On 2017 Jul 14, Randi Pechacek commented:

Marcus Leung, first author of this paper, gave some background on a microBEnet blog post.

On 2015 Apr 08, DOMINIQUE BERGMANN commented:

An anonymous peer noted that panels 6D-E are identical. This is an error on our part and we have identified the source of the error and the raw data that corresponds to the genotypes pictured. Table 2 contains quantified data related to the information in panel 6D-E and is correct. All conclusions in the paper are based on the data in Table 2. We are working to correct this error with the journal.

On 2014 Sep 02, Haoquan Wu commented:

Around 7 years ago, we have shown an extensive degree of heterogeneity at the end of mature miRNA, which can affect detection by real-time PCR employing stem-loop primer mediated RT-PCR.

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001020

On 2015 Feb 06, Pavel Baranov commented:

This paper provides an invaluable dataset of context dependent translation initiation start (TIS) efficiency. While the optimal (Kozak) TIS context has been determined a long time ago, estimating the power of individual TIS based on the contexts was usually qualitative, at best it was based on position weight matrices where the weight of each individual nucleotide was taken into account independently of other nucleotides. By systematically randomizing the context surrounding AUG codons Noderer et al measured TIS efficiencies of all 11-mers containing AUG codons. The dataset is available in Supplementary Table 2.

On 2014 Oct 03, Friedrich Thinnes commented:

To finalize three-dimensional VDAC structure: Focus on Native VDAC will be indispensable

This study, from my point of view, marks a great moment of VDAC research:

1) It points another time to the relevance of cell membrane-standing VDAC-1 for the pathogenesis of Alzheimer´s Disease via apoptosis.

2) It gives strong support to the cell membrane-expression, more precisely plasmalemmal lipid raft-integration of vertebrate VDAC-1. Furthermore, the data concerning the posphorylation of VDAC-1 in correlation to regulation of channel opening/closing argue in favor of its involvement in cell volume regulation and thus apoptosis. They are in line with much evidence indicating that plasmalemmal VDAC-1 forms the channel part of a volume regulated anion channel complex (VRAC/VSOAC).

3) From here, VDAC-1 in the plasmalemma must be “fully closed” = collapsed = N-terminus accessible outside the barrel = closed for anions and cations, and there is evidence that the N-terminal part of native VDAC-1 can be reached by antibodies even in detergent solutions (Benz et al., 1992; Thinnes and Burckhardt, 2012).

4) Applying canonical incorporation into black membranes, detergent-solubilized native phosphorylated mammalian VDAC-1 as well as recombinant channel preparations from E. coli inclusion bodies show only “open” = anion-selective = N-terminal stretch inside the barrel and “closed” = cation-selective = semi-collapsed = N-terminal stretch inside the barrel channel phenotypes (Teijido et al., 2012). “Fully closed” = collapsed = N-terminus accessible outside the barrel VDAC-1 states thus cannot be studied by this approach. The same holds true for more recent crystallization-based approaches. However, upcoming laser-based approaches may work just on native VDAC-1 in solutions; thus improvements to get detergent solubilized native VDAC preparation may pay to keep on the schedule.

--Benz R, Maier E, Thinnes FP, Götz H, Hilschmann N (1992) Studies on human porin. VII. The channel properties of the human B-lymphocyte membrane-derived “Porin 31HL” are similar to those of mitochondrial porins, Biol. Chem. Hoppe Seyler. 373: 295–303. --Thinnes FP, Burckhardt G (2012) On a fully closed state of native human type-1 VDAC enriched in Nonidet P40. Mol Genet Metab 107: 632-633. --Teijido O, Ujwal R, Hillerdal CO, Kullman L, Rostovtseva TK, Abramson J (2012) Affixing N-terminal α-helix to the wall of the voltage-dependent anion channel does not prevent its voltage gating. J Biol Chem 287: 11437-11445.

On 2015 Aug 14, Oliver T Mytton commented:

We note the comments for Kaiser et al. We are in the process of compiling a full response. When this is ready we will post it on the BMC Public Health website, linked directly to the article.

Oliver Mytton Honorary Specialty Registrar, Centre for Diet and Activity Research (CEDAR), MRC Epidemiology Unit, Cambridge.

On 2015 Jul 08, Kathryn Kaiser commented:

Having recently published a paper on the same topic [1], we read this paper (published August, 2014) by Mytton and colleagues (the authors) with great interest. We identified several important issues that vitiate confidence in the paper by the authors [2].

Errors of calculation, stated protocol violations, and documentation:

Our first concern pertains to differences in calculations in a study we both included, as well as the irreproducibility of the standard errors (SEs) reported in the Figure 2a forest plot of effects on body weight in [2]. First, we believe there is a calculation error in how the authors derived the effect size and variance in [2] for Whybrow, 2006 [3], which we both included. For clarity, we note the correct citation for the Whybrow paper is 2006, though it is sometimes cited as 2007 in [2]. Per [4], in order to reduce the potential for the over influence of studies where there are two experimental groups (300 grams per day and 600 grams per day in the case of Whybrow, 2006 [3]) compared to only a single control group, the control number (in this case, n=14) should be divided in half and rounded if necessary to the nearest integer for meta-analysis. We both calculated the same mean differences (ours reported as standardized mean differences), but substantially different confidence intervals. In the original report of outcomes [3], the effects on body weight for both treatment groups were not significant, whereas in Figure 2a [2], the confidence intervals are represented as showing a statistically significant effect for both treatment groups.

After further examination of standard errors for the other studies in included by the authors, and being unable to recreate several of the values reported in the paper [2], we recomputed the meta-analytic statistics from the source papers after making corrections to these values (see list below for all corrected estimates). For the overall analysis, this resulted in a smaller effect [-0.54 (-1.05,-0.03)] than reported in [2]. In a more appropriate analysis and using corrected standard errors, we removed an outlier [5] that the authors had identified in the “Comparison with other studies” section on page 9 of [2]. This study had a drop-out rate of over 52 percent, did not conduct an intent-to-treat analysis, and included only 9 subjects in the final analysis. When removing this paper as suggested in [2] (but not done in Figure 2), the overall effect is -0.31 (-0.70, 0.07), p=0.11, with a drop in heterogeneity from 72 to 53%.

Recalculated forest plot of included studies, excluding [5] are as follows (Mean Difference in Kg, 95% Confidence Interval, Standard Error): Smith-Warner 2000: (-0.09, -0.40 to 0.22, 0.16); Whybrow 2006 (600g group): (-0.62, -1.52 to 0.28, 0.46); Whybrow 2006 (300g group): (-0.77, -1.73 to 0.19, 0.49); Basu 2010: (-0.90, -1.88 to 0.08, 0.50); Peterson 2011: (0.40, -0.05 to 0.85, 0.23); Dow 2012: (-0.50, -1.30 to 0.30, 0.41); Christensen 2013: (-0.90, -2.17 to 0.37, 0.65).

Concerning study inclusion based on the stated protocol, the authors were not consistent in the application of their criteria. The authors opted to include one study of individuals with newly diagnosed diabetes [6], despite their exclusion criterion of studies with “subjects with a medical illness that was liable to lead to weight loss or weight gain.”[2] Excluding only this study also results in non-significant findings [excluding [6]: -0.51 (-1.06, 0.03); excluding both the outlier [5] and [6] results in: -0.27 (-0.67, 0.13)]. In other words, correcting the apparent errors of inclusion and effect size calculations changes the conclusions as stated in the paper [2].

Other points of note:

Although some differences between our papers are the result of simple methodological differences, such as our limitation to longer than eight week studies versus the authors limitation to four weeks, other differences are more substantial. We note that the authors [2] did not use the term “obesity” in their list of keywords, which is an important omission in a review looking at weight outcomes. In addition, the authors included three studies that investigated changes in only a single fruit or vegetable, while we opted to include studies where the intervention was a variety of fruits and vegetables, in order to evaluate the more general public health message that is common today. This difference in inclusion simply represents a nuanced difference between our analyses, but it is important that searches are designed to adequately answer the question at hand. For instance, in applying the stated criteria given by the authors, we found three examples [7-9] of other studies of a single fruit or vegetable that were not included by the authors but appear to meet criteria, calling into question the thoroughness of the literature search.

Lastly, in trying to reproduce the authors work, we noted that the list of included studies in Table 1 includes Singh, 1992 [10] while the final analysis does not. The reviewer comments and author replies posted on line at the BMC Public Health journal website indicate the authors followed the reviewer recommendation to remove the Singh paper from the analysis, yet it remains in the published table and appears to also be counted in the number in the PRISMA diagram, Figure 1. According to the forest plots in Figure 2, 7 studies were included for weight outcomes and 5 studies were included for energy intake outcomes. Figure 1 does not match the included studies in the search results reported.

Conclusion:

It is clear that the report presented by the authors has several concerning errors, is not consistent within itself, and does not support the conclusion the paper states that these interventions, “…result in either a small reduction in body weight or reduced weight gain relative to controls.”[2] We respectfully request that the authors reply to these concerns in order to correct the record.

Kathryn A. Kaiser, Ph.D. Andrew W. Brown, Ph.D. David B. Allison, Ph.D. Office of Energetics, Nutrition and Obesity Research Center, School of Public Health, University of Alabama at Birmingham

References

\1.\ Kaiser KA, 2014 \2.\ Mytton OT, 2014 \3.\ Whybrow S, 2006 \4.\ http://handbook.cochrane.org/ \5.\ Weerts SE, 2011 \6.\ Christensen AS, 2013 \7.\ Lehtonen HM, 2010 \8.\ Thies F, 2012 \9.\ Vafa MR, 2011 \10.\ Singh RB, 1992

On 2014 Oct 21, Paula Rahal commented:

Acknowledgements

We thank Dr. Daniel Seabra and Dr. Eliney Ferreira Faria for their collaboration in collecting the samples and Dr. Ligia Maria Kerr for pathological review. This work was supported by São Paulo State Research Foundation - FAPESP (2010/01661-2) and CNPq.

On 2015 Nov 13, Lydia Maniatis commented:

The theory and method of this study simply doesn't hold up to scrutiny. Very serious problems include:

Methods

1. The authors claim to be motivated by previous findings of “large individual differences in lightness constancy with respect to changes in surface slant...” Yet they have only two observers per condition, and one of the observers did not complete all of the replications. Large individual differences are an indication that stimuli are ambiguous to observers, who consequently have to make quasi-guesses. In this type of situation there tends to be both intra- and inter-individual variability. Here, the small number of observers does not make either form of variability detectable.

2. They describe having subjected observers to an “induction procedure (Allred & Brainard, 2009; Doerschner, Boyaci & Maloney, 2004) to familiarize them with the difference between surface reflectance...and the amount of light reflected from a surface [luminance]...” Surface reflectance is not perceived, but inferred (the perceptual correlate is lightness), and luminance has no perceptual correlate. The relative luminances of surfaces in context and their geometrical relationships conspire to produce local impressions of both lightness and apparent illumination. You cannot train observers to perceive luminance, which, again has no perceptual correlate (it does not correspond to white, gray, black; virtually any luminance can take on any of these perceptual values).

An example can help make clear why such a “induction procedure” is theoretically and methodologically hollow. This is the video version of the Adelson checkerboard illusion, in which we see a square being moved from apparent shadow to apparent plain view, and changing apparent color. We can watch the video over and over again without thereby learning to see the square as constant in both its luminance and its reflectance.

1. Asking subjects to rate the similarity in lightness of surfaces on a scale of 1 to 30 seems quite unrealistic.

2. The mathematical acrobatics necessitated by the general shoddiness and ambiguity of the methods are moot.

Theory

The authors state that “Changing the orientation of an object's surface with respect to a directional light source affects the luminance of the reflected light [they mean "the luminance of the surface"] even when the light emitted by the illumination sources is held constant.” That's true.

However, the next statement is incomplete and misleading: “The visual system can stabilize lightness with respect to such geometrical effects...” This statement needs to be highly qualified. The visual system can achieve such “lightness constancy” (requiring disambiguation of reflectance and illumination) only if the image conditions support it. An isolated surface (as that used by the authors), whether fronto-parallel or slanted, will not offer much or any such support. Thus, it should not be surprising that the authors found "essentially no lightness constancy with respect to changing the slant of a test surface..." and that "the dissimilarity data were well-accounted for by a one-dimensional perceptual representation..."

While, again, this outcome was highly predictable, they authors are mystified by it. Noting that their results differ from those of Logvinenko and Maloney (2006), they confess that "We do not know the reason for this difference." But as they note, in that study the illumination differences were not only actual, they were (more importantly) APPARENT (a fact that can be ascertained on the basis of observation), because the setting was less spare and more informative. This is something that should have been considered before, not after data collection.

