and in our experience as observers, percepts certainly aren't "noisy." The reference to deduction of "rough estimates of noise" is contingent on this same untested, untestable, and empirically implausible assumption.

In addition, the idea that the formed, conscious percept, the product of complex processing with outcomes that resemble inferential logic, (such as, for example, lightness constancy (which has been shown even in chicks)) can be used to detect the neural properties of particular sets of neurons (here photoreceptors) is untenable.

Explicitly, the authors assume that "the discrimination thresholds are set by photoreceptor noise, which is propagated into higher order processing." This is a huge assumption, given what is known about the very different receptive field properties of neurons as we go up the hierarchy, and in view of the fact that these higher levels affect the activity of lower levels via feedback. It implies, in effect, that we can ignore, suppress or control for the participation of all other neural populations and interactions in this system. It is a claim that clearly requires a bit more effort at argument, rather than the casual assumptions, expectations, references to "established" or "standard" methods, etc. being offered here.

I see the bun, the catsup, the mustard and the onion rings...but where's the beef?

Another fascinating though not original aspect of this article is that it assumes that if we fit two arbitrary "models" to a dataset, and one of them fits this dataset better than the other, then the former is "more probable" than the other. This is the "Bayesian" way (though I don't think Bayes himself would agree). The fact that both models are too vague even to test doesn't interfere with these probabilities, which are in any case "subjective" and thus reflect the authors' (and the reviewers and editor's, I guess) personal beliefs.

On 2015 Jan 25, David Mage commented:

We (Mage and Donner) agree 100% with the authors' conclusion that there could be a commonality of the terminal mechanisms of SIDS and SUDEP. Our research on SIDS, based upon the universal 50% SIDS male excess for equal numbers of males and females at risk, predicts that the SIDS causation is X-linked resulting in acute anoxic encephalopathy (See our abstracts 9076695, 15384886 and 24164639). We suggested that a presently unknown gene locus on the X with a protective dominant allele with frequency p = 1/3 is involved. It would code for a protein enzyme that would allow the respiratory control neurons in the brainstem to shift from aerobic oxidation to anaerobic oxidation during a transient episode of acute cerebral anoxia. The XY male would be at risk of not having this dominant ability with frequency q = 2/3 and the XX female would be at identical risk with frequency q x q = 4/9. For equal numbers of male and female infants at risk the ratio 2/3 to 4/9 = 1.5, giving the expectation of 3 males dying of SIDS for every 2 females dying of SIDS. The authors report that for their cited 85 cases of prone SUDEP that there were 51 male and 34 female patients which is exactly in the ratio of 3 male to 2 female as we predicted. They propose that the main mechanism of SUDEP in their study is the peripheral hypoventilation and suffocation secondary to complete or partial airway obstruction which could lead to potentially terminal acute cerebral anoxia. We suggest that this may be a fertile field for further investigation because, to our knowledge, there is no other hypothesis in the medical literature that can predict that both SIDS and SUDEP would have the same 50% male excess.

On 2015 Feb 06, Ryan Radecki commented:

Post-publication commentary: "Prednisone … for Pneumonia?"

The utility of antibiotics for eradication of bacterial pathogens from the lower respiratory tract is a given. Use of steroids – also known for their immunosuppressive properties – not so much.

But, one can imagine clinical utility for steroids in acute infection. Not every function of the immune system results in desirable patient-oriented effects. Immunologic host responses include release of many inflammatory cytokines responsible for organ dysfunction, and steroids are already part of accepted therapy for several specific manifestations of pneumonia. Based on prior results in smaller trials, these authors suspected use of steroids might be of benefit – both in mortality and in time to symptom resolution.

http://www.emlitofnote.com/2015/01/prednisone-for-pneumonia.html

On 2017 Sep 18, Konstantinos Farsalinos commented:

Although Jensen et al. mentioned in the 2015 NEJM research letter that the health risks of formaldehyde hemiacetal inhalation are unknown ("How formaldehyde-releasing agents behave in the respiratory tract is unknown..."), they made a calculation that the formaldehyde-attributable cancer risk from e-cigarette use is 5 to 15 times higher than from long-term smoking. These two statements are clearly contradictory, and the calculation of any cancer risk from formaldehyde hemiacetal emissions is invalid since no such risk has been established for these compounds. It is simply based on an unsubstantiated assumption that "inhaling formaldehyde-releasing agents carries the same risk per unit of formaldehyde as the risk associated with inhaling gaseous formaldehyde...". Additionally, the latter statement was further misinterpreted by the media that reported the cancer risk of e-cigarettes (total risk, not formaldehyde-attributable risk) being 5 to 15 times higher than smoking. This is extremely important because, according to Fowles and Dybing (http://tobaccocontrol.bmj.com/content/12/4/424.long), the formaldehyde-attributable cancer risk is less than 1% of the total cancer risk of smoking. Prof Peyton, one of the authors of the NEJM research letter, now claims that formaldehyde hemiacetals are different from gaseous formaldehyde. But then, why did he and his co-authors use the hemiacetal measurements to present the cancer risk of e-cigarettes relative to tobacco cigarettes, considering that literature data for gaseous formaldehyde emissions from tobacco cigarettes were used in the comparison?

In the recently published paper (https://www.nature.com/articles/s41598-017-11499-0), Salamanca et al. are comparing the results of an experimental method (NMR) with the results of an established and validated analytical method used to identify and measure aldehyde emissions. In our study, using a validated and widely accepted method, we found 89% higher levels of formaldehyde at 5 V than the level of formaldehyde hemiacetal reported by Jensen et al. in the NEJM research letter. Therefore, we did not underestimate formaldehyde levels. We did not reject the findings by Jensen et al. that, high formaldehyde levels can be produced when blindly testing e-cigarettes at inappropriately high power settings relevant to the atomizer used (in fact we found more, but in general our findings support their observations). However, this cannot be used as an indication that e-cigarette users are exposed to such extreme formaldehyde levels in their daily routine.

I would also like to remind that we used a product which is outdated and inefficiently designed, a design that has been abandoned since 2012. These products are not even available in the market in the European Union anymore. Recent atomizers release formaldehyde at levels orders of magnitude lower than the one we tested. Even if we assume, despite the lack of evidence, that formaldehyde hemiacetals carry the same health risk as formaldehyde and that DNP derivatization underestimates the total formaldehyde exposure, still e-cigarettes developed over that last 3 years (at least) emit by far lower formaldehyde than tobacco cigarettes. Finally, the comparison between tobacco and electronic cigarettes in formaldehyde emissions is based on measurements performed using the same analytical method (DNPH derivatization) for both products. The assumption about underestimation of formaldehyde by DNPH derivatization applies equally to tobacco cigarettes and electronic cigarettes (tobacco cigarettes contain a lot of PG and VG); thus the relative difference remains the same.

Recent studies have found that e-cigarettes release formaldehyde at levels corresponding to > 1900 cigarettes consumed per day (e.g. https://www.ncbi.nlm.nih.gov/pubmed/27461870). All these studies, which have been accompanied by press statements and wide media coverage, need to be replicated under verified realistic conditions. Science is defined by replication, and we hope that journal editors will accept the challenge of publishing studies that reject findings previously reported in the same journal. In any case, all our replication studies will be published in peer-reviewed journals.

In conclusion, our purpose was to replicate an experiment using a specific e-cigarette device, atomizer and liquid under verified realistic conditions in order to verify or reject the conclusion that e-cigarettes carry 5 to 15 times the cancer risk of tobacco cigarettes. Our study clearly identified that this statement is false, and this was evident even when we used a very outdated and inefficient e-cigarette product. Therefore, our study was a valid replication experiment that presented the clinical relevance of previous findings.

On 2017 Sep 14, David H Peyton commented:

We appreciate the feedback on our manuscript, and respect the concerns of the posters here as well as those of the authors of the 2017 Food Chem Toxicol. paper by Farsalinos et al., cited by Clive Bates below. We share in the common goal of maximizing harm reduction. However, the claim that the aforementioned manuscript embodies a “replication” of our study in NEJM is false and misleading. Unfortunately, this misunderstanding continues to propagate prior misinformation disseminated about our work published in the 2015 NEJM paper. As clearly stated in our 2015 manuscript, we discovered and measured the levels of formaldehyde hemiacetals, not gaseous carbonyl formaldehyde. The 2017 study by Farsalinos et al. only measured levels of carbonyl formaldehyde. The ongoing omission of this critical detail, as reflected in the posts below as well as in the recent claimed “replication” of our work, has led the authors (yet again) to the conclusion that high levels of formaldehyde are detectable by e-cigarette users only under “dry puff” conditions. As we have described in a very recent manuscript (https://www.nature.com/articles/s41598-017-11499-0), 35-45 % of the formaldehyde hemiacetals are not converted to the detectable form of formaldehyde using DNPH impinger or related sorbent tube methods. Accounting for an approximately 40% loss of detectable formaldehyde at a power level of 4V, which the authors described as “realistic use conditions,” affords levels of > ca. 1400 ug of formaldehyde consumed. This is essentially the same level of formaldehyde from smoking combustible cigarettes, again according to data in the manuscript by Farsalinos and co-workers. This means that the cancer risk from formaldehyde in the e-cigarette that was investigated in the so-called replication study, even at this modest power level, is the same as that for combustible cigarettes. In other words, e-cigarettes do not emit very high formaldehyde levels only in conditions that are averse to users. In addition, discussing ‘dry puffs’ with the study subjects and increasing the power levels in a non-random way do not seem best practice for generating unbiased human subject study results. Our further studies of the chemistry and health effects of e-cigarettes are ongoing, and will be reported in due course, in peer-reviewed journals.

On 2017 Sep 02, Clive Bates commented:

This study has now been replicated and its obvious flaws confirmed.

Farsalinos KE, Voudris V, Spyrou A, Poulas K. E-cigarettes emit very high formaldehyde levels only in conditions that are aversive to users: A replication study under verified realistic use conditions. Food Chem Toxicol. 2017; . [link]

Conclusion

The high levels of formaldehyde emissions that were reported in a previous study were caused by unrealistic use conditions that create the unpleasant taste of dry puffs to e-cigarette users and are thus avoided.

The high levels of formaldehyde reported by the authors occur only in "dry puff" conditions that would be too acrid and aversive for human users to withstand. The authors completely missed this human control mechanism yet went on to calculate cancer risks based on conditions that humans would never experience. The resulting media storm was deeply misleading and the result of the authors inappropriate reporting of cancer risks based on unrealistic exposures.

This research letter should have been withdrawn or corrected by the NEJM once the flaws were pointed out. Given the controversy and impact on public health arising from people being given misleading information about health risks, the journal should have proactively commissioned a replication study or at least published this one.

A correction by the authors could at least admit that cancer is a human condition and that the cancer risk calculations have no validity given that humans are not exposed toxins generated under dry puff conditions.

At the time of writing the paper has 138 academic citations and 386 media articles. How many people will have been misled into making decisions harmful to their health by this paper?

On 2016 Apr 20, Clive Bates commented:

The fundamental weakness in this study is a failure to control the vapourising temperature to the levels experienced by real human subjects, rather operating temperatures that create a sensory experience that can only be tolerated by laboratory machines. The artifically high operating temperatures account for the high levels of formaldehyde and similar detected in the study.

At high temperatures, e-cigarette vapour undergoes thermal decomposition and aldehydes, such as formaldehyde, will form. For human subjects, there is a control feedback that avoids this exposure - namely that the aerosol takes on a horrible harsh taste and the e-cigarette user stops vaping. This phenomenon is widely understood among users and is known as 'dry puff' or 'dry hit' and it is always avoided. No such control feedback exists for laboratory machines.

This is one of several studies that ignores this important effect - something that could have been easily remedied by involving human users to verify the puffing protocol and device configuration and voltage settings create a realistic human vaping experience.

While it is not incorrect, merely pointless, to measure and report the decomposition of vapour in these unrealistic conditions, it is not legitimate to go on to make and publicise calculations of cancer risk, which can only arise from human exposures.

A critique of this short but damaging paper was published in the journal Addiction with an exchange of letters (Disclosure - I am a co-author): Bates CD, Farsalinos KE. Research letter on e-cigarette cancer risk was so misleading it should be retracted. Addiction 2015;110:1686–7. doi:10.1111/add.13018 Link to critique in Addiction

A detailed critique and case for retraction under the COPE guidelines was provided and published as supplementary material in Addiction. Link to Complaint under the Code of Conduct of the Committee on Publication Ethics

Forty experts wrote to the New England Journal of Medicine to express concern about the paper. Their letter was also published as supplementary material in Addiction. Link to letter

The New England Journal of Medicine has not provided a substantive response to this complaint or offered a route for escalation at the journal, as it should do under COPE. The full engagement with NEJM is described here with a annotated document file here - Google Doc

On 2015 Sep 14, Ivan Oransky commented:

Critics have called for this paper to be retracted, and there has been extensive correspondence about it in Addiction: http://retractionwatch.com/2015/09/11/researchers-call-for-retraction-of-nejm-paper-showing-dangers-of-e-cigarettes/

On 2015 Mar 15, IGSJC - The International General Surgery Journal Club commented:

Author Tom Varghese (@TomVargheseJr) joined IGSJC and moderator Amalia Cochran (@AmaliaCochranMD) to discuss "Nonsteroidal Anti-inflammatory Drugs and the Risk for Anastomotic Failure" on Twitter March 10-11, 2015, from 5-9pm PDT. For the remainder of the month of March, JAMA Surgery has opened access to the article (available at http://j.mp/IGSJC-201503), and all are still welcome to contribute to the discussion on Twitter using the #IGSJC hashtag.

A transcript of the journal club chat is available at: https://storify.com/AmaliaCochranMD/march-2015-igsjc

On 2015 Mar 09, IGSJC - The International General Surgery Journal Club commented:

Author Tom Varghese (@TomVargheseJr) will be joining IGSJC and moderator Amalia Cochran (@AmaliaCochranMD) to discuss "Nonsteroidal Anti-inflammatory Drugs and the Risk for Anastomotic Failure" on Twitter March 10-11, 2015, from 5-9pm PDT. For the month of March, JAMA Surgery has opened access to the article (available at http://j.mp/IGSJC-201503), and all are welcome to contribute to the discussion.

For tips on getting started, read http://igsjc.wordpress.com/guide-for-new-twitter-users/. Then, read the article, and return to Twitter on March 10 to take part!

Search on Twitter for #IGSJC (https://twitter.com/hashtag/igsjc) for the latest info and to join the chat.

On 2015 Jul 18, Jan Tunér commented:

Not arguing against the results of this paper, but a problem with most studies trying to treat tinnitus with laser light is the lack of proper diagnosis of the patients. Quite a few of these have a somatosensory background (the problem is basicly muscluar). Irradiation into the ear will then have no effect.

References: Bjorne A, Agerberg G. Reduction in sick leave and costs to society of patients with Ménière´s disease after treatment of temporomandibular and cervical spine disorders: A controlled 6-year cost-benefit study. Cranio. 2003; 21 (2): 136-143. Bernhardt O, Gesch D, Schwahn C, Bitter K et al. Signs of temporomandibular disorders in tinnitus patients and in a population-based group of volunteers: results of the Study of Health in Pomerania. J Oral Rehabil. 2004; 31 (4): 311-319. Levine RA, Abel M, Cheng H. CNS somatosensory-auditory interactions elicit or modulate tinnitus. Exp Brain Res. 2003; 153 (4): 643-648. Tullberg M, Ernberg M. Long-term effect on tinnitus by treatment of temporomandibular disorders: a two-year follow-up by questionnaire. Acta Odontol Scand. 2006; 64 (2): 89- 96.

On 2015 Jan 22, Larry Parnell commented:

This paper was my selection for Paper of the Week for 19-23 Jan 2015.

On 2015 Feb 18, Arnaud Chiolero MD PhD commented:

Primordial prevention is a key strategy for initiating prevention of cardiovascular diseases early in life. This commentary gives an elegant point of view on some experimental evidence on childhood intervention to improve cardiometabolic health.

On 2016 Mar 28, Mones Abu-Asab commented:

The positive aspect of this review is that it highlights phylogenetics as “likely to have an important impact on patient care.” However, this is a rambling review on metastasis without a critical evaluation of the literature on the topic or good conclusions on how phylogenetics would make a difference in studying metastasis. It doesn’t discuss some experimental work on metastasis (e.g., PMID: 18755941), or analytical phylogenetic approaches that discuss metastasis or the conceptual framework of cancer progression (e.g., PMID: 21319991, 23548567).

On 2015 Feb 08, Arnaud Chiolero MD PhD commented:

Contrary to statements of several experts, this study suggests that cardiovascular health of children may not have worsened despite the increase in obesity.

On 2015 Jan 30, Alvaro Alonso commented:

It is well-known that food-frequency questionnaires (FFQ) are suboptimal tools to assess dietary sodium intake. Thus, not finding an association between FFQ-assessed sodium and the study endpoints is expected, but does not provide any useful information about the health effects of sodium intake.

On 2015 Feb 08, KEVIN BLACK commented:

This is a fascinating clinical problem. The following quote may be of interest:

"[T]hree of 20 patients with both TS and OCD met criteria for bipolar disorder [53]. Given a population prevalence for bipolar disorder of <1%, the 15% prevalence here is remarkable. However, a study from Spain found that 12% of 90 consecutively examined patients with TS had either bipolar disorder or schizoaffective disorder, and an additional 21% had bipolar II disorder or cyclothymia [54]. The majority of these patients (80%) also had OCD, and many of them reported fewer tics during depressive episodes and more with manic episodes [54]. This surprising prevalence of manic illness in TS+OCD patients is probably not due to excessive symptom reporting in general, as this group reported less panic disorder or agoraphobia than the other groups [53]. Further studies of this difficult to treat subgroup are warranted. ...

"53.    Coffey BJ, Miguel EC, Biederman J, Baer L, Rauch SL, O'Sullivan RL, et al. Tourette's disorder with and without obsessive-compulsive disorder in adults: are they different? J Nerv Ment Dis 1998; 186:201-206. ...

"54.    Berthier ML, Kulisevsky J, Campos VM. Bipolar disorder in adult patients with Tourette's syndrome: a clinical study. Biol Psychiatry 1998; 43:364-370."

-- from Black KJ, Mink JW: Neuropsychiatry of movement disorders. Curr Opin Psychiatry 1999; 12(3):313-319.

On 2015 Feb 25, Gerard Ridgway commented:

In relation to the failure "to replicate all the group differences in GMV reported in previous studies", it would be very helpful to extract GMV for the previously reported coordinates (or clusters) and to report the magnitudes of the group differences on the new data, for example like the plot in Figure 2 does for the significant cluster here. It would be more compelling to support an assertion of an absent effect on such an extracted summary by demonstrating that the confidence interval is reasonably narrow (i.e. confined to trivially small group differences) rather than just that the region is not significant (in this case, after correction for multiple comparisons).

It is also not made clear whether the methods closely match the unreplicated studies. For example, the smoothing of 4mm here seems very low for a VBM study (particularly one using cluster-extent inference), and it would be interesting to know whether previous studies have used similarly low smoothing, or more typical values such as 8mm. As another example, did all previous studies adjust for total intracranial volume? In cases like this, where there is a significant group difference in TIV, I think both the adjusted and unadjusted analyses can be of interest.

On 2015 Jan 26, David D Leedahl commented:

We appreciate this comment. The patient had an extensive list of allergies to antimicrobials, which included multiple beta lactams (penicillin, ertapenem, aztreonam) in addition to several additional antibiotics from other classes. The decision to initiate a desensitization protocol to ceftaroline on the general medical floor outweighed the risk of potentially inducing an allergic reaction. Whether or not the patient would have experienced an allergic reaction to ceftaroline without completing a desensitization protocol, we agree, is uncertain.

On 2015 Jan 20, Eric Macy commented:

An IgE-mediated allergy to ceftaroline was not confirmed in this patient by either skin testing or challenge. Only a very small minority of even clinically significant penicillin allergy confirmed individuals will have IgE-mediated reactions to any specific cephalosporin. Only a small minority of individuals with multiple drug intolerance syndrome have any underlying IgE-mediated allergy to any of the medications they have noted reactions associated with.

On 2016 Jan 20, Stephen Royle commented:

The analysis of cell migration presented in this paper was done using an early version of software which is now maintained at http://github.com/quantixed/CellMigration

On 2015 Mar 13, Albert Erives commented:

Animal genomes do not encode the eukaryotic ClpB chaperones, Hsp104 and Hsp78 (See: Metabolic and chaperone gene loss marks the origin of animals: evidence for hsp104 and hsp78 sharing mitochondrial enzymes as clients, PMID: 25710177). The so-called CLPB gene annotated in the human genome is a partial bacterial clp family sequence fused to an ankyrin-repeat containing domain. Phylogenetic analysis of this gene from humans and other animals puts it outside of the clpB/HSP104/HSP78 family (see Fig. S2 in the above reference).