But lightness facts and relevant literature are not of interest to these authors, who dispense with the matter early on, saying: “This form of lightness constancy [the slant surface form?] is not well-understood.” And on to data collection.

On 2014 Oct 06, Leonid Teytelman commented:

Dear Authors,

We have published an analysis in S. cerevisiae, showing expression-dependent artifactual ChIP enrichment at highly expressed loci (Teytelman L, 2013 "Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins"). As you know, our finding raises the question of whether HOT regions may also be influenced by the same artifact.

It is great that you have considered our work and have thoughtfully responded to our analysis. Below, I would like to continue this discussion in an effort to better understand the artifact, its causes, and whether it may be contributing to the enrichment at the HOT loci.

1. “we have demonstrated that there is no correlation between our non-specific binding controls (IgG) and our measured transcription factor occupancy;”

Considering our results with no-tag control experiments, an IgG may fail to control for the artifact. It would be great if you could instead perform a GFP ChIP-Seq, similarly to what we have done in yeast.

2. The regions determined in ref. 41 have very low enrichment (twofold or less) of non-specific immunoprecipation in anti-GFP antibody controls over input DNA evaluated using a non-standard sliding-window approach. Importantly, immunoprecipitation/input ratios at this level are typically not considered enriched for binding in modern peak-calling procedures. For example, the median immunoprecipitation/input ratio for our human RNA Pol II experiments is 20-fold, and only 0.033% of human RNA Pol II peaks contain an immunoprecipitation/input ratio ≤ twofold.

The mean is low, but in both anti-GFP experiments, there are loci with 3-5x enrichment (figure 4D). Most importantly, while the anti-GFP enrichment at the hyper-ChIPable loci is low, please note that the level of enrichment is variable from protein to protein (2-5X for Sir proteins, but often >10X for Cse4).

3. Thus, it is essential to note that the term ‘hyper-ChIPable’, coined by ref. 41, is quite misleading, as a correctly performed ChIP experiment will evaluate statistically enriched regions, with higher immunoprecipitation/input ratios. The so-called hyper-ChIPable regions in ref. 41 are not binding regions as determined under ChIP-seq best practices. Hence, when statistical peak-calling was performed in ref. 41 (using the established MACS peak-caller) to evaluate signals only at significantly enriched regions (Supplementary Table 1) only 17 (<7.5%) of the 238 claimed ‘hyper-ChIPable’ regions were called significant by all three Sir proteins. In fact, 68% of their 238 regions do not contain a binding site for any Sir protein as determined by MACS, despite even very liberal settings used (P < 10−5, no fold enrichment cut-off). Thus, the data of ref. 41 contradict its own major claim that all three Sir proteins showed enrichment at the 238 sites.

By reporting the 238 sites with >2fold enrichment of Sir2, Sir3, and Sir4, we are in fact being extra-demanding in terms of the threshold. We are stringently requiring all three proteins to be enriched above a threshold at the locus. So a target with 5x enrichment of Sir2 and 1.8X enrichment of Sir3 would not pass this cutoff. A typical ChIP study will focus on a single factor at a time. Had we done that, we would have many more artifactual targets for each silencing protein, with many at 5x or higher enrichment. Furthermore, the level of the artifactual signal varies from protein to protein or experiment to experiment. For example, the Cse4 signal at highly-expressed loci can give 10x or higher enrichment.

4. Furthermore, as indicated in Supplementary Table 3 of ref. 41, the Sir2, Sir3 and Sir4 ChIP-seq experiments were performed only once each, which raises the question as to whether enrichment of Sir proteins at the 238 sites is reproducible. More rigorously, even for the remaining 17 genomic loci, their status as hyper-ChIPable is questionable as each region would first have to be established as a reproducible binding site in replicate experiments for each individual Sir protein. If you consider that Sir2, Sir3 and Sir4 ChIP-seq constitutes three replicates of Sir proteins, their data show that most of their claimed sites were not reproducibly enriched.

Most of our artifact-cause analysis focuses on genome-wide data, not on the 238 sites. The 238 Sir-enriched euchromatic loci were a launching point for the analysis, but most of the paper looks comprehensively at the link between expression and ChIP levels. Figures 3, 4, and 5 are all on genome-wide correlations between Pol II/III and ChIP.

As for reproducibility, we see the same peaks, with often 10x enrichment, in Ste12, Cse4, two distinct GFP experiments, and each of the three Sir ChIP-Seq datasets. The same exact loci come up in the Sir3 paper from Oliver Rando’s group (Radman-Livaja M, 2011).

5. In addition to the analytical differences outlined above, other potential sources for the marked differences between our data and the Sir-enriched regions of ref. 41 are deviations from a typical ChIP protocol. In particular, ref. 41 employed a significantly longer cross-link time (1 h as opposed to the typical 10–20 min). This might contribute to formation of large non-specific protein–DNA complexes, which can in turn increase non-specific immunoprecipitation.

Though not discussed in the manuscript, we have in fact performed experiments to investigate if the crosslinking concentration contributed to the misleading signal. We performed ChIP with the 1 hour crosslinking at room temperature at the following formaldehyde concentrations: 0.0625%, .125%, .25%, .5% and 1%, but did not find a proportionate decrease in the hyper ChIPpable signal with the decreasing formaldehyde concentrations. Moreover, the presence of hyper-ChIPability in the Snyder datasets (Cse4, Ste12), ours (Sir2, 3, 4, GFP), and Rando (Sir3) make it clear that the problem is not in some unusual protocol steps in our hands.

We also note that we initially performed the Sir ChIP-Seq experiments because of our interest in the Sir protein biology. Because the Sir proteins do not directly interact with the DNA, we used longer crosslinking times. This is not unique to our work.

In summary, much more work is needed to pinpoint the cause of the artifact and to evaluate whether some or all of the signal at highly expressed genes in many other reported ChIP studies could be artifactual. Much more work is necessary to develop the best controls and corrections for the artifact. However, the artifact we report is not minor and is not a consequence of the methodological details of our manuscript.

Also, please note the following papers, published almost in parallel with ours, on this topic:

Park D, 2013 "Widespread Misinterpretable ChIP-seq Bias in Yeast" (Different analysis methods but the same conclusions in S. cerevisiae, analyzing an entirely different set of factors with ChIP-Seq experiments.)

Kasinathan S, 2014 "High-resolution mapping of transcription factor binding sites on native chromatin" (Questions specificity of standard ChIP in S. cerevisiae and at HOT regions of Drosophila. This work possibly provides a solution to the artifact with a modification of the ChIP technique.)

Also, the following discussion of our work on PubPeer may be useful.

On 2014 Oct 14, Hilda Bastian commented:

Thanks for drawing attention to this interesting article. Dancer SJ, 2009 argued that what's done now in an outbreak is "a veritable blunderbuss approach." Dancer's own cross-over study of enhanced cleaning addressed MRSA, although the study was too small to identify a definite impact on infection (Dancer SJ, 2009).

Environmental strategies were included in a systematic review of measures to reduce the spread of VRE (with a search for evidence up to June 2012)(De Angelis G, 2014). De Angelis found only two studies, concluding that no definite impact on infection had been identified (Hayden MK, 2006; Williams VR, 2009).

This new retrospective study (Everett BR, 2017) seems to be the second looking at the implementation of this particular set of strategies. The first was undertaken by the team that developed the method and run the associated consultancy service (Watson PA, 2012).

Both routinely and during outbreaks, Dancer SJ, 2009 concluded, "there is a lot of work still to do to establish cleaning as an evidence-based science." That still seems to be the case.

On 2014 Oct 10, Paul Watson commented:

This is an excellent article showing a simple way to dramatically lower all hospital acquired infections with single cleaning algorithm from Steiros. The final infection rates are extremely low with immediate cost savings that I have not seen with any other infection control process. As a surgeon and an infection control practitioner, I am especially excited about the zero infection rate for total knee arthroplasties in this paper. This is something that we all strive for and has been achieved by few. Thanks for the article.

On 2014 Sep 30, Ryan Radecki commented:

Post-publication commentary:

"Emergency PCI for STEMI is Dead?"

This somewhat befuddling study tries desperately to create a problem where there probably truly wasn’t – but as soon as the conflict-of-interest disclosures come up, it’s clear why.

This is the 1-year outcomes from STREAM, a prospective, open-label, parallel-group trial enrolling participants with acute STEMI, but unlikely to undergo primary PCI within 1 hour of diagnosis. Participants were then randomized to either still undergo emergency PCI, or to fibrinolysis followed by urgent or emergency rescue PCI. The initial 1-month composite outcome, despite an excess of deaths secondary to intracranial hemorrhage in the fibrinolysis group, did not demonstrate any disadvantage to fibrinolysis with delayed PCI. There was actually a 2% absolute decrease in the composite outcome favoring fibrinolysis – and thus the 1-year follow-up, hoping this small advantage in morbidity would translate into a measurable mortality advantage....

http://www.emlitofnote.com/2014/09/emergency-pci-for-stemi-is-dead.html

On 2014 Sep 10, Vojtech Huser commented:

Data on how often patients use infobutton (when they login to PHR) is a very valuable contribution.

On 2017 May 17, Mihai Paduraru commented:

In relation to this article I published a Critical observation, due to the too many similarities to a randomized controlled trial by Wang et al.: https://doi.org/10.1016/j.ijsu.2017.01.019 (http://www.sciencedirect.com/science/article/pii/S1743919117300201)

On 2017 Jul 14, Randi Pechacek commented:

Nicholas Osborne and Richard Sharpe, co-authors of this paper, wrote a joint guest blog post on microBEnet explaining the interesting background for this paper.

On 2014 Aug 27, Daniel Kripke commented:

Despite much useful material, the report of Owens and colleagues about insufficient sleep was marred by insufficient evidence-based definition of insufficient sleep. “Sufficient sleep” was “defined as ≥8 hours,” but why? Consider that in a Japanese nation-wide sample of adolescents grade 7 through 12, ≥7-<8 hours was associated with the best General Health in a number of grade groups of boys and girls and overall ≥8-<9 hours was not significantly superior in predicting mental health [1]. Consider that in a unique lifetime study of mortality risk, girls sleeping 2 hours less than the average for age 16 had the same mortality as those sleeping the average, whereas mortality was about the same for boys sleeping 1 hour less than the average [2]. More evidence concerning optimal sleep durations for adolescents is needed. Should we recognize that much of the evidence-absent advocacy for children to sleep more has been financed by hypnotics manufacturers, and has been accompanied by a sharp increase in hypnotic prescriptions for children? 1. Kaneita Y, et al. J Clin Psychiatry 2007;68:1426-1435. 2. Duggan KA, et al. Health Psychol 2014, in press.

On 2014 Aug 30, Michelle Lin commented:

This is the link where the multimodal discussion was held: http://www.aliem.com/multiple-mini-interviews-annals-em-resident-perspective-article/

On 2014 Sep 23, Samir Ounzain commented:

Very nice editorial describing this work by Patrick Hofmann and Reinier Boon can be found here;

Hofmann P, Boon RA. Non-coding RNA enhances cardiac development. J Mol Cell Cardiol. 2014 Sep 18. pii: S0022-2828(14)00286-7. doi: 10.1016/j.yjmcc.2014.09.005. [Epub ahead of print] PubMed PMID: 25240640.

On 2015 Mar 18, Airton Stein commented:

This article is not my publication - Airton Tetelbom Stein from Brazil

On 2016 Oct 24, Sean Ekins commented:

Some more recent slides on other social media tools and also using twitter for collaborations in science http://www.slideshare.net/ekinssean/pros-and-cons-of-social-networking-for-scientists

On 2014 Dec 17, Sean Ekins commented:

Some slides to expand on this are here http://www.slideshare.net/ekinssean/beyond-the-10-simple-rules

On 2014 Dec 15, Sean Ekins commented:

Also added to summary of our 2014 papers http://www.collabchem.com/2014/12/15/a-year-in-publications-2014/

On 2014 Dec 02, Sean Ekins commented:

paper mentioned here http://www.collabchem.com/2014/08/25/plos-paper-going-viral/ and here http://www.collabchem.com/2014/08/21/anatomy-of-a-plos-computational-biology-paper/

On 2014 Aug 22, Gözde Ozakinci commented:

I have to say - I wish it was clear where the study was done in the abstract.

On 2014 Aug 29, Jonathan Eisen commented:

I wrote a blog post about this paper. See here

On 2017 Oct 01, Misha Koksharov commented:

Of interest, depletion of an individual MRTF isoform (either MRTF-A or MRTF-B) almost completely suppressed induction of several contractile proteins despite the presence of the other MRTF isoform. This most likely indicates that both isoforms cooperate in expressional up-regulation of contractile proteins and that a certain threshold concentration of MRTF is required in order to stimulate the EMyT.

During my previous postdoc I've observed similar effects of silencing MRTF-A and MRTF-B separately in U2OS cells when using bioluminescent real-time reporters ("SRF-luciferase") as an output. Before me it was also noticed by Gerber et al, 2013: Fig. 4. We have used the same siRNA SmartPools from Dharmacon: (siRNA sequences are in the links) against human MRTF-A, MRTF-B and SRF (and in some experiments - against mouse SRF).

However, after a more close investigation with individual siRNAs from these pools, I came to a conclusion that the observed necessity of both MRTF forms was caused by off-target effects of some of the individual siRNAs (at least in my system):

a) Three of the MRTF-A siRNAs also reduced mRNA levels of MRTF-B. Only one was true anti-MRTF-A siRNA (and this one also gave the highest reduction of MRTF-A mRNA levels) but it didn't reduce the reporter induction in U2OS cells in marked contrast to the pool.

b) One of the anti-MRTF-B siRNAs caused some general off-target effects in addtion to MRTF-B knockdown: considerable cell death, blocked not only SRF but also some other pathways. Its effect on SRF-luc persisted even when the target site in MRTF-B was mutated to prevent the targeted silencing. Three other siRNA prevented most of the SRF reporters's induction and it was reduced further by adding the good single anti-MRTF-A siRNA.

The overall conclusion was that at least in U2OS cells the MRTF-A is not essential in these reporter settings but shows a minor partial contribution if MRTF-B is depleted.

I'll probably include this data in the later paper but for now I hope these notes will be helpful for people using these MRTF/SRF SmartPools. I strongly recommend to check the effects of individual siRNAs in them.

By the way, one of the siRNAs in human/mouse SRF pools also showed noticeable off-target cellular toxicity (and it was not even the strongest in reducing SRF mRNA levels). Given that each of the 3 pools I've worked with had some kind of off-target effect, this confirms the warnings of Jackson AL, 2010 that this can happen quite often. :( Thus, it seems that in general it is highly desirable to check all the individual siRNAs in commercial pools for target/off-target effect and have at least 2-3 different siRNAs against the same gene confirming the knockdown phenotype.

On 2014 Oct 18, David W Brandes, MS, MD, FAAN commented:

I am very happy to see more published information about this topic. I have been speaking professionally abut this topic for 10 years and it is becoming increasingly recognized as to it's importance in the management of MS patients. This article confirms the often unrecognized frequency of this issue. When MS patients say they "are tired all the time," we need to think about the details. Physical fatigue, cognitive fatigue, psychological fatigue (depression) and sleepiness may all be part of this complaint.

On 2014 Oct 31, Francisco Felix commented:

One of the major concerns about the data in this paper is the prior probability of the results reflecting the truth. Antineoplastons have no scientific basis better than homeopathy or psychic surgery. Hence, the prior probability associated with the results of this paper should be so small that a bayesian correction of its estimates (by the way, they were not even reported!) must invalidate its results. Try again (actually, don't!).

On 2017 May 01, Doug Berger commented:

LACK OF BLINDING IN THIS STUDY WAS A SERIOUS METHODOLOGIC FLAW. ADDITIONALLY, FACULTY SUPERIOR OF LEAD AUTHOR STEVEN HOLLON, DR.STEPHAN HECKERS, EDITOR OF JAMA PSYCHIATRY, BOTH AT VANDERBILT DEPT. OF PSYCHIATRY REQUIRES REPORTING AS CONFLICT OF INTEREST

This study by Hollon et al. compared an antidepressant medication-only arm with a combined cognitive therapy/antidepressant arm and concluded that the cognitive therapy/antidepressant combination enhanced the recovery rates compared with antidepressant alone, and that the magnitude of this increment nearly doubled for patients with more severe depression.

We opine that for subjects with greater severity, there could have been both antidepressant efficacy, as well as more hope and expectation as bias in the group who knew openly that they had received combined cognitive therapy/medication as a possible treatment. This can lead to an erroneous conclusion of greater efficacy for the combined group. The large subject number in this study could also easily lead to an erroneous finding on statistical testing as a small amount of bias in the subjects adds-up.

In addition, it goes against clinical trial logic to compare the unbilnded-cognitive therapy/medication group to the unblinded medication-only group.This is because, as all the study arms were unblinded, the combined cognitive therapy/medication group has an advantage over the medication-only group. The combined group does not filter any hope or expectation bias that may be lurking in the cognitive therapy arm, while the medication-only group engenders no different hope or expectation than the medication arm in the combined group. It is thus logically invalid to compare the cognitive therapy-medication arm that can have an unfiltered cognitive therapy-positive bias from the unblinded nature of receiving cognitive therapy tasks by cognitive therapy trained therapists vs. the medication-only arm which has the same bias possibility as the medication in the combined group, but lacking any possible positive bias from the combined cognitive therapy arm. Medications are required to show efficacy when compared in a double-blind study that includes a blind-placebo control as these controls are necessary to filter bias of any hope or expectation of efficacy. Neither blind controls nor blinded placebo were used in the design of the Hollon et al. study here.

Dr. Hollon should have also noted the conflict of interest in that the Director of his dept. Dr. Stephan Heckers was also on the Editorial Board of JAMA Psychiatry when this paper was submitted and was the Editor-in-Chief of JAMA Psychiatry when it was published.

The paper was retracted once for multiple errors, and it should be withdrawn completely because of poor clinical trial logic in making claims from unblinded subjects and treaters, and in addition due to conflict of interest in having the Editorial Director (Stephan Heckers) of the publication (JAMA Psychiatry) also as head of the Faculty of the lead author's (Hollon) affiliation at Vanderbilt University.

The full comment on the Hollon et al. study above can be read here;

Double blinding requirement for validity claims in cognitive-behavioral therapy intervention trials for major depressive disorder. Analysis of Hollon S, et al., Effect of cognitive therapy with antidepressant medications vs antidepressants alone on the rate of recovery in major depressive disorder: a randomized clinical trial. By D. Berger. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4863672/

On 2016 Apr 22, Ivan Oransky commented:

This paper has been retracted and replaced: http://retractionwatch.com/2016/04/21/authors-retract-replace-highly-cited-jama-psych-paper-for-pervasive-errors/

On 2014 Aug 22, Hilda Bastian commented:

It's great to see such a thorough and rigorous body of work on this subject. This group provides a good overview of the portion downsizing issue, and the limited evidence base on interventions, at Vermeer WM, 2014.

A key part of the intervention in this trial (Poelman MP, 2015) is the interactive web-based PortionSize@warenessTool. Its development and trialing is described at Poelman MP, 2013, with these elements: background reading, an interactive flash game with photos of popular food products in the Netherlands, a flash game where you can upsize/downsize portions on screen, self-test score, information on portions for children and more.

It would be helpful if details about the availability of this intervention could be provided (e.g. where it can be viewed, if the code is open source, and if the license allows translation). The TIDieR checklist (Hoffmann TC, 2014) - the template for intervention description and replication - is a good framework for this. More details on the components of interventions is important for enabling better practice (Glasziou P, 2010).

On 2014 Aug 22, Kamel Hamzaoui commented:

excellent paper

On 2014 Sep 23, Jorge Zorzopulos commented:

Own to the importance of the Ebola outbreak dramatically described in this paper, scientists having any likely tool that could help should have a proper channel to offer it for humanitarian reasons. However, I found extremely difficult to find such proper channel. Could the authors clarify this situation. Thank you very much Dr. Jorge Zorzopulos Buenos Aires Argentina

On 2014 Aug 23, George McNamara commented:

Terrific: renewable meat! Anolis looks pretty small, good for 'bite size renewables'. If Komodo dragons use the same pathways - or can be ZFN/TALEN/Cas9-sgRNA'd to do so, renewable Dragon meat!

On 2016 Jan 08, Anne Niknejad commented:

"Two MAGL isoforms are reported in the GenBank (long isoform 1; 313 amino acids, accession code NP-001003794 and short isoform 2; 303 amino acids, accession code NP-009214) (Labar et al., 2010)."

Error, correct references should be:

NP_001003794 303 aa linear PRI 15-MAR-2015

http://www.ncbi.nlm.nih.gov/nuccore/NM_001003794.2

this sequence corresponds to the canonical UniProt sequence isoform 1 (http://www.uniprot.org/uniprot/Q99685)

and

NP_009214 313 aa linear PRI 15-MAR-2015

http://www.ncbi.nlm.nih.gov/protein/6005786

this sequence corresponds also to UniProt isoform 1 but with initial Methionine as in UniProt isoform 2, that means longer

• METGPEDPSS

(303 aa + 10 aa = 313 aa)

On 2015 May 17, Maya Guglin commented:

Yes - adjusted for all classes of HF meds. Thanks for reading me so thoroughly! I think this is a really interesting topic.

On 2015 May 17, David Keller commented:

Was the effect of CHF on type 2 DM adjusted for the use of thiazide diuretics, which promote insulin resistance?

Thiazide diuretics are used widely by congestive heart failure (CHF) patients, either alone or in combination with loop diuretics (for synergy). Thiazides are well known to decrease insulin sensitivity and lead to impaired fasting glucose and frank type 2 diabetes. Was this medication side-effect adjusted for in the calculations showing that CHF increases the risk for type 2 diabetes ??

On 2014 Aug 27, Ryan Radecki commented:

Post-publication commentary:

Hypoxia & Overtreatment in Bronchiolitis

“Treat the patient, not the number” works for many things in medicine – asymptomatic hypertension, hyperglycemia, and anemia, among others. However, hypoxia is less frequently dismissed as clinically irrelevant.

And, that perfectly explains the results in this study, which evaluated clinician dependence on oxygen saturation to guide disposition in pediatric bronchiolitis....

http://www.emlitofnote.com/2014/08/hypoxia-overtreatment-in-bronchiolitis.html

On date unavailable, commented:

None

On 2014 Aug 26, Jorge H Ramírez commented:

"Why hasn’t the intensity of scrutiny on financial interests been extended to non-financial interests ie, the ones that are far more prevalent and more strongly associated with publication misconduct?" -- Woolley K.

Response:

Financial conflicts of interests & their association with less retractions is not definitive evidence of more research misconducts by persons like me (i.e., non-financial conflict of interests).

Two open questions:

(1) How many requests of retractions for human research sponsored by pharmaceutical companies are not even openly discussed because libel laws? Re: I don't know. http://www.senseaboutscience.org/pages/keep-libel-laws-out-of-science.html

(2) How many requests of retractions are not even eligible for open scientific debate? Re: I only know one which was requested & retracted by myself. However, question thread remains open.(1)

Addendum to the original PubMed commons post: I just found an article describing similar cases to the situation described (yesterday) in the second question above. URL: http://www.bmj.com/content/341/bmj.c6985

Jorge H. Ramírez. MD, MSc, PhD. Professor of Pharmacology Universidad del Valle http://chaoticpharmacology.wordpress.com/about/ Twitter: @jorgehernnramre

References

1. Ramirez, Jorge H (2014): Requested (Jul 29, 2014) & Retracted by the author (Aug 23, 2014): "Conelly S, et al. Dabigatran versus Warfarin in Patients with Atrial Fibrillation. N Engl J Med 2009; 361:1139-1151"] - Question Thread Open. figshare. http://dx.doi.org/10.6084/m9.figshare.1144305 Note: raw data is available in this fileset

My conflict of interests related to this response are described in the following URLs: http://www.bmj.com/content/348/bmj.g1888/rr/763197 http://www.bmj.com/content/347/bmj.f4386/rr/763130 http://www.bmj.com/content/347/bmj.f1880/rr/763200

On 2014 Aug 25, Karen Woolley commented:

.

“No competing interests to declare”…oh really?

The intensity of scrutiny of financial competing interests (eg, pharmaceutical industry sponsorship of research) is understandable – there have been enough high-profile cases of financial interests associated with publication misconduct to warrant concern. But…high-profile does not equate to high-frequency…indeed, the evidence (yes, evidence) on publications retracted for misconduct reveals that most retractions for misconduct occur for publications WITHOUT declared industry support (Woolley KL et al., Curr Med Res Opin 2011). A quick look at Retraction Watch supports the ongoing problem with non-financial interests and publication misconduct. The editors of PLoS Medicine highlighted the dangers of non-financial competing interests years ago:

“Like all human activity, academic research and scientific publishing are inherently subjective, imperfect, and prone to bias, corruption, and self-interest. Indeed, because professional affinities and rivalries, nepotism, scientific or technological competition, religious beliefs, and political or ideological views are often the fuels for our passions and for our careers, private competing interests are perhaps even more potent than financial ones.” The PLoS Medicine Editors (2008) Making Sense of Non-Financial Competing Interests. PLoS Med 5(9): e199. doi:10.1371/journal.pmed.0050199

When authors disclose “no competing interests to declare” what does that mean? Who is checking that nothing means nothing? Why hasn’t the intensity of scrutiny on financial interests been extended to non-financial interests ie, the ones that are far more prevalent and more strongly associated with publication misconduct?