On 2015 May 29, Hongjian He commented:

One our main criticisms of this publication was that there was no detailed reporting of number of needles used per treatment, acupuncture sites targeted per treatment, and specific treatment duration and electric stimulation for each patient. Dr. Hinman responded by stating:

"Dr. He suggests lack of acupuncture standardization, treatment infrequency, and no electrical stimulation may explain our findings. However, when comparing acupuncture with sham treatment, a meta-analysis1 found no evidence that needle number or placement; use of electrical stimulation; or number, frequency, or duration of treatments influence acupuncture outcomes"

We looked at the article Dr. Hinman cited (MacPherson et al., PLoS 2013) and she did not do a good job of reading it. They showed that number of needles used per treatment was statistically significantly correlated with effect size. The electrical stimulation had a significantly stronger effect. She incorrectly summarized the study results. Therefore, Dr. Hinman’s statement that needle numbers, length of treatment and electric stimulation have no effect on acupuncture outcomes is incorrect. We still await a sufficient explanation for why these specifics were not reported in her study.

On 2015 Jan 27, William Grant commented:

The paper on hypovitaminosis D by Daniel Podd [1] is useful in drawing attention to the problem but has a number of factual errors. This comment suggests a number of corrections.

Ref. 1 states that vitamins D2 and D3 have equivalent biological activity. Vitamin D researchers disagree on this point [2]. A recent meta-analysis of vitamin D trials found that supplementation with vitamin D3 lowered mortality rate significantly by 10% while supplementation with vitamin D2 increased mortality rate by a non-significant 3 to 15% [3].

Regarding lower vitamin D levels for those who are obese, another suggestion is that volumetric dilution rather than fat sequestration that explains the findings [4]. However, both may be important.

Regarding cancer, the findings for vitamin D levels and breast cancer are very similar to those for colorectal cancer [5]. The problem in determining this has been that prospective observational studies with longer than three-years follow up have not found a significant inverse correlation between vitamin D levels and breast cancer incidence rates [6]. The reason is that breast cancer develops very rapidly, which is why mammography is recommended annually. Case-control studies consistently find strong inverse correlations between vitamin D levels and breast cancer incidence rates [6]. The evidence that solar UVB and vitamin D reduce the risk of cancer incidence and mortality rates for many types of cancer is strong based on geographical ecological studies and laboratory investigations of the mechanisms [7]. Vitamin D plus calcium supplementation has also been found to reduce risk of breast cancer [8].

Regarding vitamin D production from solar UVB: it is very difficult to impossible to make vitamin D in winter for about six months in locations pole ward of about 40 degrees [9]. When the solar elevation angle is below 45 degrees, it is difficult to make vitamin D since most of the UVB is scattered out [9]. Thus, time of day and season are very important considerations regarding vitamin D production. Also important is skin pigmentation. Black Americans have vitamin D levels about 40% lower than white Americans [10].

Regarding calcium supplementation, 1.5 to 2.0 g/d is too high. There is increased risk of cardiovascular disease with higher calcium intake [11]. A recent meta-analysis found that dietary calcium intake of 800 to 1000 mg/d was associated with the lowest risk of cardiovascular disease, with intakes above 1200 mg/d associated with significantly increased risk [12].

Regarding the best time to take vitamin D, it is with the largest meal of the day [13]. That way vitamin D stays in the intestines longer.

Regarding frequency of dosing, daily may be optimal. A meta-analysis found "Supplementation of intermittent, high dose vitamin D may not be effective in preventing overall mortality, fractures, or falls among older adults." [14]. By intermittent, they meant once or once a month. Others have shown good results for weekly dosing. Daily dosing is similar to being in the sun daily, which is the physiological way to produce vitamin D.

While there is mounting evidence that vitamin D has important health benefits [15], there is also mounting evidence that some of the benefits attributed to vitamin D may, in fact, be due to other effects of solar UVB exposure. Non-vitamin D effects of UV exposure have been found for multiple sclerosis [16, 17], hypertension/cardiovascular disease [18] and intestinal cancer [19]. Thus, midday sun exposure may be a better option for increasing vitamin D levels when possible.

An additional critique of Ref. 1 can be found at http://vitamindwiki.com/tiki-index.php?page_id=6196

References 1. Podd D. Hypovitaminosis D: A common deficiency with pervasive consequences. JAAPA. 2015;28(2):20-26. 2. Heaney RP, Recker RR, Grote J, et al. Vitamin D3 is more potent than vitamin D2 in humans. J Clin Endocrinol Metab. 2011;96(3):E447-52. 3. Chowdhury R, Kunutsor S, Vitezova A, et al. Vitamin D and risk of cause specific death: systematic review and meta-analysis of observational cohort and randomised intervention studies. BMJ. 2014;348:g1903. 4. Drincic AT, Armas LA, Van Diest EE, Heaney RP. Volumetric dilution, rather than sequestration best explains the low vitamin D status of obesity. Obesity (Silver Spring). 2012;20(7):1444-8. 5. Grant WB. Relation between prediagnostic serum 25-hydroxyvitamin D level and incidence of breast, colorectal, and other cancers. J Photochem Photobiol B, 2010;101(2):130–6. 6. Grant WB. Effect of interval between serum draw and follow-up period on relative risk of cancer incidence with respect to 25-hydroxyvitamin D level; implications for meta-analyses and setting vitamin D guidelines. Dermatoendocrinol. 2011;3(3):199-204. 7. Moukayed M, Grant WB. Molecular link between vitamin D and cancer prevention. Nutrients. 2013;5(10):3993-4023. 8. Bolland MJ, Grey A, Gamble GD, Reid IR. Calcium and vitamin D supplements and health outcomes: a reanalysis of the Women's Health Initiative (WHI) limited-access data set. Am J Clin Nutr. 2011;94(4):1144-9. 9. Engelsen O. The relationship between ultraviolet radiation exposure and vitamin D status. Nutrients. 2010;2(5):482-95. 10. Ginde AA, Liu MC, Camargo CA Jr. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169(6):626-32. 11. Bolland MJ, Grey A, Reid IR. Calcium supplements and cardiovascular risk: 5 years on. Ther Adv Drug Saf. 2013;4(5):199-210. 12. Wang X, Chen H, Ouyang Y, et al. Dietary calcium intake and mortality risk from cardiovascular disease and all causes: a meta-analysis of prospective cohort studies. BMC Med. 2014;12:158 13. Mulligan GB, Licata A. Taking vitamin D with the largest meal improves absorption and results in higher serum levels of 25-hydroxyvitamin D. J Bone Miner Res. 2010;25(4):928-30. 14. Zheng YT, Cui QQ, Hong YM, Yao WG. A meta-analysis of high dose, intermittent vitamin D supplementation among older adults. PLoS One. 2015;10(1):e0115850. 15. Pludowski P, Holick MF, Pilz S, et al. Vitamin D effects on musculoskeletal health, immunity, autoimmunity, cardiovascular disease, cancer, fertility, pregnancy, dementia and mortality- a review of recent evidence. Autoimmun Rev. 2013;12(10):976-89. 16. Knippenberg S, Damoiseaux J, Bol Y, et al. Higher levels of reported sun exposure, and not vitamin D status, are associated with less depressive symptoms and fatigue in multiple sclerosis. Acta Neurol Scand. 2014;129(2):123-31. 17. Wang Y, Marling SJ, Beaver EF, et al UV light selectively inhibits spinal cord inflammation and demyelination in experimental autoimmune encephalomyelitis. Arch Biochem Biophys. 2014;567C:75-82. 18. Liu D, Fernandez BO, Hamilton A, Lang NN, et al. UVA irradiation of human skin vasodilates arterial vasculature and lowers blood pressure independently of nitric oxide synthase. J Invest Dermatol. 2014;134(7):1839-46. 19. Rebel H, der Spek CD, Salvatori D, et al. UV exposure inhibits intestinal tumour growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet. Int J Cancer. 2015;136(2):271-7.

Disclosure I receive funding from Bio Tech Pharmacal (Fayetteville, AR), MediSun Technology (Highland Park, IL), and the Vitamin D Council (San Luis Obispo, CA).

On 2018 Jan 05, Alexander Wolf commented:

The authors decorated the vertical axis of Figure 1b with four numbers ("10","0.0","0.01","0.001") next to four equally spaced markers, reminiscent of a logarithmic scale. The plot being fold change, I can't help to get the feeling these numbers are meant to be "100","1","0.01","0.0001" ? Might the authors please explain or clarify this ?

On 2015 Jan 21, Wichor Bramer commented:

I think the article is a very interesting overview of all quality assesment methods currently available, however, I strongly advise a thorough editing round by a native speaker, because the quality of the English language is rather low.

On 2015 Jan 23, Madhusudana Girija Sanal commented:

The Cancer Stem Cell-Bauble!

Does cancer stem cell exist after all? Yes or no because the definition is in the eye of the beholder! Cancer is well defined, stem cells are well defined, but cancer stem cell is unfortunately not the sum of these two and that is where it is poorly defined. Protein markers will come and go on the surface or inside. Currently it is like the definition of beauty or social justice! After all (most, if not all) cancers are a dynamic state of cells with respect to their epigenetic and genetic status during their course of evolution, selection and survival. So, what is NOT a cancer-non-stem cell today can become cancer stem cell tomorrow and vice versa detected by the micro and macro environment and the evolutionary pressure. Cells which have a "very loose" epigenetic stability (on a background of one or more 'oncogene' mutations) are the ideal candidates for cancer "stem" cell. This may be a better definition for those who really want to see that cancer stem cells do exist and the see that concept of cancer stem cells survive! Or wait for the bubble to burst!

On 2015 Jan 29, Matthew Shirley commented:

"A somatic activating mutation in guanine nucleotide-binding protein alpha-q (GNAQ) that up-regulates the expression of mutant Gαq protein"...

I believe the authors meant that the mutation increases the activity, not the expression of the mutant GNAQ protein product.

On 2015 Feb 09, Benoit Kornmann commented:

Superresolution microscopy has become an indispensable tool in cell biology. Several approaches exist, all aimed at seeing smaller and smaller objects.

Here the approach to superresolution microscopy is different. Instead of trying to image small objects, the idea is to 'inflate' the object before imaging, such that it becomes big enough for standard microscopy.

The procedure starts like a standard immuno-fluorescence, but before imaging the sample, it is infused with a resin. During polyerization, the fluorophore that is on the secondary antibody becomes covalently linked to the polymer.

All proteins are then digested away and the polymer -- now bearing an imprint of the sample in the form of bound fluorophores –- is dilated to an extended conformation by desalting, leading to an isotropic enlargement of the imprint. The imprint can then be imaged at superresolution using a standard microscope.

Such a method is, of course, limited to fixed samples, but brings several advantages. For instance, the deproteination of the sample reduces scattering, allowing the imaging of thick samples.

On 2015 Apr 01, Mihaela Gadjeva commented:

The paper does not properly cite published work in the field of NETosis and P. aeruginosa susceptibility to NET-induced killing. The authors seem unaware of published work (Distinct susceptibilities of corneal Pseudomonas aeruginosa clinical isolates to neutrophil extracellular trap-mediated immunity. Shan Q, Dwyer M, Rahman S, Gadjeva M. Infection and Immunity PMID: 25047845-available at the time of submission of the above manuscript) that documents in vivo NET production during ocular infection with P. aeruginosa and demonstrates that P. aeruginosa produces outer membrane vesicles (OMV) in response to NET-induced stimulation. Contrary to the claim that no reports have shown before in vivo NET production in response to P. aeruginosa-induced infection, our work available online in July 2014, presented evidence that P. aeruginosa induced NETs in vivo at the ocular surfaces following ocular infection. Further, authors reported that P. aeruginosa secreted OMVs in response to DNA pressure without citing our work on NET-induced P. aeruginosa OMV production. It is clear that in addition to new data, this paper reports findings that agree and confirm our results and should be presented as such, rather than as data reported for the first time.

On 2015 Aug 12, Lydia Maniatis commented:

There are many problems with this paper, but the most concrete is that the authors' theoretical position is contradicted by fact.

They are assuming that, if we replace a black check on a checkerboard with a grey one, such that the latter is adjacent to white checks, it will be subject to contrast, and thus appear darker than a grey check replacing a white check, which will (supposedly) lighten due to the adjacent black checks.

Simply based on the fact that contrast tends to occur in the context of a figure-ground relationship, which here is lacking, we might question and test this assumption, rather than treating it as given. DeValois and DeValois (1975) created the corresponding demonstration, which may be viewed here http://www.shapirolab.net/Graphics/Icons/Checker Board Icon.jpg. The check surrounded by white checks lightens, and vice versa. The fact that the checks that the authors' are assuming are undergoing contrast are actually undergoing assimilation completely undermines their arguments.

(Adelson makes the same incorrect assumption about checkerboard contrast here: http://web.mit.edu/persci/people/adelson/checkershadow_description.html)

Beyond this, the authors are attributing the relative lightness of pair of checks appearing to differ in illumination to contrast (incorrectly, as discussed above) and of a pair ellipses to assimilation, when both effects can (only) be explained by the same process of compensation for illumination.

On 2015 Jan 31, Stuart RAY commented:

This is an important analysis. The Lao story is a great illustration of evolution in an isolated epidemic, while the other isolates' lacking evidence for recombination highlights the remarkably rare frequency with which HCV recombines. Kudos to the authors for this contribution, including the sequences deposited into public databases.

On 2016 Apr 20, Chandan Kumar commented:

In COSMIC, a mutation in MEN1, p.T541A (c.1621A>G, refers to 22 cases of hemangioblastoma from this study. This must either represent a hotspot mutation inadvertantly missed out from the study, or an experimental artifact, that got submitted and went on to populate COSMIC database. If confirmed to be a hotspot, this would be really interesting to follow up and potentially mechanistically associate with VHL loss. If however, it might just be an NGS artifact, may be helpful to withdraw from COSMIC. Thanks.

On 2015 Jul 07, Bill Cayley commented:

A good example of when "less" is "more": https://lessismoreebm.wordpress.com/2015/07/07/comparing-non-sterile-to-sterile-gloves-for-minor-surgery-a-prospective-randomised-controlled-non-inferiority-trial/

On 2015 Jan 15, David Mage commented:

SIDS is not "inextricable." It only appear so to the non-mathematically inclined because the cited "Triple-Risk Hypothesis" cannot predict the mathematical form of the universal SIDS age and gender distributions.

Perhaps SIDS can be unlocked by explaining why SIDS throughout the world has a fixed Johnson SB (4-parameter lognormal) age distribution and a binomial (pMale = 0.61) gender distribution, that requires a mathematical insight that is not often found in the SIDS community of pediatricians and pediatric pathologists. For example, the reader can see the three articles described here, as well as our other SIDS articles in PUBMED:

"The Consistent 50% Excess Male Infant Mortality from Sudden Infant Death Syndrome (SIDS) and Other Respiratory Diseases is Evidence of an X-Linkage." David T Mage and E. Maria Donner, Review Article: J Mol Genet Med 2014, 8: 123 doi: 10.4172/1747-0862.1000123

"All sudden and unexplained infant respiratory deaths may result from the same underlying mechanism." David T. Mage, E. Maria Donner, Mechtild Vennemann, Peter Fleming, Katia Sol-Church, Rebecca Drake, Sam Gulino. Scandinavian Journal of Forensic Science 2012; 18(1):2-10.

"Is excess male infant mortality from sudden infant death syndrome and other respiratory diseases X-linked?" Mage DT, Donner EM. Acta Paediatr. 2014 Feb;103(2):188-93. doi: 10.1111/apa.12482. Epub 2013 Dec 20. PMID: 24164639

On 2016 Feb 18, Abdulla A-B Badawy commented:

None

On 2016 Feb 29, Simon Young commented:

In the J Psychopharmacol article under discussion Badawy and Dougherty “concluded that the ATD formulation of Young et al. (1985) may involve not only serotonin depletion, but also that of DA and possibly also NA, because it also induces a decrease in the [Phe + Tyr]/[BCAA + Trp] ratio, thereby decreasing Tyr availability to the brain.” Their concern was that “This could impact catecholamine-specific behaviours and those influenced by catecholamines”. They now argue that brain tyrosine may be increased, rather then decreased as argued in the article, because while some studies find a decrease in the ratio others find an increase, and because CSF tyrosine shows small increases at later times after administration of the 100g ATD mixture, when CSF is sampled for 12 hours Carpenter LL, 1998. This raises two important issues.

(a) The fact that the ratio increases in some circumstances and decreases in others supports my contention that relatively small changes in the ratio do not provide useful information.

(b) Badawy and Dougherty fail to respond to the fact that CSF HVA does not change after administration of the ATD mixture Carpenter LL, 1998, indicating that synthesis of dopamine (and hence synthesis of its metabolic product norepinephrine) is not altered. The small increase in tyrosine with no increase in dopamine synthesis is not surprising as human brain tryptophan hydroxylase is regulated not only by substrate availability but also by product inhibition and phosphorylation Almås B, 1992.

Badawy and Dougherty agree that protein intake can alter cognition, and presumably would agree that the “whey protein fraction glycomacropeptide” that they suggest using in ATD studies also may influence cognition. They do not respond to the point I made that normal physiological mechanisms (e.g. food intake and diurnal rhythms) can alter cognition. Therefore, the issue is not whether cognition will change over time after the administration of a control amino acid mixture, but whether such a change is outside the normal physiological range. As long as an ATD mixture and its control mixture differ only in tryptophan levels any change mediated by factors other than changes in tryptophan will occur after control and ATD treatments. Therefore, the difference in the effects of the two mixtures will be due only to changes in tryptophan. Badawy and Dougherty suggest “that it is prudent to use a control formulation that exerts a zero effect on all ratios”. As discussed above I do not feel that small changes in ratios are reliable indicators of anything occurring in the brain, an assertion not disputed by Badawy and Dougherty. Even if one did believe in the validity of small changes in ratios, the discussion above illustrates that ratios can vary from one study to another (both between different labs and also within labs). Therefore, Badawy and Dougherty’s suggestion “to use a control formulation that exerts a zero effect on all ratios” is probably not realistic.

I still believe that the criticisms of Badawy and Dougherty of the 100g ATD mixture are not based on experimental evidence.

On 2016 Feb 18, Abdulla A-B Badawy commented:

We thank Young for his second response and agree with him on many of his statements. We also confirm in agreement with Young that our major concern is the specificity of his control formulation for ATD (and also that of the control formulation for ATPD). Young quotes the increase in CSF [Tyr] reported by Carpenter LL, 1998 as evidence against a decrease in the Tyr ratio after ATD. Others have in fact reported increased Tyr ratios after ATD (see Booij L, 2005) and we have never questioned these findings in studies using the ATD test formulations. The data reported by Carpenter LL, 1998 Booij L, 2005 demonstrate that the ATD test formulation does increase Tyr availability to the brain. In our opinion, this confirms the lack of specificity of the ATD formulation for serotonin. We now propose further that the Tyr and Phe contents of the ATD formulation of Young SN, 1985 should be decreased. The level of such a decrease should be determined in conjunction with our proposed lowering of the BCAA content. To prevent a decrease in Tyr availability to the brain after acute Trp loading (ATL), investigators will need to optimise the ATL formulation by titrating levels of Trp, Tyr and Phe along with those of BCAA.

Regarding the control formulation, our point is that, while decreases in ratios of 30-40% may not influence behaviours such as mood and cognition in normal subjects, they may do so in subjects at risk, e.g. depressed patients, who already have a ~ 30% lower [Trp] and [Trp]/[CAA] ratio (Badawy AA, 2013). Thus, a low-dose ATD (25g) given to remitted depressed patients is associated with altered cognitive function, but not mood (Booij L, 2005; Haddad AD, 2009) and it was suggested (Booij L, 2005) that the cognitive changes observed are more sensitive markers of 5-HT function than symptoms, unless different mechanisms apply. We maintain our position that it is prudent to use a control formulation that exerts a zero effect on all ratios and this is best achieved by lowering the BCAA content from the usual 30% to 18%.

Our suggestion of lowering the BCAA content of both the control and test formulations, which can, in addition to normalising the relevant ratios, minimise the effects of BCAA on behaviour, including cognitive function, is not without support. BCAA are nitrogen donors for the synthesis of glutamate and GABA and can thus influence cognition by modulating glutamatergic function (see references in Badawy AA, 2015). Enhanced cognition by BCAA in athletes has been widely reported. Dietary BCAA ameliorate injury-induced cognitive impairment (Cole JT, 2010). Young rightly states that food intake can also enhance memory and it is interesting in this regard that cognition is enhanced by a high protein meal, which has been attributed to increased levels of BCAA and Phe (Jakobsen LH, 2011). Objective cognitive function is variously influenced acutely by the balance between carbohydrate and protein within a few hours following food intake, with cognitive performance being enhanced by a high protein or a balanced meal, but not by a high carbohydrate meal (Fischer K, 2002). These latter authors also attributed this enhancement to changes in LNAA ratios almost certainly involving the release of BCAA from proteins. In our opinion, enhancing cognition by the BCAA in the ATD control and test formulations could impact mood changes and it is therefore desirable to minimise this confounder in ATD studies by lowering the BCAA content.

We, and no doubt also Young, hope that our exchanges will stimulate the psychopharmacological research community to consider the various issues discussed in our comments in future studies.

On 2016 Feb 08, Simon Young commented:

I thank Badawy and Dougherty for their comments. The main points of disagreement concern the specificity of the 100g ATD mixture, and whether the relevant control mixture might lower serotonin synthesis and therefore mood, cognition and behavior. The arguments that Badawy and Dougherty make on both points depend on the plasma ratio of the relevant amino acid precursors to the LNAA. They suggest that the 100g ATD mixture lowers dopamine synthesis, as the ratio of Tyr or Phe + Tyr to the LNAA, while varying greatly between different studies, declines by up to 60%. They do not comment on the study I cited in my original comment showing that CSF tyrosine remained unchanged for the first few hours after the amino acid mixture, but rose slightly between about 5 and 10 hours after the mixture Carpenter LL, 1998. CSF levels of the dopamine metabolite homovanillic acid (HVA) remained unchanged. Measurement of biogenic amine precursors and metabolites has been used to study human CNS metabolism for over 50 years, and while it is not a perfect method it is generally accepted as a valid method by the scientific community, as indicated by the huge number of articles using this technique in the literature. This raises the question why the conclusion from plasma amino acid ratios differs from the CSF study concerning the specificity of the 100g ATD mixture.

The ratio of Trp or Tyr or Phe + Tyr to the LNAA is known to be only an approximation of the rate of transport of an amino acid across the blood-brain barrier, as it does not take into account the different affinities of the different amino acids for the transporter. This can be done for rats using a calculation based on Michaelis-Menten kinetics Smith QR, 1987, but it cannot be done for humans as the affinities of the different amino acids for the transporter in human brain are not know. Badawy and Dougherty point out that there is a correlation between CSF Trp and the plasma [Trp]/[LNAA] ratio. However, after ATD the Pearson’s correlation was r = 0.41 Moreno FA, 2010. R2 was 0.168, indicating that less than 20% of the variance in the plasma [Trp]/[LNAA] ratio is related to CSF Trp values. The inaccuracy of the plasma ratios as a measure of changes in brain amino acids explains why they suggest that the 100g ATD mixture causes a decline in brain tyrosine and dopamine synthesis, while the CSF measures indicate that this is not so. While the ratios of Trp, Tyr, or Phe + Tyr to the LNAA may be useful to demonstrate that ATD or APTD was effective in depleting precursor levels in any participant, where the effects are very large, these plasma measurements do not provide reliable information when the changes are smaller.

Badawy and Dougherty suggest that the control mixture should have a lower level of the branched chain amino acids (BCAA) than the control mixture used with the 100g ATD mixture because (i) this will decrease the lowering of the [Trp]/[LNAA] ratio, and (ii) reducing the BCAA content “can also minimise the BCAA effects on cognition and hence potential modulation of behavioural outcomes”. While I am not aware of any evidence for BCAA influencing behavior in humans, I suggest that all control mixtures used in ATD studies are likely to alter cognition. Meals can enhance memory, and that includes meals of carbohydrate, fat and protein Kaplan RJ, 2001. If protein meals can enhance cognition then amino acid mixtures may also have this effect. In addition there may be changes in cognition, from baseline to 5 hrs after amino acid administration, related to other factors such as the participants’ diurnal rhythms Blatter K, 2007, possible stress related to participation in the study, and so on. The issue that is not whether there will be changes in cognition in the control participants, but whether factors that may alter cognition, other than tryptophan availability, are different in the control and ATD conditions. The BCAA in the 100g ATD mixture and the relevant control mixture are the same, and it is therefore an appropriate control mixture.

The other main issue raised by Badawy and Dougherty concerns the lowering of the [Trp]/[LNAA] ratio in control mixtures. They mention a decline of 8% in this ratio with their recommended formulation and of around 40% with the control mixture for the 100g depletion mixture. On the other hand after ATD with the 100g mixture the decline in the [Trp]/[LNAA] ratio is well over 90% Predmore DB, 1976. As mentioned above, given that the [Trp]/[LNAA] ratio is a relatively poor index of tryptophan transport into brain, smaller changes in the ratio are not reliable. However, even if there were a decline in tryptophan availability for transport into brain of 40% would this alter serotonin function? Competition between the different amino acids for the transporter implies that the transporter is relatively close to saturation with amino acids. In this circumstance the actual change in transport of amino acids will be less than the change in plasma availability of the amino acids, as measured by plasma levels. Furthermore, tryptophan hydroxylase is about half saturated with tryptophan in human brain Young SN, 1981 so the decline in serotonin synthesis will be less than the decline in brain tryptophan. Will a relatively small decline in serotonin synthesis lead to a decrease in serotonin function? Little is known about this issue in human brain. However, for a decline in serotonin synthesis to alter serotonin function there must be a decline in serotonin release. This presumably implies a decrease in the serotonin content of vesicles. Another possibility is that a decline in synthesis leads to decreased catabolism of serotonin with no change in the vesicle serotonin content. Furthermore, given the presence of serotonin autoreceptors regulating, among other factors, the rate of firing of serotonin neurons, could a decrease in serotonin cause compensatory changes such an increase in neuronal firing? The factors mentioned above presumably explain why a very large decrease in plasma tryptophan availability is needed to see a lowering of mood Van der Does AJ, 2001, a change presumably mediated by lowered serotonin function. Thus, while the control mixture used with the original 100g ATD mixture probably does decrease the availability of tryptophan to the brain by a small extent, the decline is very much less than that needed to see a decline in mood. Furthermore, as pointed out in my first comment variations in tryptophan availability, as measured by the plasma ratio, vary over a 2-fold range under normal circumstances. Badawy and Dougherty provide no information demonstrating that the control mixture used with the original 100g ATD mixture causes any change, due to changes in serotonin function, in mood, behavioral or cognition. Badawy and Dougherty’s hypothesis could be tested by varying the tryptophan content of the control mixture to see if the original control mixture causes different outcomes in measures of mood, behavior or cognition, relative to a mixture that is identical except that it contains enough tryptophan to keep the [Trp]/[LNAA] ratio constant over time. I would be interested to see the results of such a study.

In relation to side effects, Badawy and Dougherty mention again one of their studies where there were side effects, and attrition of women, with the 100g ATD mixture. However this is only one study. They do not mention the fact that others had no attrition in studies on women with the 100g ATD mixture, e.g. Yatham LN, 2001. To say that one mixture is significantly better than another as far as side effects or attrition is concerned this would have to be demonstrated using a direct comparison of the different mixtures in the same study. I would welcome such a study comparing the original 100g ATD mixture, and Badawy and Dougherty’s glycomacropeptide based mixture.

In summary, I do not think the criticisms of Badawy and Dougherty are based on firm evidence.

On 2016 Jan 23, Abdulla A-B Badawy commented:

Abdulla A-B Badawy and Donald M Dougherty We acknowledge Professor Young’s important contribution to this field and our recommendations regarding the use of amino acid mixtures are based on his formulation. We proposed lowering the branched-chain amino acid (BCAA) content by 40% (from 30% to 18%) to normalise the [Trp]/[CAA] and the [Tyr + Phe]/[BCAA + Trp] ratios in the control formulation, with appropriate deletions or additions for depletion and loading, on the basis of a review Badawy AA, 2010 of many studies demonstrating Trp depletion by the control formulation of Young SN, 1985 and our experiments with various concentrations of BCAA Badawy AA, 2010. The decrease in the [Trp]/[CAA] ratio by the control formulation led investigators to increase its Trp content from the usual 2.3g (per 100g) to 2.9, 3.1, 4.1 or 4.6g (referenced in Badawy AA, 2015). However, the Trp ratio is still decreased with the 3.1g addition and increased by the larger additions. Young argues this point by quoting data obtained with proteins rather than amino acid mixtures, and although his formulation is based on the composition of human milk, it is still an amino acid formulation. The use of a low ATD formulation dose (25g) as a conservative control may reduce type 1 error, but its BCAA content can influence cognitive behaviour and thus minimise the effect of the larger BCAA dose. Reducing the BCAA content not only normalises the Trp and Tyr ratios Badawy AA, 2010, but can also minimise the BCAA effects on cognition and hence potential modulation of behavioural outcomes. Regarding our criteria for an ideal formulation, we agree with Young that his is the only ATD method validated by CSF measurements. By “robust”, we meant biochemical and not behavioural or other parameters. In the study by Moreno FA, 2010 however, CSF [Trp] did not correlate with serum Trp, but rather with the [Trp]/[LNAA] ratio. No data on serum Tyr or Phe were provided and the sham control treatment lowered the [Trp]/[LNAA] ratio by 25% at 5h. In our study Badawy AA, 2010 the corresponding decrease with the same formulation was 36% at 5h and slightly stronger (40-45%) at earlier time intervals. By contrast, only an 8% decrease in the ratio is observed with our recommended formulation. <PMID: 19896342 > calculated the ratio based on amino acid concentrations in µg/ml rather than µM. Because Trp has the largest molecular mass among LNAA, their calculation will have exaggerated [Trp] and minimised [LNAA], thereby minimising the decrease in the ratio. Our “tolerable” side effects criterion applies to somatic bodily symptoms rather than mood or cognition and drop-outs in our study involved only females receiving the 100g formulation for ATD and ATL (acute Trp loading) Dougherty DM, 2008 .Ours is therefore the only study demonstrating that a 50g dose of the ATD or ATL formulation does not cause attrition. This female sensitivity has previously been reported Sobczak S, 2014 and many studies have demonstrated the impact of somatic symptoms on attrition Dougherty DM, 2008. As Young suggests, 5-HT receptors and bright light may be factors and these and other potential mediators clearly require investigation. We also agree with Young that other factors including interactions between participants and with research staff can impact behavioural outcomes. Our approach is specific for the biochemical effects of the formulations and has to be considered along with other strategies.<br> Finally, we should like to address the question of specificity of the Young SN, 1985 formulation for serotonin. From the data in Table 3 by Leyton M, 2000, the [Trp]/[LNAA], [Phe]/[LNAA], [Tyr]/[LNAA] and [Phe + Tyr]/[BCAA + Trp] ratios are decreased by the control (balanced) formulation at 5h by 41%, 40%, 29% and 38% respectively. Many other studies have reported decreases in control formulations in the [Trp]/[CAA] ratio of up to 61% and in the [Phe + Tyr]/[BCAA + Trp] ratio of 40-60% (see Badawy AA, 2010). Thus, more studies have demonstrated decreases in the above ratios, compared to the 3 quoted by Young demonstrating no change. A recent ATD and ATPD (acute Tyr and Phe depletion) study Hildebrand P, 2015 in which the content of BCAA was 21.8% (closer to our recommended 18% than the traditional 30% of Young SN, 1985) showed that the control formulation did not alter the [Trp]/[CAA] ratio. Thus, until we establish why ratios are decreased in some, but not other, studies, it is prudent to aim for accuracy and ensure that ratios are not altered by a supposedly balanced control formulation. In subjects with normal but borderline ratios, a 30-40% decrease in ratios may tilt the balance to a state of depletion sufficiently to influence behavioural measures and their quantification in test subjects. In the study by Hildebrand P, 2015, the amino acid mixture was administered based on body wt: a further important aspect of standardization of the methodology. The control formulation for ATD and ATPD used by these latter authors included only 8 amino acids (instead of the 15 used by Young), which were the 3 BCAA plus Trp, Phe, Met, Threo and Lys. This control formulation with a Phe content of 9.24 g/70 kg, (compared with 5.7g in the <PMID: 3931142 > formulation) decreased the Tyr ratio by ~36% at 5h, thus illustrating the ability of Phe loading to decrease Tyr availability to the brain. This is one potential mechanism of the defective catecholamine synthesis in phenylketonuria (PKU). We do not doubt the low cerebral activity of Phe hydroxylase in PKU, although rates could be as high as 15-18% of those in controls van Spronsen FJ, 1998. We merely suggested an additional mechanism of decreased catecholamine synthesis, that of possible inhibition of cerebral Tyr hydroxylase (TH) by any excess Tyr that could be formed from Phe by TH. Human recombinant TH exhibits an equal Vmax for Tyr and Phe, but its Km for Phe is 8-fold higher than for Tyr Martínez A, 1993. Compared with controls, PKU patients exhibit a 6.4- and 4.6-fold higher plasma and CSF [Phe] respectively, a 1.8-fold lower plasma, but a 2.3-fold higher CSF, [Tyr] Ratzmann GW, 1984. Thus, whereas the plasma to CSF ratio of Phe in PKU resembles that of controls, the corresponding ratio for Tyr is significantly lower in PKU. The authors suggested that this may result from intracellular changes in brain metabolism and we suggest that Phe hydroxylation by TH may be responsible for this relative CSF Tyr elevation. Inhibition of catecholamine synthesis in PKU could therefore also result from substrate inhibition of TH by the likely excess of Tyr formed from Phe as suggested by us and indeed, as suggested by Young, by Phe inhibition of TH.

On 2016 Jan 10, Simon Young commented:

Badawy and Dougherty raise a number of important points in this paper, but there are number of issues that need discussion.

1. The authors suggest that the original 100g amino acid mixture used for acute tryptophan depletion (ATD), which remains the most commonly used mixture in ATD studies, lacks specificity and causes a lowering of dopamine and possibly norepinephrine. The authors say the 100g mixture lacks specificity because the mixture causes a decrease in the plasma [Phe + Tyr]/[CAA] ratio (an index of the transport of the catecholamine precursors into brain) thereby decreasing Tyr availability to the brain. However, they neglect to mention a study that investigated this issue through measurements on cerebrospinal fluid (CSF). Samples of CSF were taken before and after healthy volunteers received the 100g tryptophan-deficient amino acid mixture Carpenter LL, 1998. CSF tyrosine remained unchanged for the first few hours after the amino acid mixture, but rose slightly between about 5 and 10 hours after the mixture. CSF levels of the dopamine metabolite homovanillic acid (HVA) remained unchanged. Why do tyrosine and HVA not decline if the [Phe + Tyr]/[CAA] ratio decreases? Rat brain tyrosine hydroxylase can hydroxylate both phenylalanine and tyrosine, but it is not known whether this is true for the human brain enzyme. However, results from patients with phenylketonuria (PKU), who have very high phenylalanine levels, suggest that the activity of the human brain enzyme towards phenylalanine is not functionally significant. Thus, untreated PKU patients with phenylketonuria have low CSF tyrosine and HVA levels. When patients are treated with a low phenylalanine diet, or with tyrosine, CSF HVA levels increase Lykkelund C, 1988 Lou HC, 1985. Thus, the appropriate ratio to use as an index of tyrosine availability for catecholamine synthesis is [Tyr]/[CAA]. Badawy has stated that [Tyr]/[CAA] declines in a similar way to [Phe + Tyr]/[CAA] after ATD Badawy AA, 2013. However, others have reported that the control and tryptophan-depleted amino acid mixture cause the [Tyr]/[CAA] ratio to decrease slightly Leyton M, 2000, or not at all Golightly KL, 2001. These latter results are consistent with the CSF studies mentioned above. Although the conclusion is not definitive, the results available at this time suggest that the original 100g amino acid mixture does not decrease catecholamine synthesis and there is no convincing evidence it lacks specificity. Thus, there is no need to decrease the CAA in the 100g formula.

2. The authors suggest that the control mixture (the 100g ATD mixture plus 2.3g tryptophan) is inappropriate because it lowers the plasma [Trp/CAA] ratio. A study in which healthy participants were fed diets containing different levels of protein, and their blood was taken during the course of a day, demonstrated that the plasma [Trp/CAA] ratio varies over a two-fold range depending on the protein content of the meals and diurnal variation Fernstrom JD, 1979. The original ATD control mixture is based on the amino acid profile in human milk, so its effects would not be unphysiological to any important extent Young SN, 2013. There is a big separation between the effect of the control and depleting amino acid mixtures, as the level of CSF tryptophan after the depletion mixture is only 16.3% of the level after the control mixture Moreno FA, 2010. Some studies have used a 25g ATD mixture as a control for the 100g ATD mixture and no mood changes were seen after the control treatment e.g. Booij L, 2005. Thus, using a control mixture that causes a small decline in the [Trp/CAA] ratio does not seem to be a problem and, being a conservative control, may decrease the chance of a type I error.

3. The paper suggests various criteria for the ideal amino acid depletion and loading formulations. One of these is “Robust and reproducible changes in the study parameters”. Presumably the “study parameters” refer to biochemical changes rather than changes in mood, cognition or other outcomes. The main parameters the paper refers to are changes in plasma levels of amino acids, and in particular ratios such as [Trp/CAA]. The [Trp/CAA] is a rough index of the uptake of Trp into brain, which will be rough index of the brain tryptophan level, which is only one of several factors that regulates brain serotonin synthesis. A more valid method to obtain information is to look at measures related more directly to brain tryptophan levels and brain serotonin synthesis. An index of these can be obtained by the measurement of tryptophan and 5-HIAA in CSF. The original 100g ATD mixture has been shown to decrease human brain tryptophan and serotonin synthesis in 5 studies using measurements on CSF Carpenter LL, 1998, Moreno FA, 2000, Moreno FA, 2010, Salomon RM, 2003, Williams WA, 1999, and to decrease human brain serotonin synthesis in one study using a positron emission tomography method Nishizawa S, 1997. Currently no other method for ATD has been validated in this way. Another criterion is “Freedom from, or acceptable (tolerable), side effects not leading to attrition”. In discussing this issue the authors fail to mention two important issues. First, serotonin receptors in the gut and brain are involved in nausea and emesis Hasler WL, 1999. Second, bright light, relative to dim light, decreases side effects after ATD aan het Rot M, 2008. This suggests that central changes may be partly responsible for nausea, and that a greater lowering of serotonin, which may be more likely to reveal effects of ATD on mood, behavior or cognition, may also be more likely to induce nausea. Furthermore, there is currently no direct comparison of the 100g mixture with any alternative mixture, so direct evidence that the 100g mixture causes greater side effects is lacking.

4. Standardizing the amino acid formulation for ATD is only one factor that will help to increase the comparability of research reported in different studies. Some important factors have not always been controlled, while others cannot be controlled across studies. For example, the expectations that participants will have, that might influence their responses, could be influenced by the wording of the consent form and the demeanor and empathy of the person interacting with the participants. Other factors can be controlled. For example, bright light can reverse mood effects of ATD aan het Rot M, 2008 so studies should not be carried out in rooms with windows that can let in sunlight, and the light level should be standardized at a particular level. I suggest 200-300 lux. Between taking the amino acid mixture and being tested there is a period of several hours. If the participants are allowed to interact with others during this period this may influence their mood. For most people agreeable interactions are associated with better mood and quarrelsome interactions are associated with worse mood Côté S, 1998. Therefore interactions between research participants and research staff should be minimized. This can be done by keeping research participants by themselves in a room, and minimizing their interactions with research staff, while giving them access to relatively affectively neutral reading material and movies to avoid boredom between giving them the amino acid mixtures and testing. This has been done in my laboratory, e.g. Benkelfat C, 1994, and I suggest that this should also be standard practice in ATD studies.

In conclusion, the original ATD mixture is the only mixture that has been shown to lower serotonin synthesis in human brain, and criticisms of the method are not well supported. Currently it remains the best candidate for a standard mixture to use in ATD studies, although future studies may change this conclusion.

On 2015 Jan 15, William Grant commented:

Vitamin D may explain some of the effect of night shift work and risk of diabetes among African-American women

The paper by Vimalananda and colleagues found that "Long duration of shift work was associated with an increased risk of type 2 diabetes. The association was only partially explained by lifestyle factors and BMI. A better understanding of the mechanisms by which shift work may affect the risk of diabetes is needed in view of the high prevalence of shift work among workers in the USA." [1] In this comment, I outline the evidence that low 25-hydroxyvitamin D [25(OH)D] concentrations due to sleeping during daytime may explain some of the observed effect.

There is evidence from observational studies that low 25(OH)D concentrations are associated with increased risk of diabetes mellitus type 2 [2]. The mechanisms that may explain how vitamin D reduces risk include effects on pancreatic beta cell dysfunction, impaired insulin action and systemic inflammation [3].

Solar UVB exposure is the largest contributing factor to vitamin D production. Those with darker skin produce vitamin D from UVB exposure less efficiently than those with pale skin. As a result, black Americans have significantly lower mean 25(OH)D concentrations than white Americans [4]. This difference has been cited as an important reason for black-white health disparities in the United States [5]. The effect of shift work on risk of diabetes was also discussed in a recent letter to the editor [6].