Our risk-management strategy for reducing the risks of interest-driven biases is rather risky. When will we prioritise strategies to manage the most prevalent and dangerous interests – the non-financial ones?

Professor Karen Woolley PhD Certified Medical Publication Professional (Twitter: @kwproscribe)

Disclosures

Non-financial: Advocate for ethical publication practices, regardless of sponsor (for-profit, not-for-profit), Director of the International Society for Medical Publication Professionals and Chair of its Asia-Pacific Advisory Committee; active member of other not-for-profit organisations providing education on ethical publication practices. Author of peer-reviewed publications on ethical publication practices.

Financial: Employee of ProScribe - part of the Envision Pharma Group (providing ethical publication planning and medical writing services to authors and sponsors worldwide - we do NOT ghostwrite)

On 2014 Sep 02, Hilda Bastian commented:

An excellent overview of the need for studying humor in science communication, and the academic challenges in it. While there’s some more evidence than that gathered here, I think Hauke Riesch’s conclusions about the uncertainties of benefit and harm are spot on.

I found the review on studies of humor in teaching he points to (Banas, 2011) helpful as well. From children through to continuing education and the communication of science among peers (Rockwood K, 2004), there’s a lot to learn here.

In describing the varying results of studies, Riesch doesn’t explicitly address a key confounder in communication research: the quality of the intervention. It’s hard to make sense of bodies of evidence in this field without quality assessments and being able to see the interventions (Glasziou P, 2010). Skill in using humor may account for some of the heterogeneity. And learning about the skills necessary for effectiveness – and how to acquire them – are key issues in this field, too.

Riesch addresses well the potentially alienating and stereotyping effect of science humor, as well as the potential benefits of social group cohesion. In addition, though, satire in peer-to-peer communication and for policy-related issues is also a critical element of humor in science communication, as it is in other areas of community life (Zyglis, 2003).

I welcome the author’s desire to “open a discussion” on humor in science communication. But this article being behind a paywall isn’t going to help that process. It would be great to know if the author is engaging with discussion in any other forum.

I’ve blogged about the science of humor, and humor in science, in response to this article at Scientific American.

On 2015 Jul 12, Weijun Liu commented:

There is a comment on this paper in Pubpeer, possible duplicated data published in their later Cripsr/cas9 E6 paper.

On 2016 Nov 25, Darya Vanichkina commented:

I was trying to reproduce the results of this study, but was unable to do so.

The information for overall survival, TCGA subtype and treatment status is not available in the Supplementary tables, or as part of the Chinese Glioma Genome Atlas.

Would it be possible for the authors to provide this information, perhaps in GEO or via the Chinese Glioma Genome Atlas web portal?

On 2014 Dec 03, Rafael Najmanovich commented:

We predicted back in 2012 (Chartier M, 2012) as part of a large scale analysis of rare codon cluster that such clusters may play a role in the molecular recognition of the nascent protein for intracellular targeting and membrane insertion. It is unfortunate that the authors were not aware of our publication.

On 2014 Sep 07, Jonathan Eisen commented:

I wrote a blog post about this paper: Awesome science paper 1st lines example regarding microbes on the Archimedes Palimpsest.

On 2014 Sep 04, Kenneth Witwer commented:

This review covers an important area of research, as saliva is obtained non-invasively and extracellular vesicles including exosomes may be enriched in useful biomarkers. Unfortunately, several apparent errors are repeated throughout the review.

-The type of extracellular vesicle known as an "exosome" is not the same entity as the nucleic acid processing complex of the same name. A confusion of the two seems evident here. Readers will need to consult the sources to determine what information is and is not relevant to extracellular vesicles.

-Intra- and intercellular vesicle trafficking are also conflated, with the Suedhof/Schekman/Rothman Nobel Prize research described as focused on exosomes. Secretory and secreted vesicles are certainly related, but they are distinct.

On 2015 Feb 27, Geriatric Medicine Journal Club commented:

This study looks at atypical antipsychotics and the risk for acute kidney injury in older adults. This paper was critically appraised at the September 2014 Geriatric Medicine Journal Club (follow #GeriMedJC on Twitter). A full transcript of the discussion can be found at http://gerimedjc.blogspot.com/2014/09/to-two-articles-critically-appraised.html?spref=tw Our colleagues from the Nephrology Journal Club (follow #NephJC on Twitter) also chimed in. In general, growing concern regarding the use of atypical antipsychotics.

On 2016 Aug 12, Carrie Price commented:

Mandatory?

On 2014 Dec 05, Joanna L Sharman commented:

Links to endothelin receptors and ligand entries from this review can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12874/full

On 2014 Sep 01, Ferenc Zsila commented:

The authors claim that in relation to physiological conditions, cancer cells have substantially lower, acidic intracellular pH.

Abstract: "...under acidic conditions (pH 5.0) mimicking the intracellular pH-conditions."

In page 364: "Specifically, the intracellular pH of tumor cells (pH 5.0-6.5) is usually much lower than physiological pH conditions (pH 7.4), which has encour-aged researchers to develop pH-responsive prodrug formulations to improve therapeutic efficacy."

In contrast to this statement, numerous data show that just the opposite is true: the intracellular pH of cancer cells is modestly alkaline (pH~7.4). Not the intra- but the extracellular milieu of tumours is acidic. Intracellular acidosis leads to cell death.

Reference: Webb BA, 2011

On 2014 Sep 01, Amanda Capes-Davis commented:

The authors describe KB as an epidermoid cell line. Although this term is widely used, it is not accurate for this cell line. KB is known to be cross-contaminated with HeLa and thus is a cervical adenocarcinoma cell line. It is not an oral squamous cell carcinoma (epidermoid carcinoma) as was originally thought.

KB is a widely used model for multidrug resistance. To help scientists who might use KB for other applications, it would be helpful if authors can refer to KB as a HeLa derivative, and to avoid the terms epidermoid, squamous cell carcinoma, or oral.

I note that the authors performed authentication testing. The information above would help readers to make sense of their test results. For a list of known cross-contaminated cell lines, see http://iclac.org/databases/cross-contaminations/.

On 2016 Apr 22, Kenneth Witwer commented:

My co-authors and I found no uptake of milk miRNAs, examining the same samples reported in this paper as well as public sequencing data from these samples, Shu J, 2015. The findings encompass the two miRNAs reported by Baier et al, which are homologous in human and cow and could not be assigned to source or human by the methods of the paper, and a variety of additional miRNAs, even uniquely bovine miRNAs. It appears that both previously offered hypotheses for the observations of Baier, et al are now unsupported: the exogenous uptake hypothesis and my hypothesis, glucose-mediated regulation of endogenous RNA, Witwer KW, 2014. Instead, as also supported by rigorous experiments with genetically manipulated mouse models, Laubier J, 2015, Title AC, 2015, milk miRNAs, like other dietary RNAs Witwer KW, 2014, Dickinson B, 2013, Snow JW, 2013, do not appear to reach biologically meaningful levels in circulation, whatever this might mean, or indeed be taken up at all through the intact mammalian gut.

On 2016 Sep 10, Morten Oksvold commented:

Before reading or citing this article, please read the reports regarding the Macchiarini case from Karolinska Institute:

http://ki.se/sites/default/files/karolinska_institutet_and_the_macchiarini_case_summary_in_english_and_swedish.pdf

http://ki.se/en/news/macchiarini

http://ki.se/en/news/external-inquiry-finds-paolo-macchiarini-guilty-of-misconduct

On 2016 Feb 14, Md. Shahidul Islam commented:

None

On 2014 Sep 15, Amanda Capes-Davis commented:

The authors use the cell line HEp-2 to look at sensitivity to cisplatin. Please be aware that HEp-2 is cross-contaminated with HeLa, a cervical adenocarcinoma cell line. Results obtained from HEp-2 do not relate to laryngeal carcinoma.

Cell lines can be tested for cross-contamination through consensus methods such as short tandem repeat (STR) profiling. For a list of known cross-contaminated cell lines, see http://iclac.org/databases/cross-contaminations/.

On 2014 Nov 11, Serge Ahmed commented:

This comment is a follow-up of my previous comment about the difficulty in interpreting this study that contradicts most previous similar studies. After a careful analysis of this paper and after collecting all elements of methods “ectopically scattered” through the text, I think I finally arrived at a satisfactory explanation for why most rats preferred cocaine over sweet water in the present study. Briefly, everything was made to make access to sweet water reinforcement less direct and more difficult than access to cocaine reinforcement, thereby biasing choice towards cocaine!

More specifically, rats had to go through an unusually long chain of behavioral events before getting access to sweet water. A similar chain was not required for cocaine delivery. First, once rats turned the wheel on the operant panel, they had to cross the cage to reach a magazine on the opposite panel inside which there was a retractable drinking spout that delivered sweet water. This arrangement introduces a spatial and thus a time gap between responding and sweet water reinforcement. Both gaps are known to reduce conditioning. Second, once rats have reached the magazine, they did not have directly access to the drinking spout that delivered sweet water. They had first to insert their head into the magazine to make the retractable drinking spout appears. This behavior amounts to a second operant response which thus defines with the first response (i.e., wheel turning) an operant chain. In addition, once rats inserted and maintained their head in the magazine, the drinking spout was not continuously available but came “back and forth in the magazine during 50s.” This is a rather unusual method of fluid delivery (note: the frequency and duration of these back-and-forth movements are not indicated in the Methods).

Thus, to repeat, everything was made in the present paper to make access to sweet water reinforcement more difficult and less direct than access to cocaine reinforcement, thereby biasing choice towards cocaine. This unusual approach may be appropriate for addressing some scientific questions but it is misguided and inappropriate for studying the vulnerability to cocaine addiction which was the main goal of the present paper. If one wants to pursue such a goal, one better tries to make access to cocaine reinforcement equal to or more difficult than access to the nondrug option and not the other way around! Indeed, if one sufficiently weakens the nondrug option, then one will eventually reach a point where most individual rats, even the non-addicted ones, will prefer the drug! To take an extreme example, if one provides rats with ready access to cocaine but ask them to play piano or climb Mt Everest to get access to sweet water, they will surely choose cocaine over sweet water. This is not surprising, this is just trivial! In contrast, if rats take cocaine despite and at the expense of an equally or a more accessible potent nondrug option, then one has got something much less trivial and probably more relevant for studying the vulnerability to cocaine addiction.

On 2014 Aug 22, Serge Ahmed commented:

This study is interesting and also quite embarrassing. It is interesting because of the important questions that it asks. It is embarrassing because it shows that when given a choice, most rats prefer cocaine over sweet water – a finding that is strictly the opposite of what we and others have found over the past few years. Of course, contradiction and refutation are the “game of science”. We should not be embarrassed by them and instead welcome them.

My embarrassment comes from the fact that these opposite outcomes were obtained by a former master student of mine – Nathalie Vanhille who is the first author of this study – using a choice protocol initially developed in our lab. When Nathalie was working in our lab using this protocol, she observed that most rats preferred sweet water over cocaine – the opposite of what she now reports in this study despite the use of an identical choice protocol.

But were the choice protocols really identical? Of course not! Like always, the devil lurks into the details and details can sometimes matter a lot! Apparently, this study differs from our previous choice studies in the way rats were given access to sweet water. In our study, access to sweet water was pretty straightforward. Rats had to press a lever to fill a nearby receptacle with sweet water. Then they could obtain additional volumes of sweet water during 20s by continually licking the receptacle. In this study, however, access to sweet water was really contrived for reasons that remain unclear until one reaches the middle of the Discussion. In fact, despite my best efforts and those of other members of the team, we were unable to get a clear final picture of how rats get access to sweet water in this study.

So here is my challenge for the interested readers and researchers: I would really appreciate if someone could help me figure out how exactly rats get access to sweet water in this study.

Thank you!

On 2014 Sep 10, Suleyman Serdar Koca commented:

I thank the comment first of all. The evaluation of serum IL-33 levels was not the primary aim of the present study. It was analyzed in limited patients to determine whether IL-33 gene polymorphisms affect serum IL-33 level. However, the detected relations of serum IL-33 level with the clinical signs were reported. Behçet’s disease has complex ethiopathogenesis. Genetic basis of the disease is divergent. Not only patients with different ethnic origins but also each patient has different disease burden. Therefore, the results of cytokines may be divergent in the different cohort.