Thus, raising 25(OH)D concentrations to above 75-100 nmol/L (30-40 ng/mL) may reduce the risk of developing diabetes mellitus type 2 [2] as well as many other adverse health outcomes [8].

References 1. Vimalananda VG, Palmer JR, Gerlovin H, Wise LA, Rosenzweig JL, Rosenberg L, Ruiz Narváez EA. Night-shift work and incident diabetes among African-American women. Diabetologia. 2015 Jan 14. [Epub ahead of print] 2. Song Y, Wang L, Pittas AG, Del Gobbo LC, Zhang C, Manson JE, Hu FB. Blood 25-hydroxy vitamin D levels and incident type 2 diabetes: a meta-analysis of prospective studies. Diabetes Care. 2013;36(5):1422-8. 3. Mitri J, Pittas AG. Vitamin D and diabetes. Endocrinol Metab Clin North Am. 2014;43(1):205-32. 4. Ginde AA, Liu MC, Camargo CA Jr. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169(6):626-32. 5. Grant WB, Peiris AN. Possible role of serum 25-hydroxyvitamin D in Black–White health disparities in the United States. J Am Med Directors Assoc. 2010;11(9):617-28. 6. Grant WB. Low vitamin D concentrations may contribute to the increased risk of diabetes mellitus related to shift work. Occupat Environ Med. 2015;72:161. 8. Pludowski P, Holick MF, Pilz S, Wagner CL, Hollis BW, Grant WB, Shoenfeld Y, Lerchbaum E, Llewellyn DJ, Kienreich K, Soni M. Vitamin D effects on musculoskeletal health, immunity, autoimmunity, cardiovascular disease, cancer, fertility, pregnancy, dementia and mortality- a review of recent evidence. Autoimmun Rev. 2013;12(10):976-89.

Disclosure I receive funding from Bio-Tech Pharmacal (Fayetteville, AR) and MediSun Technology (Highland Park, IL).

On 2015 Jun 18, NephJC - Nephrology Journal Club commented:

This study, along with another study in the same area, was discussed on May 27th and June 3rd 2015 in the open online nephrology journal club, #NephJC, on twitter.

Introductory comments are available at the NephJC website. The discussion was quite animated, with more than 50 participants, including nephrologists, fellows and residents.

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

The highlights of the tweetchat were:

• The participants thought true diuretic resistance requires higher doses and correct combinations of diuretics (e.g. furosemide, metolazone and spironolactone), and were hence not convinced with the data presented. Similar concerns were expressed about the severity of heart failure, and the low rate of device usage (e.g. defibrillators)

• Given the negative outcomes from a similar trial with ultrafiltration, CARESS-HF, the discussants were not impressed with the hypothetical increased sodium removal compared with diuresis.

• However, there was unanimity that peritoneal dialysis is a promising approach, and that this should be tested in a properly designed randomized trial. There was much discussion around possible inclusion criteria, comparators (standard care or left ventricular assist devices) and feasibility of arranging peritoneal dialysis. One such trial is already underway in UK and those results will be keenly followed.

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

On 2015 Jan 15, William Grant commented:

Differences in 25-hydroxyvitamin D concentrations may explain Much of the black-white-Hispanic differences in breast cancer-specific survival in the United States

In the paper by Iqbal and colleagues, it was found that black women had higher probability of small-sized breast cancer tumors presenting with distant metastases than whites, as well as the highest proportion of triple-negative breast cancers [1]. Black women also had the highest hazard ratio for death for women with stage I breast cancer and tumors less than 2.0 cm in diameter, with Hispanic women having hazard ratios intermediate between those for blacks or whites. The conclusion, "Much of the difference could be statistically accounted for by intrinsic biological differences such as lymph node metastasis, distant metastasis, and triple-negative behavior of tumors." overlooked the fact that there might be explanations for these "intrinsic differences". In this comment, I outline the evidence that differences in 25-hydroxyvitamin D [25(OH)D] concentrations explain much of the findings.

First, there is strong evidence from geographical ecological studies that breast cancer mortality rates are inversely correlated with solar UVB doses [2]. Solar UVB is the primary source of vitamin D for most people. Since those with darker skin make vitamin D less efficiently than those with lighter skin, in the U.S., whites have the highest mean 25(OH)D concentrations, Hispanics intermediate concentrations, and blacks the lowest concentrations [3]. It was noted that these differences seem to explain the black-white cancer-specific and all-cause differences in survival after diagnosis of 13 types of cancer including breast cancer after consideration of socioeconomic status, stage at diagnosis, and treatment [4]. A recent review also found that higher 25(OH)D concentrations were significantly inversely correlated with breast cancer survival [5]. There is also strong evidence from case-control studies that breast cancer incidence is inversely correlated with 25(OH)D concentration [6, 7]. It is pointed out in that paper that nested case-control studies do not find significant inverse correlations because breast cancer tumors develop so rapidly that 25(OHD from blood drawn at the time of enrollment loses relevance as time increases.

Second, some of the findings noted in Ref. 1 are related to 25(OH)D concentrations. Those diagnosed with triple-negative breast cancer have been found to have lower 25(OH)D concentrations [8-10] and the actions of vitamin D on triple-negative breast cancer have been modeled [11].

While more research is required to fully understand the role of UVB and vitamin D in reducing risk of breast cancer and increasing survival after diagnosis, there is enough support for the role of vitamin D related to breast cancer that women who want reduce their risk of developing breast cancer and increase their chance of surviving after diagnosis should be advised to increase their 25(OH)D concentrations to above 75-100 nmol/L, which could take 1000-4000 IU/d vitamin D3 (cholecalciferol) in the absence of adequate UVB exposure [12]. Doing so would also have benefits for many other health outcomes [12].

References 1. Iqbal J, Ginsburg O, Rochon PA, Sun P, Narod SA. Differences in breast cancer stage at diagnosis and cancer-specific survival by race and ethnicity in the United States. JAMA. 2015;313(2):165-173. 2. Moukayed M, Grant WB. Molecular link between vitamin D and cancer prevention. Nutrients. 2013;5(10):3993-4023. 3. Ginde AA, Liu MC, Camargo CA Jr. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169(6):626-32. 4. Grant WB, Peiris AN. Differences in vitamin D status may account for unexplained disparities in cancer survival rates between African and White Americans. Dermatoendocrinol. 2012;4(2):85-94. 5. Mohr SB, Gorham ED, Kim J, Hofflich H, Garland CF. Meta-analysis of vitamin D sufficiency for improving survival of patients with breast cancer. Anticancer Res. 2014;34(3):1163-6. 6. Grant WB. 25-Hydroxyvitamin D and breast cancer, colorectal cancer, and colorectal adenomas: case–control versus nested case–control studies, Anticancer Res. 2015 Feb;35(2):in press. 7. Grant WB. Effect of interval between serum draw and follow-up period on relative risk of cancer incidence with respect to 25-hydroxyvitamin D level; implications for meta-analyses and setting vitamin D guidelines. Dermatoendocrinol. 2011;3(3):199-204. 8. Rainville C, Khan Y, Tisman G. Triple negative breast cancer patients presenting with low serum vitamin D levels: a case series. Cases J. 2009;2:8390. 9. Peppone LJ, Rickles AS, Janelsins MC, Insalaco MR, Skinner KA. The association between breast cancer prognostic indicators and serum 25-OH vitamin D levels. Ann Surg Oncol. 2012;19(8):2590-9. 10. Yao S, Ambrosone CB. Associations between vitamin D deficiency and risk of aggressive breast cancer in African-American women. J Steroid Biochem Mol Biol. 2013 Jul;136:337-41. 11. LaPorta E, Welsh J. Modeling vitamin D actions in triple negative/basal-like breast cancer. J Steroid Biochem Mol Biol. 2014;144 Pt A:65-73. 12. Pludowski P, Holick MF, Pilz S, Wagner CL, Hollis BW, Grant WB, Shoenfeld Y, Lerchbaum E, Llewellyn DJ, Kienreich K, Soni M. Vitamin D effects on musculoskeletal health, immunity, autoimmunity, cardiovascular disease, cancer, fertility, pregnancy, dementia and mortality- a review of recent evidence. Autoimmun Rev. 2013;12(10):976-89.

Disclosure I receive funding from Bio-Tech Pharmacal (Fayetteville, AR) and MediSun Technology (Highland Park, IL).

On 2017 Feb 06, Ryan Armstrong commented:

I would like to invite the authors to comment on a very critical misstep in their concluding remarks. When comparing the efficacy of two groups/treatments, by failing to reject the null hypothesis it is not acceptable to then accept it. In this case, the authors methodology fails to determine a difference between oil pulling and chorhexidine, but they go on to claim that they are equally efficacious.

If this critical (and misleading) error is not corrected, I would suggest removal of the article and more stringent scrutiny applied to the publisher (who I have contacted some time ago).

On 2015 Apr 22, Stuart RAY commented:

Regarding HCV the authors of this review state, "During pregnancy, pregnant women should be seen regularly by a gastroenterologist to monitor liver biochemical tests and viral load." No citation for this recommendation is cited, and I don't think it's part of the consensus guidelines http://hcvguidelines.org. It seems sensible to monitor liver enzymes for a variety or reason; in contrast, the utility of monitoring HCV RNA levels during pregnancy is dubious. Women with HIV and a high HCV RNA level are at higher risk of transmitting to the child, but it's not clear how monitoring HCV RNA level ("viral load") will have any impact on medical care. Monitoring of HCV RNA levels is only recommended immediately before, during, and shortly after HCV treatment - a different topic.

If there is a sound basis for the authors' recommendation to monitor HCV RNA levels ("viral load") during pregnancy, please provide it.

On 2015 Apr 22, Hans-Peter Müller commented:

See comment here: http://www.ncbi.nlm.nih.gov/pubmed/25687197. Authors response: http://www.ncbi.nlm.nih.gov/pubmed/25697931

On 2015 Jul 10, David Keller commented:

"Natural" skin products made from organic castor bean oil may be no safer than petroleum jelly

Castor bean oil shares many properties with petroleum jelly (petrolatum), including the ability to form a stable oily shield on dry,burned or injured skin. Unlike petrolatum, castor bean oil does not contain traces of hydrocarbons of the type associated with harm to Parkinson's patients. However, castor beans contain ricin, a highly neuro-toxic, naturally occurring lectin. A dose of purified ricin powder the size of a few grains of table salt can kill an adult human (1). Castor beans must be heated and cooked in order to extract the oil, and this heating is also relied upon to denature the ricin in the beans, allegedly making it harmless.

Skin products which contain improperly distilled petrolatum may be contaminated with toxic hydrocarbons; "organic and all-natural" products made from improperly extracted castor bean oil may contain traces of neurotoxic ricin. Thousands of cosmetics, prescription skin medications and over-the-counter creams and ointments contain petrolatum or castor oil. One hopes that the FDA is inspecting the production facilities rigorously.

Reference:

1: Homeland Security News, Frequently Asked Questions about Ricin. Accessed 7/10/2015. http://www.nationalterroralert.com/ricin/

On 2015 Jan 19, David Keller commented:

Petroleum jelly can be contaminated with polycyclic aromatic hydrocarbons; what risks are posed?

Petrolatum (also called petroleum jelly) is commonly applied to the skin as an emollient to treat dryness, chapping, sunburn, mild thermal burns, and "diaper rash" in babies. It is also used as a vehicle for topical ointments and cosmetics. Mineral oil, a related product also known as liquid petrolatum, is actually ingested as a laxative. Both of these preparations start out containing polycyclic aromatic hydrocarbons (PAHs), which must be refined out by distillation. PAHs have been found to be carcinogenic in vitro and, for this reason, the European Union has classified petrolatum as a carcinogen; it can only be sold if its full refining history is known to be satisfactory.

To what degree do PAHs and other hydrocarbon contaminants or constituents of petrolatum cross the skin to enter the bloodstream, and then cross the blood-brain barrier? Does exposure to these hydrocarbons increase the risk of Parkinson's disease? Until their safety is tested, petrolatum, mineral oil and products containing them should be avoided, and replaced with products made from vegetable oils, fats and waxes.

On 2015 Mar 16, Donald Forsdyke commented:

INTRONS FIRST. The author points out that, for the human genome, Chargaff's second parity rule (PR2) remains significant for oligonucleotides extending to 9 nt and concludes that "the phenomenon must be non-random." Furthermore, he adds:

"At present, what is important is to unravel the origin of strand symmetry, which would contribute greatly to the study of the origin and evolution of genomes. First and foremost, it is necessary, also possible, to figure out whether the phenomenon is a result of convergence of genome evolution (Albrecht-Buehler, 2006; Fickett et al., 1992; Forsdyke and Bell, 2004; Lobry and Lobry, 1999) or, on the contrary, an original trait (vestige) of the primordial genome (Zhang and Huang, 2008, 2010; Zhang et al., 2013). If strand symmetry emerged by means of direct selection, the structural feature would be functional since its appearance. Otherwise, the structural feature would not necessarily be functional, or would be exploited to have a function, if any."

It is incorrect to list Forsdyke and Bell as supporters of the "convergence" hypothesis. As elaborated in 2013 Biological Theory 7: 196-203 ("Introns first"), Forsdyke holds PR2 to be an original trait related to the role of nucleic acid structure in the correction of errors by recombination. Thus, the trait has been functional since its appearance. Indeed, the primordial genome - one long "intron" - could not have existed without it. For more on this see Evolutionary Bioinformatics (2011, Springer, New York).

On 2015 Jul 29, Bill Cayley commented:

A good example of when "Less" may be "more" - https://lessismoreebm.wordpress.com/2015/07/29/treatment-of-proximal-humerus-fractures-in-the-elderly/

On 2015 Apr 15, Ann Z Bauer commented:

We commend Frisch and Simonsen on their high quality prospective cohort analysis on ritual circumcision and the risk of autism spectrum disorder (ASD) in young boys. As the authors acknowledge, and we are pleased to see, our ecologic analysis was part of the motivation behind the performance of this study. The finding in this cohort of 342,877 boys born between 1994 and 2003, that circumcised boys were more likely than intact boys to develop ASD before age 10 (HR=1.46, 95% CI: 1.11 -1.93) concurs with our study findings.Bauer AZ, 2013 It is, however, important to note that in our study we did not focus on the psychological consequences of the circumcision procedure as the causal ASD mechanism, rather we were using circumcision exposure as a proxy for the potential paracetamol (APAP, acetaminophen) exposure that may occur with the procedure. Our study aim was to explore at a population level the hypothesis of a relationship between paracetamol exposure and ASD. The authors of this study did point out that they did not have the individual data to explore this but in light of temporal rationale and new research we believe that confounding by paracetamol exposure is a plausible explanation for the observed associations and should be part of the discussion of these findings.

Temporal Rationale: The origins of circumcision predate recorded history, yet autism is considered a relatively new phenomenon, first recognized in 1943 and escalating in incidence, beginning in the 1980’s. History CDC.gov Volkmar FR, 2014 Assuming a significant portion of the recent increase in autism incidence is real, and not strictly a function of greater recognition, something about circumcision would have to have changed during this time period for it to be a significant causal factor in autism. It is difficult to contend that the psychological stress and physical pain related to circumcision has only occurred during the past 35 years. It can, however, be demonstrated that the techniques to manage this pain and stress have changed during this time period. Research beginning in the 1980’s documented the negative consequences associated with inadequate treatment of pain in children. Anand KJ, 1987 Mather L, 1983 Schechter NL, 2008 Prior to the 1990’s many procedures, including circumcision, were generally performed without analgesics. A 1994 study by Howard et al. found that when paracetamol was given regularly for at least the first 24-hour circumcision postoperative period, infants demonstrated decreased response to pain. Howard CR, 1994 This lead to the development of circumcision pain management guidelines by the American Academy of Pediatrics in 1999 suggesting procedures including the use of paracetamol. Anonymous, 1999 The International Evidence Based Group for Neonatal Pain, which included Danish researchers, published their consensus statement on newborn pain management in 2001. This statement suggests the use of paracetamol for postoperative pain including circumcision. Anand KJ, 2001 A perplexing finding in this Frisch and Simonsen Danish study is a statistically significant association between circumcision and the development of autism in the cohort of boys ages 0-4 (hazard ratio (HR)= 1.80, 95% CI 1.25-2.60) while only a very weak association for the boys ages 5-9 (HR=1.15, 95% CI 0.75-1.77). This finding can be explained by the paracetamol hypothesis. The boys that were 5-9 years of age would have been born in 1999 or prior and, based on the timing of guideline development, would likely not have been exposed to paracetamol with the procedure, while the younger boys would likely have received analgesia.

Paracetamol Research: Three prospective cohort studies have found an association between prenatal exposure to paracetamol and adverse neurodevelopment. Brandlistuen RE, 2013 Liew Z, 2014 Thompson JM, 2014 In addition, recent animal data has shown that cognition and behavior may be altered following exposure to therapeutic doses of paracetamol during early development. A recent review by de Fays et al. summaries these finding. de Fays L, 2015

To date, most neurodevelopmental research has focused on prenatal exposure. These findings by Frisch and Simonsen and research identifying time sensitive developmental periods surrounding birth suggest the potential importance of neonate exposure. Wang SS, 2014 Although the evidence presented here is far from conclusive, paracetamol administered along with the circumcision procedure is a plausible causal mechanism for ASD that should not be dismissed and is deserving of further investigation.

On 2015 Jan 13, Dale D O Martin commented:

Take note that N-myristoylation only occurs on N-terminal glycines, hence the N- in N-myristoylation. It occurs following the removal of the N-terminal methionine co-translationally. The only examples of 'internal' myristoylation is in proteins that have been cleaved by caspases to expose new N-terminal glycines. This protein is unlikely to be myristoylated.

On 2015 Feb 27, David Keller commented:

Proposed techniques to preserve blinding in a randomized trial of ethanol capsules versus water

In reply to my letter [1], Dr. Huang writes: "We agree that a large prospective randomized controlled trial will be the most valuable path to evaluate the effects of alcohol on all-cause mortality and cardiovascular health."[2] I agree; there is voluminous epidemiological and observational evidence that mild to moderate ingestion of ethanol has benefits on cardiovascular and all-cause mortality in various populations. What we need now is a randomized interventional trial to confirm and quantify the benefits and the risks of low-dose ethanol, so that physicians can prescribe ethanol to appropriate patients with confidence.

The design of such a trial will be crucial. Dr. Huang wrote: "we are skeptical concerning the use of ethanol capsules. The blindness of the study design would not be guaranteed because the patients in the treatment arm may experience psychotic, cutaneous, or other effects."

I request clarification: what does Dr. Huang mean by "psychotic" effects? Psychosis is described among heavy abusers of ethanol, in both the intoxicated and the withdrawal states. However, the ingestion of mild to moderate doses of ethanol does not normally cause any form of psychosis or of hallucinations. Patients with any history of ethanol abuse would be excluded from a randomized trial.

Unpleasant cutaneous flushing is caused when persons with a genetic deficiency of alcohol dehydrogenase, or a mutated allele of this gene, ingest even small amounts of ethanol.[3] This reaction generally causes these persons to avoid ethanol ingestion, and they should be excluded from a randomized trial.

If there are no other objections to a randomized trial of ethanol, then I would like to propose a toast to its safe and successful completion. Cheers!

References

[1] Keller DL. Ethanol should be subjected to a randomized controlled trial. Mayo Clin Proc. 2015 Jan;90(1):160. doi: 10.1016/j.mayocp.2014.10.013. PubMed PMID:25572202.

[2] Huang C, He QQ. In reply—Ethanol should be subjected to a randomized controlled trial. Mayo Clin Proc. 2015 Jan;90(1):161. doi: 10.1016/j.mayocp.2014.10.012. PubMed PMID: 25572204.

[3] Holmes MV, et al.; InterAct Consortium. Association between alcohol and cardiovascular disease: Mendelian randomisation analysis based on individual participant data. BMJ. 2014 Jul 10;349:g4164. doi: 10.1136/bmj.g4164. PubMed PMID: 25011450; PubMed Central PMCID: PMC4091648.

On 2015 Feb 21, Jon Ebbert commented:

This article can really help the "messaging" you give to patients in clinic.

On 2015 Jul 13, CREBP Journal Club commented:

This study concluded that well conducted ecological and cohort studies in multiple settings were the most appropriate study design to quantify and monitor overdiagnosis in cancer screening programs. Establishment of a team of multinational, unbiased researchers working on internationally agreed standards for ecological and cohort studies was recommended.

The journal club agreed that cohort studies, when conducted in a methodologically sound manner, had a high potential for accurately estimating and monitoring overdiagnosis over time by providing a more “real world” view of overdiagnosis. However, we did not quite agree that it was the best approach as RCTs are known to have the least bias and a high strength of evidence compared to cohort and ecological studies. As the authors of this study have highlighted, we agree that results of the RCTs may lack generalizability due to limited external validity. Therefore we suggest that estimates from both these study designs should be used in conjunction to provide a more accurate as well as generalizable estimate of overdiagnosis in cancer screening programmes.

This review is unique in being the first study which systematically reviews different study methodologies that have been used to estimate overdiagnosis in cancer screening. Thus, we suggest that this may provide the background upon which similar studies could be conducted to estimate overdiagnosis occurring in other chronic disease screening programmes as well.

See CREBP Journal Club for more information

On 2015 Feb 08, Arnaud Chiolero MD PhD commented:

A very useful review. Look also to papers by Etzioni to have another perspective on the quantification of overdiagnosis (Ann Inter Med 2013).

On 2015 Jan 09, William Grant commented:

Differences in vitamin D status may help explain the black-white disparities in breast cancer survival rates

The paper by Coughlin discussed some of the reasons for black-white disparities in breast cancer survival [1]. Overlooked in that discussion was any mention of the role of vitamin D from solar UVB exposure and oral vitamin D intake in reducing breast cancer risk and increasing survival rates. Geographical ecological studies in the U.S. and elsewhere have found significant inverse correlations between solar UVB indices and incidence and/or mortality rates of breast cancer [2, 3] as well as many other types of cancer [3]. An important reason why black women in the U.S. have a greater risk of dying from breast cancer is that due to darker skin pigmentation, they have 25-hydroxyvitamin D [25(OH)D] concentrations about 40% lower than white women [4]. In a review of black-white disparities in cancer survival rates for 13 types of cancer, it was found that after consideration of socioeconomic status, stage at diagnosis, and treatment, blacks an average of 25% (0% to 50+%) increased relative risk of dying after diagnosis of cancer than did whites [5]. There are many other health outcomes for which blacks have poorer outcomes than whites, including cardiovascular disease and diabetes mellitus [6]. Both of these diseases have been linked to low 25(OH)D concentrations in observational studies [7,8]. Thus, any intervention programs for addressing breast cancer disparities among African American women should consider including information about the health benefits of solar UVB exposure and vitamin D supplementation to increase 25(OH)D concentrations to above 30-40 ng/mL [9].

References 1. Coughlin SS. Intervention approaches for addressing breast cancer disparities among African American women. Ann Transl Med Epidemiol. 2014 Sep 8;1(1). pii: 1001. 2. Grant WB. Lower vitamin-D production from solar ultraviolet-B irradiance may explain some differences in cancer survival rates. J Natl Med Assoc. 2006;98(3):357-64. 3. Moukayed M, Grant WB. Molecular link between vitamin D and cancer prevention. Nutrients. 2013;5(10):3993-4023. 4. Ginde AA, Liu MC, Camargo CA Jr. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169(6):626-32. 5. Grant WB, Peiris AN. Differences in vitamin D status may account for unexplained disparities in cancer survival rates between African and White Americans. Dermatoendocrinol. 2012;4(2):85-94. 6. Grant WB, Peiris AN. Possible role of serum 25-hydroxyvitamin D in Black–White health disparities in the United States. J Am Med Directors Assoc. 2010;11(9):617-28. 7. Wang L, Song Y, Manson JE, et al. Circulating 25-hydroxy-vitamin D and risk of cardiovascular disease: A meta-analysis of prospective studies. Circ Cardiovasc Qual Outcomes. 2012;5(6):819-29. 8. Song Y, Wang L, Pittas AG, et al. Blood 25-hydroxy vitamin D levels and incident type 2 diabetes: a meta-analysis of prospective studies. Diabetes Care. 2013;36(5):1422-8. 9. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 2011;96(7):1911-30.

Disclosure I receive funding from Bio-Tech Pharmacal (Fayetteville, AR) and MediSun Technology (Highland Park, IL).

On 2015 Apr 18, Dorothy V M Bishop commented:

This is an impressive paper in terms of sample size for those with aneuploidies, and the results are intriguing and important. It is particularly surprising that the asymmetries in the XXY group look so normal, when –as the authors point out - the one functional study on this group found strikingly reduced functional asymmetry in the Superior Temporal Gyrus (van Rijn et al, 2008). As the authors note, the links between functional and structural asymmetries are far from clear, and this study provides further motivation for doing studies that contrast the two.

I have just one niggling criticism, which is that the authors cite Sun et al as evidence for early asymmetric gene expression in humans. I don't think that the data from Sun et al support their conclusions, and I've explained why in a comment on PubMed Commons. http://www.ncbi.nlm.nih.gov/pubmed?term=Early asymmetry of gene transcription in embryonic human left and right cerebral cortex

On 2016 May 23, Clive Bates commented:

Robert Jackler assumes a priori that attracting adolescents to vaping through flavour descriptors is a bad thing. Saul Shiffman provides a compelling defence of their study showing that teenage interest in flavours was low and this should not concern us much. But this framing may be an over-simplification.

E-cigarette appeal may be good for health

What if the rise in e-cigarette use among adolescents is displacing tobacco smoking, and that this effect accounts for the rapid fall in teenage smoking measured in both the NYTS survey: Tobacco Use Among Middle and High School Students — United States, 2011–2014 and the University of Michigan Monitoring the Future survey, Media release: Teen cigarette smoking drops to historic low in 2015?

In that case, critics must contemplate the idea that e-cigarettes have a harm reduction function among adolescents and that their attractiveness to young people who would otherwise become smokers may be overall a public health benefit. Levy DT, 2017 shows that e-cigarettes create many beneficial pathways for the evolution of nicotine use and abstinence. It is far from clear that obstructing these pathways with policies or campaigning communications is positive for public health.

Scope for adverse unintended consequences

There is already evidence that measures designed to block youth access to e-cigarettes have adverse unintended consequences on youth smoking: see Friedman AS, 2015 and Pesko MF, 2016 on the impact of e-cigarette age restriction laws on cigarette smoking. It is not a great leap of logic to hypothesise that making e-cigarettes less attractive to adolescents would attenuate the decline in smoking that is, or should be, the primary concern in tobacco policy.

The appropriate focus is on adults

The right way to address this issue is not to try to micro-manage adolescent behaviours but to ensure that adults have attractive alternatives to smoking. Restrictions imposed with the misguided purpose of 'protecting' adolescents from very low-risk alternatives to cigarettes could have the effect of harming longstanding adult smokers - the real at-risk group. Several surveys (e.g. Farsalinos KE, 2014) have shown that non-tobacco flavours matter to adults who are using e-cigarettes as an alternative to smoking and are part of the long-term transition away from tobacco use.

Conclusion

Anyone proposing a ban on certain flavours or flavour descriptors needs to assess the risk of harmful unintended consequences - that more adolescents will take up smoking instead of vaping, and that adults smokers will find vaping a less attractive alternative to smoking and never switch, remain as dual users, or relapse back to smoking.

On 2016 Jan 10, Carl V Phillips commented:

I read with interest the exchange between Robert Jackler and Saul Shiffman about this paper and would like to comment on two bits of the exchange. In general, I believe that Shiffman’s responses were compelling, and that they effectively rebutted Jackler’s criticisms. In particular, I agree with his assessment that Jackler basically starts with a premise that the paper’s conclusions are wrong and then speculates about what made them wrong, rather than actually building a case that anything was wrong.

Jackler makes various accusations (some overt, some innuendo) about Shiffman et al. being inappropriately influenced by their sponsor. Shiffman responds by mischaracterizing these as ad hominem. Ad hominem attacks are common in this space, and anyone who departs from a strict tobacco control party line will almost inevitably be the target of them. But the term is also misused in this space, and this is an example. Claiming “Shiffman et al.’s research should be ignored because they consort with those I declare to be the enemy,” would be ad hominem, but there is only a hint of that in Jackler’s comments. Instead, he mostly claims that the research was faulty because of influences of the funder on the design of the particular research. This may be a cheap rhetorical tactic and unsubstantiated innuendo – as Shiffman argues – and it is certainly insulting to the integrity of the authors, but none of that makes it ad hominem.

Jackler introduces one valid scientific concern, and it should be extended to a most of the research in this space. He argues (my paraphrase) that the expressed lack of interest in the products, whatever flavor descriptor was offered, mostly reflects the general hostility toward tobacco products that is inculcated in this population of teenagers. Schiffman responds to the criticism as phrased, quite legitimately, by arguing this was exactly the authors’ point, that flavors are not overcoming the programmed resistance to using the products. But the comment and response skirt the real scientific concern here: All of the research in and around this point produces rough measures that only partially inform the question of interest, which is, “what product characteristics cause different choices in the real world?”

Teenagers’ responses to abstract survey questions, posed by people who presumably feel a lot like the authority figures who have been instilling the anti-tobacco message, probably do just trigger the inculcated response. This makes them a limited measure of whether a flavor option will change someone’s choice when he is presented with the opportunity to try a product. The present study is clearly more informative than anything cited in support claims that flavors have caused a torrent of underage use (let alone claims that attracting underage users is the purpose of interesting flavors, given that adults clearly prefer them; see, for example, my recent report of survey results at http://antithrlies.com/2016/01/04/casaa-ecig-survey-results/). But what was measured is only one contributor to the actions of interest.

The failure to recognize that what is measured is not the same as what is being asked extends throughout this field of inquiry. Jackler asserts that looking at flavor usage patterns would be a better measure, and Shiffman correctly points out that this would be an answer to an different question. It would tell us something that relates the question of interest, though it is even further removed than what the study measured. Yet it is quite common for opponents of product availability to claim that mere demonstrated preference for a particular product feature is evidence that the feature is causing use. Indeed, guessing at what Jackler alludes to as an “extensive body of research” about preferences for flavors of other tobacco products, this describes most of that research. Fuzzy and noisy observations that are probably associated with the question of interest can allow us to modestly update our beliefs. But commentators, including many research authors, make absolute claims, apparently oblivious to the necessary epistemic modesty.

Moreover, the common absolute claims (e.g., “this shows kids are not interested in flavors” or “this proves that flavors are attracting kids”) are absurd on their face. For any improvement in a product’s quality (such as the availability of a particular flavor), there are some combinations of individual preferences such that the improvement would tip someone’s preference about wanting to use the product. Since there are a lot of people, chances are some have that preference pattern for any substantial improvement (and this will include some “proper” and some “improper” users, if one is inclined to create such categories). The question cannot be, “are any kids motivated by the flavors?” (or by flavor descriptors, which is a somewhat different question), because the answer to that is surely yes. The question must be, “how many?” The Schiffman et al. results contradict the political claims that underage users are flocking to e-cigarettes in droves because they have heard about the particular flavors from the study, but absolute claims that have been made about the results are clearly false. Any author who seeks to make a scientific contribution in this area needs to explain, at least very roughly, how empirical results contribute to an economic model of preference and choice that can provide a quantitative estimate of the phenomenon of interest. (Anyone who wants to go further and claim that the phenomenon is substantially harmful, to whatever extent it occurs, must present separate analysis. This obviously does not follow from claims that the phenomenon is occurring, as many authors imply; it is entirely plausible that the material impact is nil or even beneficial.)

The absolutist rhetoric that dominates the policy fights in this area seeps into the science and poisons it, causing researchers to traffic in simplistic claims. Indeed, the rhetoric that causes the problems addressed above is exemplified in the second sentence of the abstract, in which the authors assert that e-cigarette use has no benefits apart from smoking cessation. This is obviously not true; if people are choosing an action, it is because it has benefits. But if the myth to the contrary is taken as a premise – effectively assuming that actions are caused by demonic possession rather than volition – it is very difficult to apply the economic reasoning sketched above. Researchers and commentators in this area give little indication they recognize that they are making claims about choices, which are volition that is a function of preferences, opportunity, and product characteristics. They need to assess how particular observations fit into a model of that process, rather than implicitly assuming that whatever happened to be measured is isomorphic to the outcome of that process.

On date unavailable, commented:

None

On 2016 Jan 08, Saul Shiffman commented:

CONTINUED FROM PRIOR POST

Jackler: Given the long history of tobacco industry manipulation of scientific research, the fact that the Shiffman et al. study was designed and sponsored by an e-cigarette brand gives reason for skepticism . . . The circumstances under which the paper arose, as illustrated by the timeline below, indicates that NJOY had a powerful motivation to commission a paper exculpating e-cigarette flavors from preferential youth appeal proximate to the time that Pinney Associates was engaged and the survey was conducted. [Followed by long discourse on conjectured commercial context . . .] . . . In summary, it appears that the Shiffman et al. paper was not hypothesis driven research, but rather a study commissioned by NJOY to create cover for their reversal on flavors driven by dropping market share.

Response: Dr. Jackler raises the point that our disclosed relationship with an entity with a commercial interest should increase skepticism among readers of the paper. The purpose of disclosure is transparency, and an invitation for readers who wish to be so to be skeptical. Skepticism is welcome. Certainty without data, not so much….. Dr. Jackler has no information whatsoever about the origin and motivation for the study. Absent any actual information, Dr. Jackler constructs a just-so story about how the research came about, in order to make an ad hominem argument. We again suggest that the appropriate approach is to focus on the science and data.

Jackler: With teens uninterested and adults minimally interested in flavors (other than tobacco and menthol), one wonders why do they bother to market them. If so unappealing to their customers, what accounts for the dramatic rise in NJOY sales after introduction of flavors such as butter crunch, peach tea, and wild berry?

Response: As cited in our paper, there is independent evidence that adult smokers who are not confirmed e-cigarette users tend to prefer tobacco flavors, as they are familiar. With more experience and transition away from smoking to exclusive e-cigarette use, preferences shift to non-tobacco flavors (Farsalinos et al., 2013). That is consistent with what our paper showed.

In conclusion, e-cigarettes are a novel and disruptive force in the tobacco environment. While many are concerned that they may ultimately be harmful, there is a strong and legitimate segment of the tobacco control community that sees e-cigarettes as a potential public health breakthrough. Both sides can agree that there are many questions yet to be definitively answered by data. It would be most constructive to spend less time on ad hominem attacks, and get on with the science.

References

Farsalinos KE, Romagna G, Tsiapras D, Kyrzopoulos S, Spyrou A, Voudris V. Impact of flavour variability on electronic cigarette use experience: An internet survey. International Journal of Environmental Research and Public Health 2013;10:7272-7282. http://www.ncbi.nlm.nih.gov/pubmed/24351746

Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3881166/pdf/ijerph-10-07272.pdf.

Federal Register. Proposed Rules: Federal Policy for the Protection of Human Subjects.

1. Available at: https://www.gpo.gov/fdsys/pkg/FR-2015-09-08/pdf/2015-21756.pdf.

Manning KC, Kelly KJ, Comello ML. Flavoured cigarettes, sensation seeking and adolescents’ perceptions of cigarette brands. Tobacco Control 2009;18:459-465. http://www.ncbi.nlm.nih.gov/pubmed/19700436

Shiffman S, Sembower MA, Pillitteri JL, Gerlach KK, Gitchell JG. The impact of flavor descriptors on nonsmoking teens’ and adult smokers’ interest in electronic cigarettes. Nicotine and Tobacco Research 2015;17:1255-1262. http://www.ncbi.nlm.nih.gov/pubmed/25566782

Singh T, Marynak K, Arrazola RA, Cox S, Rolle IV, King BA. Vital Signs: Exposure to Electronic Cigarette Advertising Among Middle School and High School Students – United States, 2014. MMWR January 5, 2016;64. Available at: http://www.cdc.gov/mmwr/pdf/wk/mm64e0105.pdf.

Truth Initiative. The Truth About: Electronic Nicotine Delivery Systems. 2015. Available at: http://truthinitiative.org/sites/default/files/ENDS Fact Sheet - 1 4 16[1].pdf.

DISCLOSURES The study in the original paper was sponsored by NJOY, Inc., a developer and marketer of electronic nicotine delivery systems. The authors have worked in tobacco control and tobacco research for as long as 40 years. In the past three years, PinneyAssociates has provided services for a range of companies, including GlaxoSmithKline Consumer Healthcare on their stop-smoking medications (Nicorette and NicoDerm CQ in the U.S.), for NJOY, Inc., and since February 2015, for Reynolds American, Inc. (RAI) on tobacco harm minimization. Our work for RAI focuses on products, regulations, and policies related to smoking cessation and harm minimization; we do not work on combustible conventional cigarettes. Some of us (JGG, SS) also are members in a limited liability corporation that owns intellectual property for an as-yet not-commercialized nicotine gum, an option for which has been acquired by Niconovum, a subsidiary of RAI.

On 2016 Jan 08, Saul Shiffman commented:

CONTINUED FROM PRIOR POST

Jackler: With such a low response rate, responders may have had a special motivation to participate. This raises the likelihood of selection bias, differences between responders and non-responders, a factor which undermines the value of surveys with a small percentage of responders.

Response: With regard to the response rate, the issue of selection bias must certainly be considered. It is important to note that in such online surveys with research panels, email invitations are blasted out, and it is not even known how many recipients got the email or saw it. Furthermore, the study closed when the target sample size was reached and it is likely additional invitees tried to enroll, but were too late. Online surveys routinely send large numbers of invitations in order to complete data collection quickly. We capped the total numbers of respondents (432 for adults and 216 for teens) in order to ensure that the combinations of product and flavors were balanced for appropriate comparisons to be made in the analyses. Most important, nothing in the invitation hinted at the subject of the survey, so it is not at all clear what bias might be introduced by the respondents' choice to complete this survey versus some other surveys they may have been invited to complete. Dr. Jackler seems to believe that a sample with a higher response rate would yield a different conclusion; we would welcome seeing such data, and hope Dr. Jackler will produce it.

Jackler: Two sorts of teens are likely to reply to such a survey: those contemplating e-cigarette use who may be attracted to the products being surveyed and those who are vehement in their intention to neither smoke nor vape and are motivated to send a rejectionist message.

Response: Dr. Jackler’s comments suggest he did not understand our research method adequately. The core of his concern about some distortion introduced in the sample would only be plausible under the circumstance where potential participants were informed (or somehow were able to deduce) the nature and focus of the survey. This information was not provided during recruitment, and even during screening, participants were asked about bottled water and ice cream in addition to tobacco product use, to mask the survey's focus. In other words, respondents could not have elected to enroll or not enroll in this survey on the basis of its content, because they did not know the content.

Jackler: A sizable fraction of teens are opposed to smoking, some with notable vehemence instilled by parents and schools. It would be expected that a fraction of teen responders felt a special impetus to do so as a means of communicating their opposition to tobacco products. The motivation of teen responders to oppose smoking is the most probable reason for the low interest in e-cigarettes which the authors erroneously interpreted as a lack of interest in flavored e-cigarettes.

Response: Dr. Jackler's point is that some teens are simply opposed to smoking of any kind, including e-cigarettes, and thus are not influenced by offering of different flavors. We agree – that's exactly the point: offering flavors seems unlikely to attract the teens not already smoking or predisposed to smoke!

Jackler: That the paper reaches the conclusion: “Nonsmoking teens’ interest in e-cigarettes was very low” is yet another reason to question the survey’s validity. This observation is at variance with 2014 CDC data showing a rapid rise in e-cigarette use among high school students coupled with a reduction in combustible cigarette use.

Response: Dr. Jackler earlier criticized our study for focusing on non-smoking teens, as opposed to all teens or smoking teens. Here, he ignores this fundamental design feature. There is no contradiction between our finding and the uptake of e-cigarettes by teens, because data repeatedly show that e-cigarettes are being taken up predominantly by SMOKING teens. The use of e-cigarettes by non-smoking teens (especially any kind of substantial use) is exceedingly low (Truth Initiative, 2015), consistent with our findings. Dr. Jackler points out that smoking prevalence is dropping even as e-cigarette use increases. One explanation is that e-cigarettes are drawing teens away from smoking to e-cigarette use, which is not inconsistent with our data.

Jackler: One interpretation of these data is that, relatively speaking, non-smoking teens prefer a variety of sweet and fruity flavors while adult smokers prefer tobacco flavor.

Response: Dr. Jackler’s alternative interpretation of our results, that the teen nonsmokers actually “prefer” a range of flavors, would seem to be utterly contradicted by the data, which (a) show very low absolute appeal of e-cigarettes among nonsmoking teens, and (b) show no statistical variation in teen interest by flavor.

Jackler: The Shiffman et al. survey offers no benefit to the minors surveyed and entails significant risks. Exposing teens to a harmful product they may not have considered using, such as use of e-cigarettes and flavored e-cigarettes, could increase the likelihood that they will try the product and develop nicotine addiction. This represents a clear risk to the future health of the minor. The existence of risk, coupled with the lack of benefit to the individual child, necessitates both institutional review and parental informed consent.