On 2014 Aug 26, Kamel Hamzaoui commented:

Recently an interesting paper was published in Brain Behv Immunology about IL-33 in the CNS

On 2014 Aug 26, Kamel Hamzaoui commented:

Interesting paper. I cannot understand how the authors found similar IL-33 levels in BD patients than in healthy controls (52 patients were studied). They also found increased levels of IL-33 in BD patients with uveitid (32 patients were studied). We found the same increase of IL-33 in BD patients with retinal uveitis. We and others found increased levels of IL-33 in serun, in cerebrospinal fluid and in EN skin lesions measured at the mRNA and immunohistology.

Our laboratory and the Korean laboratory used the same Kit Elisa, but not the authors. The authors of the paper have to study control disease such as RA, SLE, MS patients.

Thank you

On 2017 Nov 12, Gerardo Ferbeyre commented:

I have red this paper many times and I find always inspiration for experiments and new ideas. The authors condensed a tremendous body of work while keeping their main message beautiful and simple. In pancreatic cancer, inhibition of the RAS/MAPK pathway allows the emergence of a subpopulation of dormant tumor stem cell with increased dependency on mitochondria. We have reproduced many of the key characteristics of these pancreatic cancer stem cells using a different experimental model and I wonder whether this phenotype is unique to these cancer cells when they evolve or whether the cells are reactivating a primitive embryonic program of pancreatic epithelial stem cells.

On 2014 Nov 24, Guillaume Filion commented:

This article is one of the "CISCOM meta-analyses", which are very similar papers written by different authors. For more information about the CISCOM meta-analyses, check the blog post "A flurry of copycast on PubMed" at the following link http://blog.thegrandlocus.com/2014/10/a-flurry-of-copycats-on-pubmed

On 2014 Aug 27, Ryan Radecki commented:

Post-publication commentary:

"Should the 48-hour Cardioversion Window Be Revised?"

It has become generally accepted practice to treat new-onset atrial fibrillation and atrial flutter with electrical cardioversion in the acute setting – provided the known onset of atrial fibrillation is less than 48 hours. Beyond that, caution tends to be advised – whether through use of transesophageal echocardiography to rule out left atrial thrombus, or through pre- and post-procedural anticoagulation.

However, this data from a research letter in JAMA suggests – possibly we ought to be even more cautious regarding time-of-onset....

http://www.emlitofnote.com/2014/08/should-48-hour-cardioversion-window-be.html

On 2014 Sep 15, Gaetano Santulli commented:

In this elegant retrospective cohort study Gialdini and colleagues report that among patients hospitalized for surgery, perioperative atrial fibrillation is associated with an increased long-term risk of ischemic stroke, especially following noncardiac surgery. To predict cumulative rates of ischemic stroke the Authors used the CHA2DS2VASc score. Remarkably, in the low-risk categories (score≤1) there are 13 and 9 strokes among patients undergoing cardiac and noncardiac surgery, respectively. The Authors however do not provide any information about baseline electro-, echocardiographic or cerebrovascular assessment for these subjects. Thus, clinical data on potential cardiac (inter-atrial septum defect, mitral valve stenosis, cardiomyopathies) and cerebrovascular alterations, in our opinion essential for interpreting the results provided, are missing. This aspect is particularly relevant since the above-mentioned disorders are functionally related to ischemic stroke (1,2) and might exert pro-thrombotic effects also in low-risk subjects. Moreover, diabetes mellitus has been shown to be an independent risk factor for stroke, including in patients with low CHA2DS2VASc score (3). In this sense, it would be interesting to know the percentage of diabetic patients in the low-risk population. Equally important, a more comprehensive assessment of the baseline coagulation status could be useful: given the lack of data on antithrombotic therapy, acknowledged by the authors in the limitations of the study, it would be helpful to the Reader to know at least the rate of hemorrhagic stroke events.

References 1. McCabe DJ, Rakhit RD. Antithrombotic and interventional treatment options in cardioembolic transient ischaemic attack and ischaemic stroke. J Neurol Neurosurg Psychiatry. 2007 Jan;78(1):14-24. 2. Finsterer J, Stollberger C. Stroke in myopathies. Cerebrovasc Dis. 2010;29(1):6-13. 3.Marfella R, Sasso FC, Siniscalchi M, Cirillo M, Paolisso P, Sardu C, Barbieri M, Rizzo MR, Mauro C, Paolisso G. Brief episodes of silent atrial fibrillation predict clinical vascular brain disease in type 2 diabetic patients. JACC. 2013;62(6):525-30.

Celestino Sardu, MD (1), Gaetano Santulli, MD, PhD (2) (1) Second University of Naples, Naples, Italy; (2) Columbia University Medical Center, New York, NY, USA

Conflict of interest: none.

On 2015 Jan 01, Prashant Sharma, MD, DM commented:

Please note that these extremely precious specimens in these tiny humans were obtained by trained pathologists, and not neonatologists, pediatricians or radiologists.

Just as is the case for fine needle aspirates, the best bone marrow specimens with highest diagnostic yields and lowest inadequacy rates come when pathologists themselves take the specimen they are to subsequently analyze.

There are of course many other issues (logistic, training, regulatory, safety) that are tied up with this fact, but the above, in my experience, is invariable.

On 2014 Oct 27, Maria Flavia Di Renzo commented:

As mentioned in the paper and shown in Supplementary Figure 2, we also used different single shRNAs. Actually, we also used five shRNAs specific for different sequences of CDT2 and different from the sequences of the four siRNAs of the pool. We obtained regulation of CDT2 targets and commitment towards cell death in cancer cells but not in normal cells. However, the in-depth analysis of molecular targets and cell cycle was not feasible in cells stably committed to death. Thus we carried out experiments with siRNAs that resulted in similar, but transitory, biochemical and functional effects. In conclusion, we are confident that the observed phenotypes might be attributed to CDT2 suppression.

On 2014 Oct 17, Eugen Buehler commented:

The authors cite our paper (Buehler E, 2012) as evidence that “…pools of siRNA targeting different mRNAs, such as those used in libraries, results in increased off-target effects”. Unfortunately, that sentence is not supported by our paper. The method demonstrated in our paper was applied to an arrayed screen of individual siRNAs in which there were no pools, and no conclusions about pooling of siRNAs can be reached from that manuscript. Furthermore, the authors state that “The use of this siRNA pool allows avoiding too high concentration of each single siRNA and thus prevents off-target effects” and cite Jackson AL, 2010 to support this. Again, this assertion is not supported by the cited reference. Jackson AL, 2010 states that “Pooling of multiple siRNAs to the same target may help to reduce off-target silencing, due to competition among the siRNAs in the pool” (emphasis added). No evidence exists to support the claim that pools 3 or 4 siRNAs will prevent off-target effects. To the contrary, we have demonstrated previously that pooling siRNAs does not generate more reproducible phenotypes than single siRNAs (see Marine S, 2012, Figure 1, B and C) and that pools of siRNAs can and do generate many off-target phenotypes, which can then be confirmed by deconvolution of those pools (see Marine S, 2012, Figure 2). Unfortunately, the authors base most of their conclusions on experiments using a single reagent (a pool of four different siRNAs targeting CDT2) and a single non-silencing control. This means that many or all of the phenotypes they observed experimentally may be due to siRNA off-target effects (see Chung HY, 2014 for a case study in how off-target effects can result in false positives). This is not to say that their conclusions are incorrect, only that I believe that RNAi experiments require multiple independently tested reagents and/or matched controls to guard against the false positives due to off-target effects that are so frequent in these experiments.

On 2014 Aug 28, Frederick K Korley commented:

Agreed. Troponin values can no longer be used in a dichotomous fashion. Any troponin elevation is a bad troponin elevation. However, those without acute elevations may not necessarily need inpatient hospitalization. If they are discharged, they will benefit from expedited outpatient follow-up.

On 2014 Aug 27, Ryan Radecki commented:

Post-publication commentary:

"Highly Sensitive Troponins – False Positive Bonanza"

The “highly sensitive” troponin has received a great deal of publicity, hyped ad nauseum, see: “Simple test could help rule out heart attacks in the ER.”

But, as sensitivity increases – invariably, specificity decreases. However, that is not the fault of the test – it is a failure of clinicians to ask the correct question of the test. When asking “does this patient have an acute myocardial infarction?”(most commonly Type 1 MI in the ED), our training and education has been outpaced by assay technology – the test no longer provides a dichotomous “yes” or “no”.

http://www.emlitofnote.com/2014/08/highly-sensitive-troponins-false.html

On 2014 Nov 02, Swapnil Hiremath commented:

This systematic review, along with its accompanying review was discussed on Oct 21st 2014 in the open online nephrology journal club, #NephJC, on twitter. Introductory comments are available at the NephJC website. It was a great discussion, with more than 20 participants, including nephrologists, cardiologists and residents/fellows from different specialties. A transcript and a curated (i.e. Storified) version of the tweetchat are available from the NephJC website. The highlights of the tweetchat were:

• The investigators and the funding agency (AHRQ) should be commended for attempting to answer these important questions about the role of troponins in patients with chronic kidney disease.

• Most participants rely on a rise in troponin levels to help diagnose acute coronary syndrome in CKD patients, and were disappointed that no evidence was found to support (or refute) this practice.

• The adverse prognostic implications of higher troponin levels, especially in asymptomatic CKD patients was thought to be quite concerning and was the subject of much discussion, with many possible therapeutic management options being raised.

Overall, these reviews answered a few questions, but also brought out many areas where further research needs to be focused. Interested individuals can track and join in the conversation by following @NephJC or #NephJC, or visit the webpage at NephJC.com.

On 2014 Nov 02, Swapnil Hiremath commented:

This systematic review, along with its accompanying review was discussed on Oct 21st 2014 in the open online nephrology journal club, #NephJC, on twitter. Introductory comments are available at the NephJC website. It was a great discussion, with more than 20 participants, including nephrologists, cardiologists and residents/fellows from different specialties. A transcript and a curated (i.e. Storified) version of the tweetchat are available from the NephJC website. The highlights of the tweetchat were:

• The investigators and the funding agency (AHRQ) should be commended for attempting to answer these important questions about the role of troponins in patients with chronic kidney disease.

• Most participants rely on a rise in troponin levels to help diagnose acute coronary syndrome in CKD patients, and were disappointed that no evidence was found to support (or refute) this practice.

• The adverse prognostic implications of higher troponin levels, especially in asymptomatic CKD patients was thought to be quite concerning and was the subject of much discussion, with many possible therapeutic management options being raised.

Overall, these reviews answered a few questions, but also brought out many areas where further research needs to be focused. Interested individuals can track and join in the conversation by following @NephJC or #NephJC, or visit the webpage at NephJC.com.

On 2014 Aug 12, Ryan Radecki commented:

Post-publication commentary:

"Just Another Advertisement for tPA"

As with last week's coverage of the updated Cochrane Systematic Review for tPA in acute ischemic stroke, the key question is: what’s new?

The first pooled meta-analysis, published in The Lancet in 2004, included NINDS, ECASS I, ECASS II, and ATLANTIS. It was subsequently updated in 2010 to add ECASS III and EPITHET. Now, these authors have decided to add IST-3.

I am actually a huge fan of individual-patient meta-analyses. Depending on the data availability, the similarity of trial protocols, and other issues associated with heterogeneity, this is the gold-standard for aggregating data and increasing power. Individual-patient analyses also allow for more reliable exploration of subgroup effects not otherwise possible through regular meta-analyses or systematic reviews.

But, at the crux of it, a meta-analysis is only as good as the included trials – and this is a topic much debated over the last twenty years....

http://www.emlitofnote.com/2014/08/just-another-advertisement-for-tpa.html

On 2014 Aug 12, Jens Staal commented:

Interestingly SNP rs147414021 corresponds to a R149Q mutation. It will be interesting to see whether this SNP is associated to disease.

http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?geneId=10892

On 2014 Nov 24, Guillaume Filion commented:

This article is one of the "CISCOM meta-analyses", which are very similar papers written by different authors. For more information about the CISCOM meta-analyses, check the blog post "A flurry of copycast on PubMed" at the following link http://blog.thegrandlocus.com/2014/10/a-flurry-of-copycats-on-pubmed

On 2014 Nov 05, Fillip Port commented:

This paper reports on the use of the H1 promoter to drive expression of gRNAs for CRISPR/Cas genome engineering. The authors demonstrate that gRNAs expressed from H1 can efficiently modify the genome of cultured human cells in conjunction with Cas9 endonuclease. Interestingly, gRNA expression levels from H1 are lower than from the commonly used U6 promoter. Although this can negatively effect mutagenesis rates at the on-target site, it can also increase CRISPR/Cas specificity, as high activity is more likely to lead to off-target effects. This makes the H1 promoter a potentially useful tool for CRISPR/Cas genome engineering in human cells.