Response: We certainly agree that the survey did not benefit respondents, and was not presented (or intended) as a benefit to them. We do not agree that it posed any risk. The survey had no promotional value or content, and did not in any way encourage use of e-cigarettes. No graphics or branding were presented, and no claims were made. It simply presented text on a hypothetical e-cigarette flavor and asked for a rating of interest. Further, it should be noted that awareness of e-cigarettes is nearly universal among teens (Singh et al., 2016), so it was highly unlikely that the survey even introduced anyone to the concept of e-cigarettes. We note that surveys, including those fielded and sponsored by the government, routinely ask participants about smoking and their interest in smoking without any implication they have put participants at risk. Current Federal rules exempt survey research from IRB review in which respondents are anonymous, as well as research that asks about matters that do not put the respondents at legal risk or risk of embarrassment. Our study met both criteria. Of interest, 16 federal agencies have proposed IRB rules that go out of their way to further emphasize that simple surveys such as this are exempt from review (Federal Register, 2015).

CONTINUED IN NEXT POST

On 2016 Jan 08, Saul Shiffman commented:

Response to Dr. Jackler’s critique of Flavor Descriptors Paper published in Nicotine and Tobacco Research (Shiffman et al., 2015)

PLEASE NOTE: We have divided our response into several consecutive posts, to deal with the length limitations imposed by PubMed, while quoting Dr. Jackler's original comments, to allow readers to better follow the conversation.

We appreciate the continuing interest in our work and relish critical commentary and suggestions. Such a dialogue will serve to advance science and thus foster the development and implementation of public health-advancing policies.

In the post below, we have replied to the criticisms of our study raised by Dr. Jackler (most of which are redundant with those raised by Drs. Popova and Glantz, see our earlier post). We note that many of Dr. Jackler's remarks are ad hominem. We believe the science should be judged on its merits, so will not comment on Dr. Jackler's imaginings about our motives, just as we will discipline ourselves to be silent about his.

Three comments about general process and principles are worth making before we address specific issues:

1) All the issues raised by Dr. Jackler were also raised by Dr. Stanton Glantz in a peer review for the journal Nicotine and Tobacco Research, and subsequently posted by Dr. Glantz on the web. (The review was conducted single-blind, but Dr. Glantz' s posted comments are all but identical to the review, which makes clear he was the reviewer.) In accord with good peer review practice, the journal editor asked us to respond, and two editors and another (anonymous) peer reviewer were satisfied that we had responded adequately to the critiques, and that they did not fundamentally undermine the scientific quality of the work. Perhaps we are seeing the beginning of an era where peer reviewers do not accept editorial and consensus judgments, but simply repeat their critique on the web. 2) Some of the comments on methodology made by Dr. Jackler suggest methods he wishes we had used, populations he wishes we had studied, and studies he wishes we had done. We do not claim that our one study answers all questions, and cannot know whether different methods might have yielded different results – and neither can Dr. Jackler . The currency of science is empirical data, not hypothetical speculation. The real test is replication and extension. If critics believe different methods or populations would yield different results, they owe it to the science to do the work to show that. It has been 12 months since our study was published – time enough for Dr. Jackler to have collected the data he believes would be more informative. Criticism is easy; data counts.

3) Much of Dr. Jackler's critique amounts to his certainty that our study must be wrong, and therefore flawed, because it contradicts what he is certain he "knows" must be true, even though the data are not there. If the results contradict his intuition or certainty, then the methods must be flawed, and the researchers biased, and unethical, to boot. Returning to point #2, intuition and subjective certainty are nice; data are better. If there are conclusions Dr. Jackler wishes to assert, let him present definitive data.

Below, we take up each of Dr. Jackler's comments, quoting from his posting:

Jackler: In comparing the relative appeal of flavored e-cigarettes to non-smoking teens versus smoking adults, the obvious hypothesis would be that flavors would be of greater attraction to the young.

Response: It is uncertain what critique of our method, analysis, or interpretation can be derived from a statement of “the obvious hypothesis,” other than “I disagree with the results, because they violate my intuition.” Intuition is good. Data are better. The "obvious hypothesis" is sometimes wrong. We note also that a previous study, often cited in support of teen interest in flavors in cigarettes (and cited in our paper; Manning et al., 2009), shows interest in flavors is seen ONLY in teens who score high on sensation-seeking, which predisposes teens to cigarette smoking. Thus, observations or intuitions about the appeal to smokers cannot be generalized to all teens, and our data on nonsmoking teens is not incompatible with existing data on smoking teens.

Jackler: In a broader context, a finding that adolescents have no preference for sweet and fruity flavors would mean that e-cigarettes somehow are a special exception to well established consumption trends throughout the food and beverage industry.

Response: Dr. Jackler's statement ignores what we demonstrated empirically in the study – that the favors tested DID appeal to teens in foods and beverages (ice cream and bottled water), using the very methods he criticizes, even while having no effect on their interest in e-cigarettes. Again, data must trump intuition and conjecture.

Jackler: The logical way to determine the differences in e-cigarette flavor preferences between adults and teens would be to compare the actual frequency of flavor use by teen and adult e-cigarette users. Rather than use a direct method, Shiffman et al. compared a small cohort of teen non-smokers with adult smokers . . . There is reason to question the validity of comparing non-smoking teens with adult smokers as they are notably unequal groups. Nationally only approximately 15% of teens smoke whereas the entire adult survey group smoked. In this survey teen smokers were systematically excluded.

Response: Certainly the suggestion of comparing actual use patterns of adults and teens would be useful, but it would answer a different question than the one we set out to answer. As we clearly stated in our paper, we focused our research on the reported interest of the flavor descriptors on the two populations we regarded as being of most interest: current nonsmoking teens (who some assert are being lured in to use of nicotine by the appealing flavor descriptors, which would be a public health concern) compared to current adult smokers (whom one would want to find e-cigarette flavors appealing, to facilitate transition away from combustion cigarettes, a transition that we and many others regard as a public health good). Dr. Jackler is right that it would be interesting to know the flavor preferences of teen smokers, and we hope he or others will do that research. However, it is not clear that attracting a teen smoker away from using deadly combustible cigarettes to using e-cigarettes is the biggest public health worry – the big concern has been whether e-cigarettes would attract teens who are NOT smoking, hence our focus on this group.

Jackler: Shiffman et al. compared a small cohort of teen non-smokers with adult smokers via a marketing survey conducted for the sponsoring e-cigarette brand (NJOY) . . . The Shiffman et al. study consisted of an online survey inquiring about interest in products of the NJOY e-cigarette brand.

Response: Unfortunately, Dr. Jackler is simply wrong; we did not attach a brand name to any of the three types of products (e-cigarettes, ice cream, and bottled water) tested nor was the sponsor of the study mentioned to respondents. In addition, Dr. Jackler characterizes our study as a "marketing survey." It was no such thing. It was conducted for research and publication. This is yet another ad hominem argument to impute motives to us, without evidence, and, more importantly, without relevance.

CONTINUED IN NEXT POST

On 2015 Nov 01, Robert Jackler commented:

Commentary on Shiffman et. al. study which surprisingly found no preference for flavored electronic cigarette products among teens.

The paper ‘Impact of flavor descriptors on non-smoking teens and adult smokers’ interest in electronic cigarettes” by Shiffman and coworkers suffers from flaws in experimental design and data interpretation which call into question their conclusions that flavored e-cigarettes are not of differential appeal to teens.1 As discussed in detail below, their survey methodology has a high likelihood of selection bias, the study groups are too dissimilar to enable the comparisons made, informed consent for minors was not obtained despite evident risks, and commercial interest appears to have influenced the study’s outcome. In comparing the relative appeal of flavored e-cigarettes to non-smoking teens versus smoking adults, the obvious hypothesis would be that flavors would be of greater attraction to the young. The factual basis for this conjecture is the extensive body of research which found that flavored tobacco products have differential appeal to youth. This led the US Congress to ban flavored cigarettes in 2009. In a broader context, a finding that adolescents have no preference for sweet and fruity flavors would mean that e-cigarettes somehow are a special exception to well established consumption trends throughout the food and beverage industry.

Concerns over survey validity:

The logical way to determine the differences in e-cigarette flavor preferences between adults and teens would be to compare the actual frequency of flavor use by teen and adult e-cigarette users. Rather than use a direct method, Shiffman et. al. compared a small cohort of teen non-smokers with adult smokers via a marketing survey conducted for the sponsoring e-cigarette brand (NJOY). There is reason to question the validity of comparing non-smoking teens with adult smokers as they are notably unequal groups. Nationally only approximately 15% of teens smoke whereas the entire adult survey group smoked. In this survey teen smokers were systematically excluded.<br> The Shiffman et. al. study consisted of an online survey inquiring about interest in products of the NJOY e-cigarette brand. These included then current flavors (tobacco and menthol) and a list of contemplated flavors. A survey of flavor preferences for bottled water and ice cream was used as a comparison. Invitations were sent to 20,235 adults and 14,151 teens with a mere 432 adults and 216 teens enrolled. This is an under 2% response rate. With such a low response rate, responders may have had a special motivation to participate. This raises the likelihood of selection bias, differences between responders and non-responders, a factor which undermines the value of surveys with a small percentage of responders.2 A sizable fraction of teens are opposed to smoking, some with notable vehemence instilled by parents and schools. It would be expected that a fraction of teen responders felt a special impetus to do so as a means of communicating their opposition to tobacco products. Two sorts of teens are likely to reply to such a survey: those contemplating e-cigarette use who may be attracted to the products being surveyed and those who are vehement in their intention to neither smoke nor vape and are motivated to send a rejectionist message. The motivation of teen responders to oppose smoking is the most probable reason for the low interest in e-cigarettes which the authors erroneously interpreted as a lack of interest in flavored e-cigarettes. That the paper reaches the conclusion: “Nonsmoking teens’ interest in e-cigarettes was very low” is yet another reason to question the survey’s validity. This observation is at variance with 2014 CDC data showing a rapid rise in e-cigarette use among high school students coupled with a reduction in combustible cigarette use. Not surprisingly, their survey showed non-smoking teens queried on common consumer products (ice cream and flavored water) indicated widespread interest in flavors. In the adult cohort, flavor preferences for e-cigarettes, other than tobacco and menthol, roughly paralleled that of ice cream and water. (Shiffman et al. Figure 3b) That teens expressed a liking for flavored ice cream, but not e-cigarettes, may well be accounted for by responses of teens who had no interest in e-cigarette products. (Shiffman et. al. Figure 3a) It was not that these non-smoking teens did not like the e-cigarette flavors, but rather they categorically were uninterested in NJOY’s products in all flavor variations. In their flavor analysis, the paper’s data reveals a marked preference among adult smokers for tobacco flavor and menthol flavors with all other flavors (except vanilla) appearing at a fraction of their appeal. (Shiffman et al. Figure 1) Among teen non-smokers, by contrast, interest was essentially equal across flavors with no preference for tobacco or menthol flavors. One interpretation of these data is that, relatively speaking, non-smoking teens prefer a variety of sweet and fruity flavors while adult smokers prefer tobacco flavor. This interpretation of their data is the opposite the authors’ conclusions.

Inappropriate Claim of Exemption from IRB for Survey of Minors

The Shiffman et. al. survey offers no benefit to the minors surveyed and entails significant risks. Exposing teens to a harmful product they may not have considered using, such as use of e-cigarettes and flavored e-cigarettes, could increase the likelihood that they will try the product and develop nicotine addiction. This represents a clear risk to the future health of the minor. The existence of risk, coupled with the lack of benefit to the individual child, necessitates both institutional review and parental informed consent.3

The paper concludes that adult interest in flavors as modest, with the exception of classic tobacco flavor, which they observed can “ease the transition for smokers from a familiar product to a less-familiar one.” With teens uninterested and adults minimally interested in flavors (other than tobacco and menthol), one wonders why do they bother to market them. If so unappealing to their customers, what accounts for the dramatic rise in NJOY sales after introduction of flavors such as butter crunch, peach tea, and wild berry?

References:

1. Shiffman S, Sembower M, Pillitteri J, Gerlach K, Gitchell J. Impact of electronic cigarette flavor descriptors on non-smoking teens’ and adult smokers’ interest in electronic cigarettes. Nicotine Tob Res. 2015 Jan 7. pii: ntu333.

2. Groves RM, Peytcheva E. The Impact of Nonresponse Rates on Nonresponse Bias: A Meta-Analysis The Public Opinion Quarterly Vol. 72, No. 2 (Summer, 2008), pp. 167-189

3. Heath and Human Services. Institutional Review Board Exemptions: Special Classes of Subjects: Children and Minors: (http://www.hhs.gov/ohrp/archive/irb/irb_chapter6.htm)

4. Pinney Associates: About our Company. (http://www.pinneyassociates.com/overview.xml)

5. NJOY to Discontinue Flavors, Takes Additional Steps to Prevent Underage Electronic Cigarette Use. Reuters. Dec 10, 2009

6. Giovenco DP, Hammond D. Corey CG, et. al. E-Cigarette Market Trends in Traditional U.S. Retail Channels, 2012–2013. Nicotine & Tobacco Research, 2015, 1–5

7. Wells Fargo Securities. Equity Research: Vapor - NJOY’s New Product Line Positions it Ahead of the Pack. Wells Fargo Securities; 2014.

8. Richtel M. E-Cigarette Makers Are in an Arms Race for Exotic Vapor Flavor. New York Times, July 15, 2014.

9. NJOY tops US retail Vaping sales. Convenience Store news Nov 11, 2014. http://www.csnews.com/product-categories/tobacco/iri-njoy-tops-us-retail-vaping-sales

Interest Disclosure: Dr. Jackler served as a consultant to the State of California Attorney General’s office on the 2014 motion to enforce the 2010 Soterra (NJOY) consent judgment.

On 2015 Jul 09, Hanxiang Chen commented:

I'm searching for 'CRISPR HPV' these days and find some pictures in figure 3 are similar with the paper 'Disruption of HPV16-E7 by CRISPR/Cas system induces apoptosis and growth inhibition in HPV16 positive human cervical cancer cells', especially in Figure 3A, just as a reminder and please check it.

On 2015 Jan 09, David Ameisen commented:

The current link to the full text is inaccurate. Please follow this link instead: http://www.diagnosticpathology.org/content/9/S1/S3

On 2015 Sep 16, Geriatric Medicine Journal Club commented:

There is an epidemic of assessment tools and this systematic review tries to wade through the available instruments. This article was critically appraised at the May 2015 Geriatric Medicine Journal Club (follow #GeriMedJC on Twitter). The full discussion can be found at: http://gerimedjc.blogspot.com/2015/05/may-2015-gerimedjc.html?spref=tw While the results of the study disappoint in terms of identifying an instrument that can accurately predict post-ED adverse outcomes, do these tools still serve a purpose in "geriatrifying" the emergency departments?

On 2015 Jun 07, Torsten Seemann commented:

The software is available at http://hgwdev.cse.ucsc.edu/~benedict/code/Cactus.html or https://github.com/benedictpaten/cactus

On 2015 Jan 12, Larry Parnell commented:

This paper was my selection for Paper of the Week for 5-9 Jan 2015.

On 2015 Jan 08, Christophe Dessimoz commented:

A freely accessible preprint of this letter is available here: http://lab.dessimoz.org/papers/ReplyGuideTree_preprint.pdf

On 2015 Jan 09, William Grant commented:

Low UVB exposure accounts for some of the increased risk of cancer for waiters in Nordic countries

The paper by Reijula and colleagues investigated the risk of cancer for waiters in the five Nordic countries for the period 1961 to 2005 [1]. They found that all-cancer incidence rates were 46% (95% CI, 41%-51%) higher for men and 9% (7%-11%) higher for women. They attributed much of the difference to higher alcohol consumption, smoking, and occupational exposure to tobacco smoke. Overlooked in their paper was any discussion of the role of solar UVB and vitamin D in affecting risk of cancer.

I used data in their related paper from 2009 [2] to estimate the effect of UVB in reducing risk of cancer in the Nordic countries. I reasoned that those in the 54 occupational categories had different UVB exposures depending on how much working time they spent out of doors. The index I developed was lip cancer incidence less lung cancer incidence for males (females wear lipstick, so do not develop much lip cancer) [3]. As they noted, "All eight occupations with significant SIRs (standardized incidence rates) [for lip cancer] >1.20 in males include a major part of outdoor work, while the lowest SIRs are in indoor occupations." However, smoking is also a risk factor for lip cancer, hence the combined cancer incidence index. Men in the occupations with the greatest time spent out of doors had the lowest rates of most types of cancer.

Looking at the findings for waiters in Ref. 1, both male and female waiters have significantly increased incidence rates for many of the UVB/vitamin D-sensitive cancers [3, 4], although as mentioned in Ref. 1, the largest SIRs are for the smoking-related types of cancer. They also had lower rates of melanoma and the women had lower rates of non-melanoma skin cancer, indicating that they spent less time in the sun than those in other professions. Thus, waiters and others in Nordic countries whose occupations keep them largely indoors could reduce their risk of cancer by spending more time out of doors when the UVB intensity is high enough to make vitamin D [5] or taking vitamin D supplements to raise 25-hydroxyvitamin D concentrations to at least 75-100 nmol/L [6].

References 1. Reijula J, Kjaerheim K, Lynge E, et al. Cancer incidence among waiters: 45 years of follow-up in five Nordic countries. Scand J Public Health. 2015 Jan 6. pii: 1403494814565130. [Epub ahead of print] 2. Pukkala E, Martinsen JI, Lynge E, et al. Occupation and cancer - follow-up of 15 million people in five Nordic countries. Acta Oncol. 2009;48(5):646-790. 3. Grant WB. Role of solar UV irradiance and smoking in cancer as inferred from cancer incidence rates by occupation in Nordic countries. Dermatoendocrinol. 2012;4(2):203-11. 4. Moukayed M and Grant WB. Molecular link between vitamin D and cancer prevention. Nutrients. 2013;5(10):3993-4023. 5. Engelsen O. The relationship between ultraviolet radiation exposure and vitamin D status. Nutrients. 2010;2(5):482-95. 6. Garland CF, French CB, Baggerly LL, Heaney RP. Vitamin D supplement doses and serum 25-hydroxyvitamin D in the range associated with cancer prevention. Anticancer Res 2011;31:617-22.

Disclosure I receive funding from Bio-Tech Pharmacal (Fayetteville, AR) and MediSun Technology (Highland Park, IL).

On 2015 Mar 25, Janet Kern commented:

There is a statistical error in this research study. This error can be seen in Table 2 at 24 months of age. By utilizing the numbers provided in Table 2 (see below) it is evident that the difference between cases and controls at 24 months is highly statistically significant. The journal, Vaccine, was notified of the error. However, since, to date, no clarification has been issued, it is important to note that the conclusions seen in the abstract above are misleading and are the opposite of the conclusion supported by the data. The corrected results indicate that there is a statistically significant relationship between Thimerosal exposure and autism spectrum disorder.

***** At 24 months from the data provided using a t-test reveals the following:

Unpaired t test Mean of * sample 1 from summary = 804.2 (n = 189) Mean of * sample 2 from summary = 632.1 (n = 224)

Assuming equal variances Combined standard error = 71.838701 df = 411 t = 2.395645 One sided P = 0.0085 Two sided P = 0.017 95% confidence interval for difference between means = 30.882882 to 313.317118 Power (for 5% significance) = 90.07%

Assuming unequal variances Combined standard error = 72.061016 df = 394.166765 t(d) = 2.388254 One sided P = 0.0087 Two sided P = 0.0174 95% confidence interval for difference between means = 30.445864 to 313.754136 Power (for 5% significance) = 66.35%

Update 5/24/2015: When the journal Vaccine was notified of the error, it notified the authors. In response to the notification of the error, the authors changed the numbers in Table 2 of their study. The authors changed the mean and standard deviation for the controls at 24 months from 632.1 (715.1) to 676.8 (719.5). No explanation for the error or justification for the change was given.

To date, the journal Vaccine and the study authors have refused to release the study dataset for further evaluation.

On 2015 Jan 20, Rafael Najmanovich commented:

This concise review provides a welcome introduction to Normal Mode Analysis (NMA) methods. Unfortunately, the authors fail to discuss one major limitation of the elastic network models cited in the text, which is that all are sequence agnostic by means of using spring constants that are not dependent on the type of amino acids they connect.

Our group has overcome this limitation of traditional elastic network models with the ENCoM method (Frappier V, 2014) where a non-bonded interaction term in the potential makes spring constants dependent on the nature and extent of atomic pairwise interactions. This addition drastically improves the prediction of large-scale loop and domain movements upon ligand binding as compared to ANM (Anisotropic Network Model). Furthermore, this also makes it possible to use ENCoM to predict the effect of mutations on thermal stability and function. ENCoM was compared to a large number of dedicated thermostability prediction methods and shown to be among the most accurate and unbiased (see Frappier V, 2014 for details). Furthermore, this ability to use vibrational entropy differences to study the effect of mutations has allowed us to perform a large-scale comparison of mesophile/thermophile ortholog protein pairs where the structure is highly conserved (Frappier V, 2015). Whereas a number of factors contribute to the higher stability (at a given temperature) of thermophiles, vibrational entropy differences correctly classify about 2/3 of thermophiles as more stable than their mesophile counterparts (Frappier V, 2015). In one case tested, that of rubredoxin, all possible mutations in each position where performed in silico and each mutation was ranked according to the calculated vibrational entropy differences relative to the mesophile. The mutations present in the thermophile were among the top ranking. Thus, vibrational entropy differences calculated with ENCoM can be used to guide the selection of stability-coffering mutations.