However, the authors suggest that their results have much more general significance by expanding the CRISPR/Cas genome targeting space. This is because the U6 promoter initates transcription at a G nucleotide (although see comment below and reference therein), which according to the authors constrains genomic target sites to GN19NGG. This assertion is surprising as it is common practice in the CRISPR field to target sites that do not start with a G by simply adding a (often mismatched) G to the corresponding gRNA or to replace the first nucleotide with a G to create a gRNA that is mismatched at the first position. The authors acknowledge these strategies in the first paragraph of their discussion, but cite six papers as providing evidence that 5’ extensions or truncations reduce gRNA efficiency. However, these papers in fact provide evidence that 5’ extensions or truncations of a single nucleotide often have no effect on activity and when they do the effect is usually minor (modified gRNAs usually retain >80% activity). Furthermore, the authors do not cite another study that shows that small gRNA truncations retain high activity in the majority of cases and have reduced off-target effects (Fu Y, 2014). Therefore, much of the published evidence suggests that extending or truncating gRNAs by a single nucleotide is well tolerated by the great majority of gRNAs. As a result it is in principle possible to target any genomic site adjacent to a PAM motif with gRNAs expressed from a U6 promoter and hence the H1 promoter, although potentially useful, does not expand the CRISPR target space.

On 2014 Aug 12, Haoquan Wu commented:

The preferred initiation nucleotide for U6 promoter are A and G, as we have shown in our study which can be found http://www.nature.com/mtna/journal/v3/n5/full/mtna201412a.html. We also confirmed that gRNAs starting with A can disrupt the target gene at similar level as gRNAs starting with G in the study. H1 promoter is much weaker than U6, which might significantly lower the CRISPR-Cas9 system efficiency.

It is amazing that the commonly accepted conceptions about U6 promoter initiation and termination are not complete although U6 promoter has been the most commonly used promoters to drive small RNA expression. The commonly accepted conception that the continuous Ts is the termination signal for U6 promoter is also not correct.

On 2015 Sep 10, Lydia Maniatis commented:

The authors say that “we assessed whether the highest luminance is mapped onto a fixed surface reflectance (“white”) independently of target reflectance, illumination level (real or simulated), or luminance. ”

Evidently, by “illumination level” they are referring to the collection of luminances of their “Mondrians,” and by “luminance” they are referring to the luminance of individual target surfaces. It may be that, in relatively low luminance conditions, observers experienced a sense of low illumination, but neither the authors nor anyone else has, to my knowledge, tested whether such an impression does, in fact, arise. The fact that “low illumination” conditions resulted in lower lightness matches for targets actually implies the opposite, since impressions that surfaces are “in shadow” typically cause them to appear lighter than equiluminant. apparently plain view surfaces. So average luminance (regardless of how the investigators achieve this) and contrast are the variables being looked at,

As is typical in experiments using “Mondrians” or checkerboard stimuli, the authors seem to overlook the fact that these stimuli are capable of producing differential illumination, transparency, luminosity effects, despite the absence of the usual “cues.” This is evident, for example, in checkerboard stimuli used by Allred, Radonjic, Gilchrist & Brainard (2012), who tentatively acknowledge (but do not test for) some but not all of the (self-evident) effects. Anderson et al cite Radonjic, Allred, Gilchrist and Brianard (2011) as having shown that “a stimulus ratio of 5905:1 could be mapped onto an extended lightness ratio of 100:1 and concluded that such results ruled out theories that predict perceived lightness from luminance ratios or Weber contrast” and suggest that “these data cast significant doubt on the view that the visual system has any understanding of the range of reflectance values that populate natural environments.” However, it is not clear that the high-range stimuli in Radonjic et al's experiments did not produce luminosity effects. Their observers did, in fact, report luminosity for the highest luminance values in those stimuli, but the authors discounted those reports as being due to the stimuli having been presented on an “emissive screen.” However, this explanation does not seem credible given that the same luminance values did not produce luminosity reports in lower-range situations.

If there were apparent scission effects going on in Anderson et al's stimuli, then observer reports would have been affected. When a surface is completed “beneath” a shadow, and seen as homogeneous in its lightness, this does not mean that we can't see that it is also darker at the shadowed place, in the same way that amodally completed surfaces are seen and not seen at the same time. You can't disprove that an amodal completion is occurring simply on the basis of asking an observer to report on the local color in the image at an “obstructed” location. Similarly, simply asking a naive observer to make a lightness match where scission is occurring is problematic.

It is always important for readers of perception papers to be able to see the stimuli used, so it would have been good if all of the Mondrians, and not just the one, were made available.

On 2016 Sep 18, Daniel Schwartz commented:

The ROKS nomogram can be used to predict recurrence of a second kidney stone via an online calculator or via a mobile app https://www.qxmd.com/calculate/calculator_3/roks-recurrence-of-kidney-stone-2014

Conflict of interest: Medical Director, QxMD

On 2014 Aug 13, Amanda Capes-Davis commented:

Unfortunately many cell lines are misidentified and do not correspond to the tissue from which they were thought to be established. This is the case here: both HEp-2 and KB are cross-contaminated by HeLa, a cervical adenocarcinoma cell line. For a list of known cross-contaminated or otherwise misidentified cell lines, see http://iclac.org/databases/cross-contaminations/.

On 2015 Mar 28, Amit Kumar Chowhan commented:

Dear Sir,

We read the article by Srikanth S et al.¹ on ‘A comparative study of fine-needle aspiration cytology (FNAC) and fine-needle non-aspiration cytology (FNNAC) techniques in head and neck swellings’ with interest and appreciate the inclusion of multiple organs i.e. lymph node, thyroid and salivary gland located in head and neck region in the study. Although fine needle aspiration cytology is a well-established tool as a first line diagnostic modality, however, a major criticism pertains to its use in highly vascular organs such as thyroid and liver, or in haemorrhagic lesions where large quantities of blood compromise cytologic interpretation. Hence we agree with the authors when they excluded lesions of vascular origin from their study.

We agree with the authors that FNNAC allows greater ease of sampling with better control of the hand during the procedure and a good perception of the lesion consistency, enabling more precise entry into the mass and thus is more user friendly. Apart from being less traumatic and painful to the patient, as suggested by the authors, we would like to add that with this technique the patient would also be less apprehensive about the procedure, when a large syringe with needle and a syringe holder is not seen, thus making FNNAC more patient friendly. Another advantage of FNNAC is that the syringe is used to expel the material after the procedure is completed, whereas in FNA it is used to create a suction force to aspirate the cells in to the needle. A fresh sterile syringe is therefore not necessary for FNNAC, thus reducing the cost of procedure.

In their study the authors concluded that for thyroid lesions the non-aspiration technique was better than aspiration technique with respect to all the five parameters proposed by Mair et al.² i.e. background blood/clot, amount of cellular material, degree of cellular degeneration & trauma and retention of appropriate cellular architecture. In a similar study conducted by us³ on thyroid lesions, we found that the non-aspiration technique was better in relation to all the parameters except for amount of cellular material, which was better with aspiration technique. In cases of colloid goiter, brownish colloidal fluid drained out immediately on putting needle and drenched the fingers holding the needle. This problem was handled by keeping a syringe ready, which was immediately attached to the needle and the fluid collected in the syringe – to be cyto-centrifuged for better cellularity. Another problem encountered was for calcified nodules, which required vigorous aspiration, as it didn’t yield any material on non-aspiration. We therefore, would recommend first non-aspiration technique to be performed and if the material obtained is insufficient then only to go for another pass with aspiration technique.

References:

1.Srikanth S, Anandam G, Kashif MM. A comparative study of fine-needle aspiration and fine-needle non aspiration techniques in head and neck swellings. Indian J Cancer 2014;51:98-9.

2.Mair S, Dunbar F, Becker PJ, Du Plessis W. Fine needle cytology - is aspiration suction necessary? A study of 100 masses in various sites. Acta Cytol. 1989;33:809-13.

3.Chowhan AK, Babu KV, Sachan A, Rukmangdha N, Patnayak R, Radhika K, Phaneendra BV, Reddy MK. Should we apply suction during fine needle cytology of thyroid lesions? A prospective study of 200 cases. Journal of Clinical and Diagnostic Research 2014;8:19-22.

On 2014 Sep 01, Amanda Capes-Davis commented:

The KB cell line is often used to test therapeutic activity against cancer. But although KB comes from cancer, it is not oral squamous cell carcinoma. KB is known to be cross-contaminated with HeLa and is actually cervical adenocarcinoma. For a list of known cross-contaminated cell lines, see http://iclac.org/databases/cross-contaminations/.

On 2014 Sep 01, Graham Coop commented:

Here is a post on some of the difficulties of interpreting positive signals of polygenic selection found by our approach: http://gcbias.org/2014/08/07/some-thoughts-on-our-polygenic-selection-paper/

On 2014 Aug 18, David Keller commented:

Ibuprofen, but not other NSAID's, is associated with decreased risk of Parkinson's disease

Ibuprofen use has also been observed to be strongly associated with a lower risk of incident Parkinson's disease (1). Other NSAID's, such as naproxen, have not showed any such apparent protective effect (2). Given that long-term use of NSAID's "such as ibuprofen" has been observed to be associated with "reduced risk and delayed onset of Alzheimer's disease", I would be cautious about substituting a different NSAID, such as flurbiprofen, unless it has also demonstrated the same associations with reduced Alzheimer's risk, because the epidemiological studies of NSAID use and Parkinson's risk have indicated that, when it comes to the risk of neurological degeneration, different NSAID's can have vastly different effects.

References

1: Gao X, Chen H, Schwarzschild MA, Ascherio A. Use of ibuprofen and risk of Parkinson disease. Neurology. 2011 Mar 8;76(10):863-9. doi: 10.1212/WNL.0b013e31820f2d79. Epub 2011 Mar 2. PubMed PMID: 21368281; PubMed Central PMCID: PMC3059148.

2: Driver JA, Logroscino G, Lu L, Gaziano JM, Kurth T. Use of non-steroidal anti-inflammatory drugs and risk of Parkinson's disease: nested case-control study. BMJ. 2011 Jan 20;342:d198. doi: 10.1136/bmj.d198. PubMed PMID: 21252104; PubMed Central PMCID: PMC3023971.

On 2014 Aug 19, Sameer Agnihotri commented:

Dear Dr. Li, the uncorrected proof is currently online. Figure 7 will be corrected soon for the final edited version. Thank you for your observation.

On 2014 Aug 12, Mengxia Li commented:

In Figure 7, the authors listed LIG4 as BER gene by mistake which is not a typical BER gene and functioning in NHEJ.

On 2017 Oct 21, Anju Anand commented:

http://bmjopen.bmj.com/content/6/7/e011519.responses#effectiveness-of-the-assessment-of-burden-of-copd-abc-tool-on-health-related-quality-of-life-in-patients-with-copd-twitter-discussions-from-the-university-of-toronto-respirology-and-sleep-journal-club-respandsleepjc

This is a summary from our twitter online journal club on this article #rsjc

On 2014 Nov 27, Mangesh Thorat commented:

Our systematic review of harms associated with aspirin use is now published (Thorat MA, 2015). Also recently published is our detailed response (Thorat MA, 2015) to Elwood P, 2015, who suggest that we have overestimated aspirin’s harms.

On 2014 Aug 22, Mangesh Thorat commented:

Response to Hilda Bastian’s recent comment:

Thank you for the continued discussion. Individual studies like PHS did report a 5-year follow-up, which is not uncommon. Rothwell’s recent overview (Rothwell PM, 2012) did look at studies with shorter follow-up, but the central question in this overview was aspirin’s effect on incidence. The effect on incidence starts to appear at 3 years, while that on mortality takes about 5 years. On the other hand, for example, the endpoint Seshasai SR, 2012 used was mortality and not incidence and therefore they could not observe a significant reduction. Sutcliffe P, 2013 looked at all these data and treated them as equal. Additionally, they did not have access to updated WHS results that showed a significant reduction in CRC. This resulted in their excessive perception of uncertainty; it is prominently reflected in their interpretation.

We believe that most experts agree that "the evidence supporting aspirin's benefits on cancer is now overwhelming.", the differences in opinion probably only exist for the magnitude and site-specific effects (e.g. 3 of our co-authors). This is the reason we provide several sensitivity analyses that use lower magnitude of benefits, higher magnitude of harms and also lack of effect on certain cancer sites. All these show a net benefit.

We agree that long-term harms should not be easily dismissed, but we believe that the severity of harms also needs to be considered in any assessment. In our assessments, we have erred on the side of caution and very likely over-estimated the harms. Individual circumstances differ, and therefore we believe that a careful assessment by and an informed discussion with a healthcare professional is necessary.

We also look forward to the new USPSTF review as we have been informed that on this occasion USPSTF will look at the overall picture by assessing impact on all diseases/conditions affected by aspirin and not just single disease/disease group.