In summary, our group has developed ENCoM, the first coarse-grained elastic network model that is sequence dependent. This allows ENCoM to improve on the prediction of loop and domain conformational movements, predict the effect of mutations on thermal stability and function and guide the selection of mutations that affect thermal stability and rigidity with applications in protein engineering.

On 2016 Dec 16, Laura Williams commented:

Our online journal club discussed this article on January 20, 2015. https://www.youtube.com/watch?v=vlucO0nH6HI

On 2016 Sep 08, John Tucker commented:

None

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 NCT021683615. We believe the correct ID, which we have found by hand searching, is NCT02168361.

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 2016 Jun 29, Nicolas Griffon commented:

As implicit scores are ordinal, a model using logits of cumulative probabilities [Agresti A. Categorical Data Analysis. p241] would have been a more proper way to compare implicit scores before vs. after.

On 2015 Sep 10, Jeff Kiefer commented:

The authors of the publication unfortunately named their resource BioGPS when that name has already be used for another bioinformatic rescue six years ago. The resource, BioGPS, developed in Andrew Su lab was published six years ago. Wu C, 2009

On 2015 Feb 27, George McNamara commented:

This is also available as a blog with Michael's videos on the blog page, and links to Canti's 3 videos

http://blog.wellcomelibrary.org/2014/09/cells-on-film-making-movies-in-biology/

Video 3 link goes to http://search.wellcomelibrary.org/iii/encore/record/C__Rb2102100?lang=eng#attachedMediaSection and then click on the "download entire title" link (above the static image) or go directly to http://film.wellcome.ac.uk:15151/0055-0000-7566-0000-0-0000-0000-0.mp4

You may also enjoy the classic David Rogers video, digitized by Tom Stossel and colleagues, posted in several places on the internet, including,

https://embryology.med.unsw.edu.au/embryology/index.php/Movie_-_Neutrophil_chasing_bacteria

http://php.med.unsw.edu.au/cellbiology/index.php?title=File:Neutrophil_Rogers1950s.flv https://www.youtube.com/watch?v=n0FELIKQHsw http://www.neatorama.com/2010/01/21/video-white-blood-cell-hunting-slaying-bacterium

http://works.bepress.com/gmcnamara/5/ ... page has references, this is the MetaMorph STK file version, can be opened in MetaMorph, Fiji ImageJ, and some (not all) other scientific imaging programs.

http://works.bepress.com/gmcnamara/18/ ... "The Chase" (named by Mary David), this is the bigger, better, version from 2012, won ASCB video contest honorable mention. Back story and technical details available in my articles at https://mdc.custhelp.com/euf/assets/content/MetaMatters vol 2iss3.pdf https://mdc.custhelp.com/euf/assets/content/MetaMatters vol 2iss4.pdf https://mdc.custhelp.com/euf/assets/content/MetaMatters vol 2iss5.pdf https://mdc.custhelp.com/euf/assets/content/MetaMatters vol 2iss6.pdf

As a former Dictyostelium discoidium researcher I also want to give credit to Dr. Arthur Arndt, 1937 movies of the developmental cycle of Dicty, see

http://jeb.biologists.org/content/81/1/33.full.pdf http://www.filmarchives-online.eu/viewDetailForm?FilmworkID=358af36868fa2a8548b03bb2c2cbbf2a

or simply search the Internet for: arthur arndt dictyostelium movie

On 2015 Jan 09, William Grant commented:

Differences in vitamin D status may help explain racial disparities in failure-to-rescue among children undergoing congenital heart surgery

The paper by Chan and colleagues did a comprehensive analysis of outcomes of pediatric congenital heart surgery, finding that failure-to-rescue was due to inherent racial factors rather than complications related to the surgery, with blacks having the worst outcome and Hispanics intermediate between blacks and whites [1]. A possible explanation for this finding is differences in 25-hydroxyvitamin D [25(OH)D] concentrations. A paper from 2004 reported that cardiac failure in infancy due to vitamin D deficiency in two infants who avoided surgery through vitamin D and calcium therapy [2]. A recent paper noted that 84% of African-American neonates undergoing cardiac operations had vitamin D deficiency (25(OH)D <20 ng/mL) and had a mean pre-operation 25(OH)D concentration of 10±4 ng/mL compared to 16± ng/mL for Caucasians and other race/ethnicity [3]. Those with lower 25(OH)D concentrations required greater post-operation care. Another recent study found that 25(OH)D concentrations decreased by 40% in infants and children after surgery for congenital heart disease [4].

In general, white Americans have the highest mean 25(OH)D concentrations, Hispanics somewhat lower, and black Americans the lowest concentrations [5]. The differences are due to differences in skin pigmentation since most vitamin D is produced from solar UVB exposure, and the darker the skin, the lower the efficiency in producing vitamin D. Disparities in various health outcomes have been attributed to these differences [6].

While it may be difficult to obtain blood samples from those who were included in this study for measurement of 25(OH)D concentrations, that could be done for planned operations in the future. In the meantime, it would be worthwhile to consider making sure that all children planning congenital heart surgery first be supplemented with vitamin D to raise 25(OH)D concentrations to 30-40 ng/mL [7] with a daily dose depending on how rapidly it is desired to raise the concentrations [8]. It would be worthwhile to see whether the heart condition improved before scheduling the operation. Since post-operation infection such as sepsis occurs in about 3.7% of operations in the U.S. [9], and vitamin D reduces the risk of sepsis [10], reducing the risk of sepsis is another good reason to raise 25(OH)D concentrations prior to operating.

References 1. Chan T, Lion KC, Mangione-Smith R. Racial disparities in failure-to-rescue among children undergoing congenital heart surgery. J Pediatr. 2014 Dec 30. pii: S0022-3476(14)01072-5. doi: 10.1016/j.jpeds.2014.11.020. [Epub ahead of print] 2. Carlton-Conway D, Tulloh R, Wood L, Kanabar D. Vitamin D deficiency and cardiac failure in infancy. J R Soc Med. 2004;97(5):238-9. 3. Graham EM, Taylor SN, Zyblewski SC, et al. Vitamin D status in neonates undergoing cardiac operations: relationship to cardiopulmonary bypass and association with outcomes. J Pediatr. 2013;162(4):823-6. 4. McNally JD, Menon K, Chakraborty P, et al. Impact of anesthesia and surgery for congenital heart disease on the vitamin d status of infants and children: a prospective longitudinal study. Anesthesiology. 2013;119(1):71-80. 5. Ginde AA, Liu MC, Camargo CA Jr. Demographic differences and trends of vitamin D insufficiency in the US population, 1988-2004. Arch Intern Med. 2009;169(6):626-32. 6. Grant WB, Peiris AN. Possible role of serum 25-hydroxyvitamin D in Black–White health disparities in the United States. J Am Med Directors Assoc. 2010;11(9):617-28. 7. Holick MF, Binkley NC, Bischoff-Ferrari HA, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 2011;96(7):1911-30. 8. Heaney RP, Davies KM, Chen TC, et al. Human serum 25-hydroxycholecalciferol response to extended oral dosing with cholecalciferol. Am J Clin Nutr. 2003;77(1):204-10. 9. Pasquali SK, He X, Jacobs ML, et al. Hospital variation in postoperative infection and outcome after congenital heart surgery. Ann Thorac Surg. 2013;96(2):657-63. 10. de Haan K, Groeneveld A, de Geus H, et al. Vitamin D deficiency as a risk factor for infection, sepsis and mortality in the critically ill: systematic review and meta-analysis. Crit Care. 2014;18(6):660. [Epub ahead of print]

Disclosure I receive funding from Bio-Tech Pharmacal (Fayetteville, AR) and MediSun Technology (Highland Park, IL).

On 2015 Feb 25, Miguel Lopez-Lazaro commented:

Conclusion not supported by the data

The authors propose that cancer is largely caused by unavoidable mutations arising during DNA replication. This proposal is based 1) on a strong correlation between the number of stem cell divisions accumulated by a tissue and the risk of being diagnosed with cancer in the tissue, and 2) on the assumption that the number of stem cell divisions is equivalent to the number of unavoidable mutations arising during DNA replication. The authors do not report any correlation between the number of mutations in a tissue and the risk of cancer in the tissue. However, since cell division can generate mutations, they assumed that the parameters “stem cell divisions” and “mutations arising during DNA replication” are interchangeable. Recent data indicate that this assumption is incorrect:

Tissue-specific mutation accumulation in human adult stem cells during life. https://www.ncbi.nlm.nih.gov/pubmed/27698416

Cancer Etiology: Variation in Cancer Risk among Tissues is Poorly Explained by the Number of Gene Mutations. https://www.preprints.org/manuscript/201708.0103/v1

The correlation reported by the authors indicates that carcinogenesis is driven by the accumulation of cell divisions in stem cells, and not by random mutations arising during DNA replication. The implications are completely different: https://www.preprints.org/manuscript/201707.0074/v1

On 2015 Feb 06, Daniel Corcos commented:

In the abstract of the paper, there is a wrong statement : « These results suggest that only a third of the variation in cancer risk among tissues is attributable to environmental factors or inherited predispositions », seemingly supported by the following sentence : « If there is a high cancer risk of that tissue type relative to its number of stem cell divisions—then one would expect that environmental or inherited factors would play a relatively more important role in that cancer’s risk. »

Basically, this type of reasoning tends to confuse mathematical normality with health, which leads to the conclusion that the higher disease risk population is always less than half of the total population.

Estimating the percentage of cancers related to genetic inheritance is impossible.
 As an example, if a minority of individuals are protected by their genetic constitution (for instance, if they have a three time less probability of cancer), then one could rightly say that the majority of cancers are related to genetic inheritance. Pure speculation?
 Then have a look:

http://www.ncbi.nlm.nih.gov/pubmed/10506723

Now, let's suppose that two cancer types have different incidence relative to their corresponding normal tissue cell divisions. One might say, with the authors, that one is due to environmental factors, whereas the other is not. Wrong. Both could be due to environmental factors, but one would be more affected.

More generally, if it is possible to say that ten per cent of cancers are attributable to tobacco, it is impossible to say that there is a defined percentage of cancers due to environmental factors, because there is no such thing as an environment free population. The only thing we can do is to give a minimal estimate of the percentage of cancers that would be prevented by removing defined environmental factors.

In conclusion, in addition to the criticism that has been made on the methodology in this place and many others, one may wonder if this paper has anything to do with science (as a knowledge enterprise) and how it has passed peer review in Science (the journal).

https://www.researchgate.net/profile/Daniel_Corcos2

On 2015 Feb 05, Jim Brody commented:

The key paragraph in this paper is:

"A linear correlation equal to 0.804 suggests that 65% (39% to 81%; 95% CI) of the differences in cancer risk among different tissues can be explained by the total number of stem cell divisions in those tissues. Thus, the stochastic effects of DNA replication appear to be the major contributor to cancer in humans."

Thus, the authors have a parameter that quantifies the "randomness" of cancer. By definition this parameter must be between 0% and 100%. The authors are 95% confident that it lies between 39% and 81%.

I look forward to more precise measurements of this parameter.

On 2015 Feb 05, Cyrille Delpierre commented:

No proof that cancer is mainly a random process C Delpierre, R Fantin, S Lamy, P Grosclaude, T Lang, M Kelly-Irving

Tomasetti and Vogelstein (1) suggest in a recent study that the majority of the variation in cancer risk in tissues (65%) is attributable to “bad luck”. This resounding fact is underscored in their paper, and has led to widespread global media interest. The unfortunate consequence of such media interest is misrepresentation and confusion surrounding the results. Three points should be raised to appropriately consider the significance of the authors’ findings. First point: the authors compare the probability of a cancer cell appearing at the tissue level, and not at the individual level. Their study does not say anything about the differences in cancer incidence observed at the population level, between countries or social groups. The risk of developing cancer is not random at the population level. The risk at the tissue level cannot be attributed to the level of the individual human, and certainly should not lead to population-level assumptions. The authors found that 21 cancers among the 32 studied (71%) represent ‘replicative cancers’ (stochastic cancers) and 9 cancers (29%) represent ‘deterministic cancers’ (linked to environmental or hereditary of cancer types). If we consider the number of cancer cases by type, the proportions are then dramatically different. Based on the same data used by the authors on cancer incidence in 2014 (2) and information provided in the supplementary materials, the proportion of total cancers represented by replicative cancers is around 20%. Cancer types not included in the study like prostate, breast, cervical, uterine and endometrial, kidney and bladder cancers, for which environmental factors have been identified or suspected, represent around 40% of cancer cases. Consequently, the significance accorded to “bad luck” is radically different in terms of cancer cases, since only a minority of cancer cases may be due to “bad luck”, ultimately changing the conclusions of the paper in particular at a public health level regarding possible prevention strategies. It is then ambiguous to write in the last paragraph that “stochastic effects associated with DNA replication contribute in a substantial way to human cancer incidence.” Second point: we have a number of questions regarding calculations presented in the paper. After careful reading of the main article and the supplementary materials it seems that figure 1 does not represent the lifetime risk of cancer according to the total number of stem cell divisions, as written in column 3 on page 79, but plots the log of the lifetime risk for cancer according to the log of the total number of stem cell divisions (as indicated on page 11 of the supplementary materials). If so the Pearson correlation of 0.804 has in fact been calculated using the log of the two values. Consequently the explained part of variance of 65% refers to the explained part of variance of the log of the lifetime risk for cancer. There are two ways to estimate the proportion of observed differences in cancer risk among tissues explained by the observed total number of stem cells divisions: 1) Using the initial values, the linear correlation is 0.53 suggesting that this proportion is 28% instead of 65%. 2) Using a log-log model (seemingly used by the authors here), the inverse function of the log10-function should be used to calculate the errors of the model. According to our calculations the proportion is 15%. Obviously, whatever the approach used, the results refer to a significantly smaller proportion of the variation in cancer risk among tissues due to ‘bad luck’ which substantially modifies the main message of the paper. Third point: as epidemiologists it seems important to underline that correlation does not mean causation. The authors highlight that cell replication is a major factor determining the appearance of tumor cells. However, mutation is a necessary – but not sufficient - condition for developing cancer. A cancer occurs when many physiological systems fail in particular the immune system which must fail to identify and destroy a cancer cell, allowing it to replicate (3,4). The “behavior” of the immune system has not been shown to be random, but linked with a number of exogenous factors. Thus, even if mutations occur at random, cancer development cannot be considered a random process. Moreover, the assumption that tumor cells are forming at different rates in different tissues on a regular basis depending on the number of stem cell divisions is questionable. Some evidence exists indeed regarding the interconnection between epigenetic processes and mutations in cancer (5). Since epigenetic processes are likely to vary according to environmental conditions, mutation rates might vary according to environmental challenges through epigenetic mechanisms.

Cancer does not occur randomly. While tumor cell production may have an inherent stochastic nature, this is one component of an interaction between complex systems at the individual level, which are not random. At the population level, cancers are not randomly distributed between groups. Medicine and public health need to persist in finding areas of cancer prevention moving above and beyond classic risk factors that take whole systems, both biological and social, into account.

References 1. C. Tomasetti, B. Vogelstein. Science 347, 78-81 (2015) 2. National Cancer Institute, Surveillance, Epidemiology, and End Results Program. http://www.seer.cancer.gov/statfacts 3. K Ryunga, E Manabu, T Kazuaki. Immunology 121, 1-14 (2007) 4. T.J. Stewart, S. Abrams. Oncogene. 2008 Oct 6;27, 5894-903 (2008). 5. J.S. You, P.A. Jones. Cancer Cell Review. 22, 9-20 (2012)

On 2015 Feb 04, Michele Ciulla commented:

The human side of randomness.

The interest of the article by Tomasetti and Vogelstein (1) is mainly epistemological: can science, with its current setting, help us to understand the true meaning of diseases such as cancer? When we try to justify our inferences on the machine of fate, despite knowing that this machine is driven largely by randomness, well we have a problem and this is, at least in part, our partial ignorance of the phenomenon that requires, above all, a reflection on the science of certainty and uncertainty (2). From the clinical point of view, a random event, like an unexpected disease, could have other explanations related to the history of that individual patient. Before building the clinic of randomness, it might be useful to consider patients not only as cases of a statistics but like mind-body unities with a psychosocial individuality and physicians are invited to reflect on Descartes (3). When considering the series of events leading to the neoplastic drift they are possibly non-linear showing a kind of evolution which reflects the changes of the environmental pressure on the individuals and their adaptive responses. This pressure is higher exactly where the genetic program has planned to allocate generative and re-generative resources for development and to buffer environmental changes (4). Thus, tissues that undergo the greatest environmental stress and, therefore, require a greater renewal, are the ones most exposed to the risk of developing malignancies, as the article clearly shows. The boundary between health and disease moves according the reciprocal interaction phenotype-environment and each of us, it should be remembered, is a different phenotype. Who will be next? It is not a roll of the dice to decide it, we have a genetic program that goes on and an environment in continuous change, the machine of fate is just what we call living.

References

1 C.Tomasetti, B. Vogelstein, Science 347 (6217), 78-81 (2015) http://www.sciencemag.org/content/347/6217/78.long

2 E.V. Colani, Journal of Uncertain Systems 2 (3), 202-211 (2008) http://www.worldacademicunion.com/journal/jus/jusVol02No3paper05.pdf

3 G. Duncan, Journal of Medicine and Philosophy 25 (4), 485-513 (2000) http://jmp.oxfordjournals.org/content/25/4/485.short

4 M.M. Ciulla, G.L. Perrucci and F. Magrini, in Regenerative Medicine and Tissue Engineering (InTech Press, 2013), chap 26. http://www.intechopen.com/books/regenerative-medicine-and-tissue-engineering/adaptation-and-evolution-in-a-gravitational-environment-a-theoretical-framework-for-the-limited-re-g

On 2015 Jan 20, Paolo Vineis commented:

We read Drs. Tomasetti and Vogelstein paper on the strong and positive association between the frequency of stem cell division and the risk of cancer with interest (1). However, their analytical approach was limited and their interpretation of findings was misleading. First, the study was based on a relatively small number of cancer types, most of which are rare, and excluded several common cancer types such as breast, prostate, bladder, and endometrium. Second, the frequency of stem cell division over time or across region is expected to change very little compared to changes in risk of cancer for the various cancer types. For example, during the 20th century in the US, risk for lung cancer increased by more than 50 fold but decreased by about ten-fold for cervix and stomach cancers (2). Liver cancer incidence rates in males (number of newly diagnosed cancer cases per 100,000 males per year) range from 2 in Iceland to almost 100 in Mongolia (3), with even larger variation if we were to consider incidence in high-risk vs. low-risk subgroups of populations. These data suggest that the degree of association between the frequency of stem cell division and the risk of cancer across tissues is unlikely to remain constant over time and across regions. Third, their statement on page 79, first column “we show that these stochastic influences are in fact the major contributors to cancer overall, often more important than either hereditary or external environmental factors” is not supported by the data. They can only say that variations in life time risk of cancer across cancer types could be explained by differences in frequency of stem cell divisions as stated on page 79 of the paper. Fourth, the inclusion of oesophageal and head and neck cancers in the “Replicative” category is questionable, since risk factors are well-known for a large fraction of these cancers. The overall conclusion that a large proportion of cancers would not be preventable is not supported by the analyses contained in the paper.

Paolo Vineis School of Public Health, Imperial College London, W2 1PG UK. e-mail - p.vineis@imperial.ac.uk Ahmedin Jemal American Cancer Society, Atlanta, GA 30303 USA

1. Tomasetti C, Vogelstein B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science. 2015 Jan 2;347(6217):78-81
2. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2015. CA Cancer J Clin. 2015 Jan 5. doi: 10.3322/caac.21254. [Epub ahead of PRINT
3. Globocan 2012, International Agency for Reaserch on Cancer. Acessed on January 9, 2015. http://globocan.iarc.fr/Default.aspx

On 2015 Jan 15, Vladimir Kuznetsov commented:

One of the main hallmarks of cancer is uncontrolled cell proliferation, ultimately leading to the death of the multicellular organism. The strong link between cell proliferation and cancer risk is well-known[1]. While studying this correlation in various human tissues, the scientists at Johns Hopkins University in the U.S. hypothesize that most of the genomic changes leading to human cancer "occur simply by chance during DNA replication rather than as a result of carcinogenic factors"[2]. According to their hypothesis, the authors observed correlation between the estimates of division rates of normal self-renewing tissue-specific cell population (termed as “normal stem cells”(NSC)) with the estimates of overall lifetime risk of cancers, studied across the USA population. In this comment, I point out that the lack of tissue-specific data quality and its incompleteness, population bias, and oversimplification of modeling can lead to ambiguity in the interpretation of the results and loss of confidence in the dichotomized classification of tissue-specific cancer risks in context of efficiency of 'primary prevention measures'. 1. The cell counts and division rates of NSC in normally slow-proliferative and non-proliferated tissues/cell subpopulation (in liver, brain, gallbladder, thyroid tissue, bone, and pancreas) might not be accurately estimated and extrapolate to CSC. 2. The correlation model does not consider directly the level of variation of the number of NSC divisions due to "random mutations arising during DNA replication in normal non-cancerous stem cells" in individuals. According to the authors, "random mutations arising during DNA replication in normal non-cancerous stem cells” leading eventually to occurrence of cancer stem cells (CSC) and tumors due to “many genomic changes occur simply by chance”, or “bad luck”. However, recent integrative genomics studies of somatic mutation spectra in different tumors defined tissue-associated non-random somatic mutation signatures, mutation clusters with specific sequence context and preferential location of the mutations in certain disease-associated chromosomal regions involved in the initiation and development tissue-origin tumor types and subtypes [3, 4]. 3. The model in [2] assumes that the proliferation rate of NSC in normal tissue and the proliferation rate of CSC are closely correlated, however the assumption may be not true [3]. 4. Based on "extra risk score"(ERS) cluster analysis, the authors provided a classification of the tumors into two classes, referred as R-tumors (occurred at random; relatively smaller ERS) and D-tumors (occurred via additional 'deterministic factors'). However, ERS did not include any additive and multiplicative factors which allow to estimate explicitly the effects of individual covariates and their interactions (for instance see [5]). Thus, clustering model does not allow in principle to estimate the significance of alternative factors due to absence of these covariates in the model risk assessment. 4.Variation of the total number of stem cell divisions during an individual’s lifetime is unknown. Therefore, the model reported in [2] says nothing about variation in the cancer risk between individuals and cannot say that ~70% of cancer cases are just "bad luck" only due to count of the rates of divisions in the limited number of normal stem cells. It was argued, that breast cancer stem cell-like cells arise de novo form non-stem-like tumor cells [3] and this could make cancer cell population more heterogeneous. Such plasticity indicates that the concept of CSC can be essentially different from that of NSC. It makes a linear correlation model proposed in [2] more difficult in context of its mechanistic interpretation.<br> 5.The authors considered the "ovarian cancer germ cell" as a representative precursor (“cell of origin”) of the cancer cells in ovaries. However, according to the literature, “ovarian cancer germ cells” (should be classified as D-tumors) and responsible for only a few percent of ovarian cancer cases. Furthermore, "ovarian cancer” can be mostly represented by some distinct secondary “cell of origin” due to migration (or metastasis) from source tissues/organs [6, 7]. Therefore, the estimations under their model assumptions become inappropriate.<br> 6.The authors used 31 normal tissues. The exclusion of the common cancers (breast, prostate and gastric cancers) and the inclusion of 5 times osteosarcomas (depended samples) could induce essential bias which further complicates result interpretation and ability to extrapolate the results into entire population in the USA. 7.Their analysis was not directly concerned with the variations of population-specific incidences or other environmental causes of cancers. For instance, according to published statistics, oral cancer (OrCa) is a heterogeneous group of cancers arising from different parts of the oral cavity, with well-defined and differentiated predisposing risk factors, prevalence, and treatment outcomes[8-10]. There is a significant difference in the incidence of OrCa in different regions of the world. In contrast with the U.S. population where oral cavity cancer represents only about 3% of occurring malignancies, it accounts for over 30% of all oral cancers in India. Due to these results, it is unlikely that these well-established observations can be explained with the prevalence of “oral cancer stem cells”[2] variations. It was estimated that 91% of OrCa cases in the U.K. are linked to lifestyle factors including smoking (57%), alcohol (30%), and infections (13%)[9]. Such knowledge provides oncologists and patients a real hope for prophylactic efforts and prevention via early detection of the OrCa in a near future[10,11]. However according to the prediction in [2], OrCa was classified as so-called R-tumors, of which “primary prevention measures are not likely to be very effective”[2]. 8. Comparison of Fig2 and Fig S1 in the main text and suppl. file, shows that so-called D class tumors includes 9 cancer types in Fig 2 whereas12 cancer types were represented in Figure S1. In Fig S1, head &neck, melanoma, and gallbladder tumors were included in the D-cluster. Also, for ovarian, testicular, and thyroid cancers which were classified by the authors[2] as R-tumors, the significance of the impact of lifestyle and diet in reducing the cancer risk has been reported[12]. For others such as pancreatic, laryngeal, lip and oral cancers which were also classified as R-tumors, the significance of smoking as significant risk factors has also been established. Therefore, there are several inconsistencies in the results of [2] when compared with current knowledge from the literature. 9.Summary: The classification of the tumors on the R (random) and D (deterministic) classes is based on indirect and unreliable measurements and to a certain extent, even inconsistent with well-established data. Risks of at least several of the R-tumor types of cancer can be significantly reduced by several environmental improvements, diets and prophylactic approaches. Therefore, the conclusion that "primary prevention measures are not likely to be effective..." for tumors arising in organs undergoing origin stem cells and their divisions could be misleading and inappropriate. The predictions of the models based on the U.S. data might not be scalable onto other countries and geographic regions. Direct detection of the NSC and CSC characteristics should be obtained and multivariate probabilistic models of cancer risk prediction should be developed and used.

References: PMID: 1: 2174724; 2: 25554788; 3: 21854987; 4: 24132290;5: ISBN 978-0-205-45938-4; 6: 24879340; 7: 24265397; 8: 24408568; 9: http://www.cancerresearchuk.org/cancer-info/cancerstats/keyfacts/Allcancerscombined/;10: 16629526; 11: 15936419; 12: 24379012

On 2015 Jan 13, Mark Burkitt commented:

The powerful correlation between the rate of stem cell proliferation and the incidence of cancer across a range of tissues, reported recently by Tomasetti and Vogelstein (1), provides important insight into the origins of cancer.

From the findings of their study, the authors appear to make the case that random mutations, occurring during stem cell divisions, are responsible for the majority of cancers. Although many environmental agents (including dietary carcinogens and various forms of radiation) are able to cause mutation, Tomasetti and Vogelstein argue that the variation in the incidence of cancer across different tissues can be explained largely in terms of the differences in the rates of cell division between tissues: the greater the number of cell divisions, the greater the number of random mutations, hence the greater the incidence of cancer. There is, however, an alternative explanation for the statistical correlation revealed by Tomasetti and Vogelstein – an explanation in which environmental factors play a more important, underlying role.

Consider the scenario in which an environmental agent (let’s say, a chemical carcinogen in the diet) causes a mutation in a stem cell. Whether or not this develops into a tumour depends on several factors, including whether the mutated (precancerous) cell undergoes proliferation before it can be destroyed by apoptosis and/or the immune system. There is also a window of opportunity for DNA repair enzymes to detect and repair the lesion before the cell divides. Therefore, from the moment a mutation arises, a race is on: can the cell be repaired or deleted before it divides? If the mutation occurs in a cell type having a high rate of cell division, it is more likely to lead to a clone of modified cells and, consequently, a tumour.

Such a view of the origins of cancer is consistent with the statistical trend reported by Tomasetti and Vogelstein: an environmental ‘factor’ acting on a stem cell in, for example, the colon is more likely to result in a tumour than the same factor acting on a cell in the leg. The rate of cell proliferation correlates with the risk of cancer so convincingly precisely because cell division is needed to ‘fix’ the mutation caused by the environmental agent – to convert the mutated cell into a tumour clone before it is repaired or deleted by the various surveillance systems.

The situation is not unlike that encountered in chemical kinetics, a central tenet of which is that the overall rate of a chemical reaction cannot be greater than the rate of its slowest step, the so-called rate-limiting step. No matter how many environmental mutagens you throw at the DNA of a cell, its rate of conversion to a tumour is determined by the rate-limiting step of the whole process – the rate of cell proliferation. If so, then environmental factors may play a greater (albeit ‘hidden’) role in the risk of cancer than might be suggested by Tomasetti and Vogelstein. It could be argued that the environmental component of cancer risk has evaded detection by the statistician’s radar.

References 1. Tomasetti C, Vogelstein B. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 2015; 347:78-81. doi: 10.1126/science.1260825

On 2015 Jan 09, GEORGE BLANCK commented:

Stochastic aspects of cancer development:

The article by Tomasetti and Vogelstein (1) has helped add another dimension to the study of cancer development, most often driven by researching intracellular signaling pathway malfunctions and microenvironment effects, including inflammation. In fact, there are many molecular aspects cancer development that are governed by a random chance component of biological processes. First, genes that form cancer fusion genes are comparatively large (2, 3), presumably due to large introns providing for many opportunities for a productive recombination that leads to a chimeric protein, requiring of course intact exons, but in most cases, no particular retention of intron sequences. Interestingly, one of the smallest cancer fusion genes, EWSR1 (2), occurs in Ewing’s sarcoma, among the rarest cancers.

The sequential order of mutations, originally thought to represent requirements for an ordered process of acquisition of cancer hallmarks, could also be (at least partially) explained on the basis of gene size (4), with metastasis suppressor genes, which cannot be readily distinguished from classical tumor suppressor genes via signal pathway exclusivity, being comparatively small (4) and thus potentially less likely to be mutated early in cancer development. It is not inconceivable that certain functions are required to precede others in cancer development. For example, lack of apoptosis remains a good candidate for preceding metastasis (4). However, stochastic functions are likely to be a major basis for sequential mutations, and this underlying factor is consistent with the more recent appreciation of signal pathway degeneracy in cancer, reflected in the many alternative pathways discovered in drug-resistant cells and by many other aspects of cancer research indicating pathway degeneracy. In short, there is less opportunity to argue for an ordered acquisition of cancer hallmarks if the underlying mechanisms do not credibly distinguish such hallmarks (4, 5).

Interestingly, cytoskeletal related proteins represent some of the largest coding regions in the human genome, and not surprisingly cytoskeletal related protein coding regions are very commonly found mutated in the cancer genome atlas (6). Thus, mutant cytoskeletal proteins may be stochastically inevitable, which raises several interesting questions related to the mutational basis of cancer development and cell shape. First, do mutant cytoskeletal related proteins have a high propensity for a dominant negative impact on the cytoskeleton, as do mutant forms of collagen polypeptides in Osteogenesis imperfecta, where cartilage polymer formation is disrupted by a mutant collagen molecule from just one allele? Second, is the oft-reported, and decades-old connection between metastasis and spherical cells (7, 8) due to mutant forms of the cytoskeleton, which provide for cell rounding and detachment and thus distant circulation? And finally, is the oft-reported, but much more recent connection between spherical cells and drug resistance (9-11) due to common, mutant cytoskeletons that essentially lead to a decreased surface area to volume ratio, in turn leading to reduced intracellular drug concentrations?

As noted (1), the more cell divisions, the more errors, due to intrinsic DNA replication error rates. It remains to be seen to what extent this conclusion is relevant to cancer development in specific settings. Cell division rates may vary with circumstance, particularly over a lifetime, due to such events as wound healing, surgery or radiation or accidents that remove replicative tissue, lymphocyte replication in infections and vaccinations, etc.

The appreciation of probabilistic functions governing cancer development should lead to fresh research avenues, as did the understanding of the roles of signal pathway malfunctions and inflammation. Can screening be organized with more refined purposes and more cost-efficiency, when accounting for the stochastic aspect of mutation occurrence and cancer development? For example, it is likely that point mutations leading to an activating oncoprotein are relatively rare and will have a relatively high probability of being followed by a mutation in a large tumor suppressor gene. On the other hand, many (apparent) cancers that represent large gene mutations may not require aggressive treatment, because the chance of a single base change leading to an activating oncoprotein, needed for aggressive cancer, may be minimal (12).

And, how do stochastic events compare to differences in DNA repair polymorphisms or other rate-limiting aspects of mutation occurrence, such as the differences in mutation rates between heterochromatin and transcriptionally active regions (13)? For example, are there long-term, disease-free smokers because of luck or because of highly efficient repair mechanisms?<br>   References to PubMed comment, January 9, 2015: 1. PMID: 25554788. 2. PMID: 19446742. 3. PMID: 23162078. 4. PMID: 22701759. 5. PMID: 25450826. 6. PMID: 25451318. 7. PMID: 6256751. 8. PMID: 7000337. 9. PMID: 24409314. 10. PMID: 24112388. 11. PMID: 24821384. 12. PMID: 25294886. 13. PMID: 25456125.

On 2015 Jan 06, Andrea Coletta commented:

As other commentators before me, I find hard to accept the authors conclusions.

Although the clustering shows a larger number of cancer types (22 vs 9) for which random mutations seems to be the major causing factor, this cannot directly lead to the conclusion that the majority of absolute number of cancers are caused by random mutations. If we compare Fig1 and Fig2 in the article we can see that the so called "minority" (9 cancer types) have an approximate average lifetime risk ~10E-1 while this is surely lower in the case of the "majority" (22 cancer types). I think that a simple weighted average would re-balance the presented figures of 1/3 vs 2/3 in "favor" of the genetic+environment causes, though I'm not sure this would be the most accurate way to follow (from a statistical point of view).

On 2015 Jan 05, Vincent Detours commented:

No one denies that cancer initiation has a stochastic component, but the conclusion that "prevention measure are not likely to be effective" for tumors arising in organs undergoing many stem cell divisions could be dangerously broad if not misleading.

The paper's investigation is limited to the variation of cancer incidence among organs within a single population. But the incidence of many cancers varies enormously among populations. For example, the incidence of esophagus cancer is 20-30 time higher in China than in the USA and 50-100 higher in subject with a history of Barett esophagus (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769895). Yet, this cancer is considered the result of 'bad luck' and unlikely to benefit from prevention measures (R-group) according to Tomasetti and Vogelstein who consider only the overall incidence in the USA. Their analysis is blind to the population-specific incidences and consequently to many environmental and genetics causes of cancer.

The variation of cancer incidence among organs spans five orders of magnitudes. Hence, a one or two orders of magnitude difference due to, say, Barett esophagus, would presumably not affect drastically the overall correlation between organ-related incidence and stem cell division. The classification in the 'deterministic' vs. 'replicative' framework proposed in the paper, however, could change dramatically. This is in fact illustrated by a few cancers for which the authors stratify incidence according to etiology, e.g. virus-associated liver and head & neck cancers vs. their non virus-related counterparts and lung cancers of smokers vs. non smokers. Likely the same could have occurred with many other cancers provided a more detailed population stratification.

Another possibility limiting the authors conclusions is that the total number stem cells divisions in an organ could itself vary from person to person under the influence of non-random genetic and/or environmental factors. A trivial example is age. It is hardly a preventable phenomenon, but other preventable factors could also play a role, tissue injury for example.

A fully developped and extended version of this comment can be found here: http://biorxiv.org/content/early/2015/08/12/024497.

On 2015 Jan 03, William Grant commented:

The paper by Tomasetti and Vogelstein reported that variation in cancer risk among tissues can be explained by the number of stem cell divisions (1). In addition, they divided cancer types into two categories: those more susceptible to environmental factors (D-tumors) and those more susceptible to stochastic effects associated with DNA replication of the tissues' stem cells (R-tumors). The authors concluded that primary prevention measures were not likely to be very effective for R-tumor types of cancer. In this comment, I point out that risk of cancers in this category can be reduced through primary prevention measures.

One way to reduce risk of many types of cancer is through higher solar UVB exposure. Many types of cancers have been found inversely correlated with indices of solar UVB doses in geographical ecological studies in the United States and several other countries including these types of R-tumor types of cancer:esophageal, gallbladder, ovarian, and pancreatic cancer (2). Also, these R-tumor types of cancer have been found inversely correlated with 25-hydroxyvitamin D [25(OH)D] concentrations in prospective observational studies: chronic lymphocytic leukemia (3), head and neck cancer (4), hepatocellular carcinoma (5), and pancreatic cancer (4). One D-tumor type of cancer has the strongest evidence for beneficial effects of UVB exposure and vitamin D: colorectal cancer.

The findings with respect to UVB exposure are generally attributed to production of vitamin D. However, they may also include some effects from mechanisms other than vitamin D. For example, in a mouse model experiment on intestinal tumors, considered an R-tumor type of cancer, both UVB exposure and oral vitamin D reduced the progression of the tumors, with UVB being more effective than oral vitamin D at approximately the same 25(OH)D concentrations (6). However, neither approach reduced the incidence rate of intestinal tumors in this mouse model with a genetic propensity for intestinal tumors. The mechanisms whereby vitamin D reduces the risk of cancer include effects on cellular differentiation, progression, and apoptosis (2). Vitamin D also reduces progression of tumors by reducing angiogenesis around tumors, and reduces metastasis as well.

Another very important risk factor for cancer is diet. In a multi-country ecological study of cancer incidence rates with respect to diet, smoking, latitude, gross domestic product, alcohol consumption, and life expectancy, high animal product consumption was a significant risk factor for three types of R-tumor cancers: ovarian, testicular, and thyroid cancer (7). Smoking was a significant risk factor for these types of R-tumor cancers: laryngeal, lip and oral, and pancreatic cancer. Animal product consumption is a risk factor for cancer in part through increasing insulin-like growth factor-I (IGF-I) (8), which increases growth tumors as shown for small cell lung cancer (9).

Thus, risk of several of the R-tumor types of cancer can be reduced by several environmental approaches including higher UVB exposure and 25(OH)D concentrations, eating fewer animal products, and not smoking. So "bad luck" can be overcome by "healthy choices".

References 1. Tomasetti C, Vogelstein B. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science 2015;347:78-81. 2. Moukayed M, Grant WB. Molecular link between vitamin D and cancer prevention. Nutrients. 2013;5:3993-4023. http://www.mdpi.com/2072-6643/5/10/3993 3. Luczynska A, Kaaks R, Rohrmann S, et al. Plasma 25-hydroxyvitamin D concentration and lymphoma risk: results of the European Prospective Investigation into Cancer and Nutrition. Am J Clin Nutr. 2013;98:827-38 4. Afzal S, Bojesen SE, Nordestgaard BG. Low plasma 25-hydroxyvitamin D and risk of tobacco-related cancer. Clin Chem. 2013;59:771-80. 5. Fedirko V, Duarte-Salles T, Bamia C, et al. Pre-diagnostic circulating vitamin D levels and risk of hepatocellular carcinoma in European populations: A nested case-control study. Hepatology. 2014;60:1222-30. 6. Rebel H, der Spek CD, Salvatori D, et al. UV exposure inhibits intestinal tumour growth and progression to malignancy in intestine-specific Apc mutant mice kept on low vitamin D diet. Int J Cancer. 2015;136:271-7. 7. Grant WB. A multicountry ecological study of cancer incidence rates in 2008 with respect to various risk-modifying factors, Nutrients. 2014;6:163-189. http://www.mdpi.com/2072-6643/6/1/163 8. Larsson SC, Wolk K, Brismar K, Wolk A. Association of diet with serum insulin-like growth factor I in middle-aged and elderly men. Am J Clin Nutr. 2005;81:1163-7. 9. Warshamana-Greene GS, Litz J, Buchdunger E, et al. The insulin-like growth factor-I (IGF-I) receptor kinase inhibitor NVP-ADW742, in combination with STI571, delineates a spectrum of dependence of small cell lung cancer on IGF-I and stem cell factor signaling. Mol Cancer Ther. 2004;3:527-35.

Disclosure I receive funding from Bio-Tech Pharmacal (Fayetteville, AR) and MediSun Technology (Highland Park, IL).

On 2015 May 02, Arthur Yin Fan commented:

The original trial by Hinman's has many methodology flaws, please read the comments under the original trial report.

On 2015 Apr 14, RheumJC - Rheumatology Twitter-Based Journal Club commented:

This article was discussed on January 29th, 2015 by participants of the inaugural session of #RheumJC, an international Twitter-based Rheumatology Journal Club. An engaging discussion on the science of the topic as well as the methodology of the study was had over 2 different one hour live "chats". There were 383 tweets (330 unique tweets, 53 retweets) by 38 unique participants from 5 different countries. Participants included 17 Rheumatologists, 7 Nephrologists, 6 Fellows in training/students, 1 Hospitalist, 1 Pharmacologist, and others.

A storify summary of the sessions can be seen at http://rheumjc.com/2015/01/storify-inaugural-rheumjc/

Interested individuals can track and join in future journal clubs by following @RheumJC or #RheumJC, or visit the webpage at rheumjc.com and sign up for announcements.

On 2016 Aug 23, Ben Goldacre commented:

This trial has the wrong trial registry ID associated with it on PubMed: both in the XML on PubMed, and in the body of the text of the article. The ID given is NCT00137370. We believe the correct ID, which we have found by hand searching, is NCT00137670.

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

Many thanks,

Jessica Fleminger, Ben Goldacre*

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

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