Response to David Colquhoun’s comment:

Please note that the NHS Choices comment has been amended to delete the unsubstantiated statement about our study being ‘not reliable’. As stated in the paper this was a benefit-harm analysis based on very recent systematic overviews by some of our co-authors, so it was not necessary to repeat them.

On 2014 Aug 18, David Colquhoun commented:

NHS Choices does rather good assessments of medical headline news. I notice that they say of this study

"While the findings of this study show promise, it is not clear whether the methods used in compiling it were systematic, so the results may not be entirely reliable."

On 2014 Aug 16, Hilda Bastian commented:

Thanks for replying, Mangesh Thorat. I didn't review the primary studies, so hadn't picked up the error in Sutcliffe P, 2013 with respect to the Women's Health Study (Cook NR, 2013). The concern remains valid, as it applies to most of the evidence.

I disagree, though, that the Sutcliffe review has a "major flaw," considering all studies equal irrespective of follow-up. Their analyses for duration of follow-up are front and center. And they specifically report on, and discuss, 20-year analyses on colorectal cancer, in coming to their conclusions.

Nor are they the only group in this field to consider studies with shorter follow-up (see for example Rothwell PM, 2012). And the Physicians' Health Study (Steering Committee of the Physicians' Health Study Research Group., 1989) had 5-year follow-up.

Many people agree with your statement that "the evidence supporting aspirin's benefits on cancer is now overwhelming." But many do not. The National Cancer Institute's recent round-up (NCI, 2014) considers perspectives on the same body of evidence. NCI highlights "mixed opinions" and "reasons for caution."

While the potential for important net benefit from daily low-dose aspirin for more people is vitally important, I don't think the issue of harms of longterm use should be too easily dismissed. People who have common conditions that are potentially affected by taking aspirin daily (like asthma (Morales DR, 2014)), or at high risk of developing ARMD from mid-life, or whose concomitant medication use may be a relevant consideration (such as with arthritis (Colebatch AN, 2011)) might well want less uncertainty about what this means for them.

Given the differing interpretations of this body of evidence, the findings of the US Preventive Services Task Force review, expected this year, will be interesting (NCI, 2014).

On 2014 Aug 15, Mangesh Thorat commented:

We thank Hilda Bastian for her comment, our response to the points raised is given below:

Sutcliffe P, 2013's systematic review is not discussed in Cuzick J, 2015 because we believe that it has a major flaw; it considered all reviews to be equal irrespective of the length of follow-up. For example, the review by Seshasai SR, 2012 that failed to show any cancer benefit had a follow-up of only 6 years. As it takes 5 years for aspirin’s beneficial effects on mortality to appear, inclusion of such data by Sutcliffe P, 2013 resulted in underestimation of beneficial effects on cancer. The updated results of WHS (Cook NR, 2013), which showed 42% reduction in CRC incidence were published almost at the same time as Sutcliffe P, 2013, and therefore were not included in this review. Sutcliffe P, 2013 based their interpretation on earlier WHS results (Cook NR, 2005), which did not show any reduction in CRC.

Sutcliffe P, 2013 also were under wrong impression that all the primary studies and meta-analyses for benefit "assessed reduction in cancer incidence and mortality retrospectively through re-analysis of RCTs of aspirin for primary prevention of CVD." Cancer incidence and mortality is one of the primary endpoints in the WHS (Cook NR, 2013). The importance of WHS lies in the fact that it not only confirmed the benefit in cancer as a primary endpoint, even with alternate day low dose, but also confirmed that there is a long lead time and a prolonged carry-over benefit. This is where the recent WHS publication (Cook NR, 2013) differs from results published earlier (Cook NR, 2005).

We also disagree with the statement that “uncertainty around the cancer estimates remains high”, a very large body of evidence from observational studies (Bosetti C, 2012; Algra AM, 2012) is consistent with the findings from RCTs and should not be ignored as done in Sutcliffe P, 2013. The evidence supporting aspirin’s benefits on cancer is now overwhelming with over 200 published studies and those with adequate follow up showing very consistent evidence for a reduced incidence and mortality of three major digestive track cancers – colon, stomach and oesophagus.

It is clear from the evidence that the harms associated with aspirin (and the cardiovascular benefits) begin at the time of use and cease with stoppage of drug use. However, cancer benefits have a lead time before becoming apparent, but these continue for a long period after stopping drug use; a long carry-over effect as seen with other preventive drugs like tamoxifen. With this understanding, mere pooling of data from meta-analyses and trials with variable treatment durations and variable post-treatment follow-up to assess benefit and harms, as Sutcliffe P, 2013 have done is not a reliable method for assessing the impact of aspirin. This is primarily where our work and therefore the results differ.

In addition, we have modelled benefits and harms of aspirin for the average risk population using actual event rates in the general population to give estimates of the impact of aspirin specifically for this group, which is the major focus of our work.

We accept that the question of aspirin’s impact on ARMD is unresolved, but ARMD is uncommon (National Eye Institute) below 70 years of age, which again is the group on which we have focussed our attention.

On 2014 Aug 10, Hilda Bastian commented:

These authors (Cuzick J, 2015) come to a more positive conclusion about the state of the evidence on routine aspirin use and cancer prevention than do Sutcliffe P, 2013 (also reported at Sutcliffe P, 2013).

Sutcliffe P, 2013 undertook a thorough and well-reported systematic review of the evidence, based on previous systematic reviews, the primary studies in them, and the relevant RCTs published post-2008, re-analyzing the primary study data. They took into account the same individual patient data and other meta-analyses on which Cuzick J, 2015's interpretation of benefit rely. (Sutcliffe P, 2013's systematic review is not discussed in Cuzick J, 2015.)

The main data included in Cuzick J, 2015 but unavailable to Sutcliffe P, 2013 appear to be an analysis of harms (where insufficient detail on the sources or selection process have been published), and a long-term follow-up report from the Women's Health Study (Cook NR, 2013). However, as Cook NR, 2013 shows a broadly similar outcome to the <10 year results (no effect on total cancers, but an effect on colorectal cancer only), this does not appear to account for the difference in interpretation of the state of the evidence by these two groups.

The main data relied on in Sutcliffe P, 2013 that differ to those in key analyses of Cuzick J, 2015 are the Physicians' Health Study (Steering Committee of the Physicians' Health Study Research Group., 1989) and the Women's Health Study (Ridker PM, 2005). These are of long-term aspirin use on alternate days, rather than daily. These two studies include around 62,000 people, and Sutcliffe P, 2013's analyses show they dominate several calculations.

Sutcliffe P, 2013 point to a critical issue: all the primary studies and meta-analyses for benefit "assessed reduction in cancer incidence and mortality retrospectively through re-analysis of RCTs of aspirin for primary prevention of CVD." They conclude that the uncertainty around the cancer estimates remains high, and the "long term all-cause mortality data does not provide a compelling case for aspirin protection against CVD and cancer mortality."

With further trials underway, the picture may become clearer in the next few years. While previous trials and analyses address the major harms associated with long-term daily aspirin use (hemorrhagic stroke and gastrointestinal bleeding), many people considering this intervention may also be concerned about additional outcomes. For example, the still-unresolved question of any potential impact on neovascular age-related macular degeneration (Klein BE, 2012, Liew G, 2013, Christen WG, 2014).

On 2016 May 10, Morten Oksvold commented:

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

https://ori.hhs.gov/content/case-summary-pastorino-john-g

On 2014 Aug 13, Amanda Capes-Davis commented:

This study looks at laryngeal carcinoma, using patient tissue and a cell line model. Please be aware that the cell line used here, HEp-2, is not from laryngeal carcinoma. HEp-2 is cross-contaminated with HeLa and is actually cervical adenocarcinoma. For a list of known cross-contaminated cell lines, see http://iclac.org/databases/cross-contaminations/.

On 2014 Aug 28, Mohammad Rostami Nejad commented:

I read with interest the paper by Rahmati et al. entitle” Correlation of Tissue Transglutaminase Antibody with Duodenal Histologic Marsh Grading” which retrospectively reviewed hospital files of 159 patients with available tTG titer and pathology reports in the gastrointestinal clinic of Firoozgar Hospital, Tehran, Iran. They reported that the mean tTG titers was significantly higher in patients with Marsh III (a-c) and more than 9 folds higher than the kit’s cut-off value. In their conclusion they said that small intestinal biopsy should always be considered in case of high clinical suspicion, regardless of the results of serologic testing and I personally not agree with this statement. New guidelines confirmed that celiac disease diagnosis in children can be made on the basis of clinical signs, serology and genetics without the need of biopsy. Therefore, in my opinion HLA typing is useful tool for this matter and can be used as diagnosis tool for celiac disease and we can avoid invasive procedures like endoscopy in these cases.

On 2014 Aug 10, Ryan Radecki commented:

Post-publication commentary:

"Bizarrely Alarmist Pediatric URI Study"

In our new Gawker and Buzzfeed-fueled, short-attention span reality, attention-grabbing headlines are essential. So, let me come up with the modern headline for news coverage of this latest article, published in Pediatrics: “Is your child's next cold a killer?”

Seriously, as covered by Medscape (subscription required): “As many as 1 in 3 children seeking treatment in the emergency department for influenza-like illnesses (ILI) at the peak of influenza season are at high risk of suffering severe complications, such as pneumonia.”

But, that’s hardly the case....

http://www.emlitofnote.com/2014/08/bizarrely-alarmist-pediatric-uri-study.html

On 2015 Mar 15, David Simpson commented:

Mass spectrometry-based proteomics dataset deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with dataset identifier PXD001528; DOI: 10.6019/PXD001528 (http://dx.doi.org/10.6019/PXD001528).

On 2014 Aug 12, Jacob Puliyel commented:

I congratulate the authors of this analysis. However their analysis begs more questions than it answers.

They found that there were 581 cases of ultrasound diagnosed intussusception per 100,000 child years, during the Rotavirus trial, which works out to be 1 intussusception in every 172 children, each year, or 1 intussusception for every 86 children who were followed up for 2 years in the study (approximately - not counting 6 weeks before vaccination).

Bhandari et al Bhandari N, 2014 have reported that 40 babies have to be vaccinated to prevent one severe rotavirus gastroenteritis episode (NNT = 40) in the 2 year of study.

The analysis by Jehangir et al Jehangir S, 2014 of data from the Rotavirus vaccine trial showed that 1 in every 86 babies in the trial: 1) developed symptoms and signs of intussusception confirmed by a study pediatrician (namely pass blood in stools, or have continuous vomiting, abdominal distension or abdominal lump) and 2) had the diagnosis of intussusception confirmed on ultra sound.

About half the cases resolved spontaneously. In field conditions, the other half will need urgent radiographic reduction or surgery and if these are not available (in remote villages where the vaccine will be administered), mortality is near 100%

Jehangir and colleagues report intussusception in the study sample, without differentiating the babies who received the study drug (rotavirus vaccine) from those who received placebo. This differentiation is crucial because the rate of intussusception in the controls can be assumed to be the natural rate of intussusception using the surveillance methods described in the study.

As the study has now been analysed after unmasking the vaccine recipients, this data on how many among the trial drug recipients and how many among the placebo recipients developed (ultrasound proven)intussusception, should be provided on the PubMed Commons. From this we can determine the NNT for intussusception (numbers of babies that need to be vaccinated to cause intussusception in 1 child) The authors need to publish data on the number of ultrasound diagnosed intussusception per 100,000 child years among those who received rotavirus vaccine and the corresponding figure for placebo recipients.

Instead of this comparison, the authors do a retrospective analysis of data on intussusception treated at their tertiary referral hospital, between 1 January 2010 and 31 August 2013.

Only babies who had intussusception that needed surgical or radiological treatment and which was confirmed on ultrasound examination, were included.

Thus only cases qualifying as intussusception at Level 1 diagnostic certainty, were included and all those whose intussusception resolved spontaneously (without medical intervention) were excluded from the retrospective study.

Under these circumstances it is meaningless to assert that all the babies selected for analysis in the retrospective study needed some intervention but only about 44% of those that were identified in the rotavirus vaccine-trial-active-surveillance, needed intervention.

The authors then go on to conclude that, as 56% of babies with ultrasound diagnosed intussusception in the vaccine trial recovered spontaneously, active surveillance is not a good method to detect adverse events following immunization, and that sentinel hospital based surveillance (for post marketing surveillance after rotavirus vaccine introduction) was better.

The rationale for this conclusion is difficult to fathom. Children who come to tertiary centers (and sentinel surveillance hospitals) with intussusception usually survive. It is the babies in remote areas, far away from roads and transport who die untreated and undiagnosed after intussusception.

The sentinel surveillance will have no record of these cases of intussusception or deaths. Their deaths will not even be counted using the WHO recommended strategy of sentinel hospital based surveillance. That is the big tragedy.

On 2014 Aug 12, Jacob Puliyel commented:

I read with interest this paper that describes efficacy and safety of the 116E rotavirus vaccine in the 2nd year of the trial. The results for the first year were published earlier in June 2014 Bhandari N, 2014. The authors need to be congratulated for this study.

However some of the data appears incongruous. According to the clinical trial registry http://clinicaltrials.gov/show/NCT01305109 one of the secondary outcome measures was to be "safety of ORV 116E for intussusception events [Time Frame: Up to 2 years of age] Safety of ORV 116E for intussusception events in comparison to a placebo will be assessed in all subjects, from day of 1st dose till the age of 2 years (24 months) + up to 14 days". The term used here is 'intussusception events' not specifically only cases with Level 1 certainty.

The multicenter trial was conducted in 3 centers at Delhi, Pune and Vellore. The study was done in 6799 infants of whom 4532 received the 116E rotavirus vaccine and 2267 received placebo. In the Vellore limb of the study 1000 received the vaccine and 500 were given placebo.

Bhandari N, 2014 reports that 8 babies developed intussusception (intussusception by Brighton Level 1 criteria) during the 2 year follow up . However Jehangir et al, in the same issue of the journal Vaccine Jehangir S, 2014 report that there were 16 cases of intussusception (diagnosed on ultrasound) in the 1500 infants followed up at Vellore. 7 of these required radiological reduction meeting Brighton criteria level 1.

It seems unlikely that there were 16 children with intussusception in Vellore center (7 meeting level 1 criteria) and there were only 11 cases in the entire trial. This would mean that there were 7 cases (meeting level 1 criteria) in Vellore among 1500 children studied and only 4 case among the 5041 children at Delhi and Pune. In view of this, I will request the authors to report in the PubMed Commons how many babies in the two group (vaccinated and placebo group) had intussusception (any level of diagnostic certainty by Brighton criteria ) and how many had Level 1, Level 2 and Level 3 certainty.

On 2014 Aug 15, Jacob Puliyel commented:

Licensing the vaccine for general use (in remote areas of India), seems impossible to justify

I commend Dr John and colleagues for this report on the trial with the 116E Indian rotavirus vaccine. However the authors limit their discussion to comparisons with the trials of Rotarix and Rotateq which recruited some 60,000 patients each. It will be more useful to compare the 116E trial safety results with the RotaSheild vaccine trials http://www.path.org/vaccineresources/files/RotaShield_Fact_Sheet_CDC.pdf.

RotaSheild trial

The RotaSheild trial recruited double the numbers recruited in the present 116E study. RotaSheild was licensed after the trial involving 14,687 patients (10,054 received the rotavirus vaccine and 4,633 received placebo). In the study there was one case of intussusceptions among the 4633 receiving placebo. This suggests that the ‘normal rate of intussusception’ was approximately 2/10,000, in that population. Five cases of intussusceptions occurred among 10,054 RotaSheild vaccine recipients. Thus there were an excess of 3 cases of intussusceptions for each 10,000 children vaccinated. All the intussusceptions were among infants who received a second or a third dose of vaccine. The difference between the vaccinated and placebo recipients was not statistically significant http://www.path.org/vaccineresources/files/RotaShield_Fact_Sheet_CDC.pdf.

116E Trial

With the 116E vaccine trial there were 6 cases of intussusceptions in 2267 controls which works out to be 2.6 cases per 1000 placebo recipients. The ‘normal rate of intussusception’ in this study was at least 10 times higher than the RotaSheild trial (where it was 0.2 cases per 1000 placebo recipients). There were 17 cases of ultrasound confirmed intussusceptions among the 4532 given the 116E vaccine which is 3.75 cases per 1000 babies vaccinated. The comparative figure for the RotaShield study was 0.5 cases/1000. In the 116E trial there was an excess of 1 case of intussusceptions for every 1000 children vaccinated with the rotavirus vaccine (compared to the RotaSheild trial where there were 3 excess intussusceptions per 10,000 vaccinated). RotaSheild vaccine was withdrawn after licensing, on account of unacceptable risk of intussusception. The risk of intussusception in the 116E trial was three times higher than with the RotaSheild trial. We are told that in the 116E trial, 50% intussusceptions diagnosed by ultrasound, resolved spontaneously John J, 2014. In the remaining 50% there is need for urgent treatment by a radiologist or pediatric surgeon. In remote parts of India, without motorable roads, let alone radiologists and pediatric surgeons, mortality will be near 100% http://emedicine.medscape.com/article/930708-overview. Such specialized care (radiological or surgical reduction of intussusception) is not available in vast swathes of India and we can assume vaccinated babies would die at home passing blood and mucus in the stools and it will be presumed they had died of dysentery and sepsis rather than intussusception caused by the vaccine.

Intussusception risks compared to diarrhea deaths avoided

Assuming only 50% ultrasound diagnosed intussusceptions need urgent treatment John J, 2014 we can assume that one child in 2000 vaccinated babies will develop this life threatening condition. The possible harm in remote areas (deaths from intussusceptions 1/2000) is not offset by benefits (diarrhea deaths avoided using the 116E vaccine).

In the first two years after vaccination, there number of infants that needed to be immunized to prevent one episode of rotavirus diarrhea of any severity was 21 Bhandari N, 2014. Assuming mortality from rotavirus diarrhea to be 1% in the first 2 years of life with community management Lal S, 1994 Kosek M, 2003 2100 babies will have to be vaccinated to prevent one death from diarrhea in the first 2 years of life.

When 2100 babies are vaccinated to prevent that 1 death from rotavirus diarrhea - 1 child will have intussusceptions and die in remote areas of the country. This is why, given the limited evidence of this 116E trial, licensing the vaccine for general use (in remote areas of India), seems impossible to justify.

On 2016 Jul 21, Jacob H. Hanna commented:

Theunissen et al. Cell Stem Cell 2014 reported absolute failure to detect human naïve PSC derived cell integration in chimeric mouse embryos obtained following micro-injection into mouse blastocysts, as was reported for the first time by our group (Gafni et al. Nature 2013). However, the authors failed to discuss that imaging and cell detection methods applied by Theunissen et al. Cell Stem Cell 2014 were (and still) not at par with those applied by Gafni et al. Nature 2013.

Regardless, we find it important to alert the readers that Theunissen and Jaenisch have now revised (de facto, retracted) their previous negative results, and are able to detect naïve human PSC derived cells in mouse embryos at more than 0.5-2% of embryos obtained (Theunissen et al. Cell Stem Cell 2016 - Figure 7) Theunissen TW, 2016 < http://www.cell.com/cell-stem-cell/fulltext/S1934-5909(16)30161-8 >. They now apply GFP and RFP flourescence detection and PCR based assays for Mitochondrial DNA, which were applied by the same group to elegantly claim contribution of human neural crest cells into mouse embryos (albeit at low efficiency (Cohen et al. PNAS 2016 Cohen MA, 2016).

While the authors of the latter recent paper avoided conducting advanced imaging and/or histology sectioning on such obtained embryos, we also note that the 0.5-2% reported efficiency is remarkable considering that the 5i/LA (or 4i/LA) naïve human cells used lack epigenetic imprinting (due to aberrant near-complete loss of DNMT1 protein that is not seen in mouse naive ESCs!! http://imgur.com/M6FeaTs ) and are chromosomally abnormal. The latter features are well known inhibitors for chimera formation even when attempting to conduct same species chimera assay with mouse naïve PSCs.

Jacob (Yaqub) Hanna M.D. Ph.D.

Department of Molecular Genetics (Mayer Bldg. Rm.005)

Weizmann Institute of Science | 234 Herzl St, Rehovot 7610001, Israel

Email: jacob.hanna at weizmann.ac.il

Lab website: http://hannalabweb.weizmann.ac.il/

Twitter: @Jacob_Hanna

On 2016 Jul 14, Jacob H. Hanna commented:

None

On 2016 Jan 21, Rupert Collins commented:

The authors may wish to revise the identification of this fish. I blasted the COI and CYTB sequences, and both were from the goldfish (Carassius auratus), and not from Piaractus brachypomus. http://www.ncbi.nlm.nih.gov/nuccore/KJ993871

On 2014 Oct 29, Benoit Kornmann commented:

The unfolded protein response (UPR) is a conserved pathway that senses stress in the endoplasmic reticulum (ER) and responds to it by eliciting a transcriptional response. Mechanistically, when the stress sensor IRE1 is activated by the accumulation of unfolded proteins in the ER, its cytosolic RNAse domain cleaves the messenger of a transcription factor (Xbp1 in mammals, Hac1 in yeast) at two precise positions, removing an inhibitory intron. The 5' and the 3' parts of the messenger are then re-ligated together, to encode a functional transcription factor that takes part in the response. While the ligase involved in the last processing step was known in yeast, it evaded identification for years in mammals.

Here, the mammalian ligase is identified using a clever trick. A synthetic construct consisting of a Cre recombinase fused to Xbp1 is not expressed in normal conditions, but upon ER stress, Xbp1 is spliced and Cre recombinase is produced. The induction of the recombinase can be easily monitored using a Cre-induced proapoptotic factor, which kills the cells. Using this synthetic circuit, the authors screen a lentiviral RNA interference (RNAi) library and identify hits that fail to induce Cre upon acute ER stress, by simply scoring for survival. They identify the ligase RtcB, which, as with the yeast UPR ligase Trl1, is also involved in tRNA splicing.

On 2014 Aug 22, Attila Csordas commented:

2 associated proteomics datasets are available via PRIDE/ProteomeXchange with identifiers PXD000287 and PXD001254:

http://www.ebi.ac.uk/pride/archive/projects/PXD000287 http://www.ebi.ac.uk/pride/archive/projects/PXD001254

On 2014 Aug 14, Peter English commented:

This is an editorial about paper http://www.ncbi.nlm.nih.gov/pubmed/25086160

On 2014 Nov 26, Marc Girard commented:

By the distance between what it demonstrates and what it claims, this paper (as well as the preceding IOM report this one is supposed to update) illustrates that the issue of vaccines safety is still a matter of serious concern for anyone endowed with a minimum of expertise in drug safety or pharmacoepidemiology.

To start with methodological consistency, one may wonder why the McHarm instrument used by the authors to assess the quality of the reviewed studies was not published in a peer-reviewed journal (the internet link given by the authors [their ref. 7] being not accessible) whereas the only investigations they included had to be published (in contrast with Cochrane reviews which, inasmuch as possible, also take into account unpublished investigations). This inconsistency in the authors’ way of referencing sources exposes their review to a number of biases which are well known in general, but reach unparalleled levels as far as vaccines are concerned. To take just one example, the Ascherio et al.’s study, which suggested a lack of neurotoxicity with hepatitis B vaccination, was triumphantly published in The New England Journal of Medicine (2001;344:327-32) (despite its worrying biases, some of them detailed in further correspondence) with an accompanying editorial celebrating the study as a milestone, whereas, in spite of the professional fame of its authors and of its intrinsic quality, a later study on the same subject by Hernan et al., which suggested opposite conclusions, was first rejected by The NEJM, The Lancet, The BMJ (personal communication), before being published in Neurology (2004;63:838-42) with an accompanying editorial contending that nothing significant had changed regarding the safety of this immunization: as a matter of fact, Hernan was a co-writer of both studies… Another example of selective assessment, extracted from Maglione et al.’s paper itself: it remains unclear why the study by Gallagher and Goodman (which suggests a link between hepatitis B vaccine and autism) would display “high risk of bias and low quality”, whereas the “protective effect [of some vaccines] against acute lymphoblastic leukemia” does not deserve the slightest word of caution… Dozens of additional examples of publication biases could be cited: this is the personal experience of anyone working in the field of vaccines that positive results or enthusiastic reviews are far more easily published than negative investigations or critical comments. Finally and as a number of respected authors have already emphasized, a major part of vaccines studies are performed or supported by manufacturers or governmental agencies responsible for previous recommendations, a situation which clearly maximizes conflicts of interests.

Regarding now experimental designs in vaccine studies, the following list of methodological defects is concerned with safety assessments (but could be easily extended if efficacy issues were also concerned).

i) During development, use of false placebos as comparators (i.e. not devoid of pharmacological effects: adjuvants, other vaccines) is a frequent practice.

ii) Compared to the supposed duration of the beneficial immunological effects of the tested vaccines, the duration of the safety studies aimed at assessing a potential for delayed immunological hazards is often ridiculously short.

iii) Required on a standard regulatory basis with any new pharmacological entities, the interactions studies are weak, scarce, if not nonexistent with vaccines, whereas most of them are now administered as combinations.

iv) Likewise, the dose-ranging studies are generally defective, accounting for impressive changes in the booster recommendations once the vaccine is on the market, which would be inconceivable for any other drug.

Overall and as far as safety issues are concerned, Cochrane reviewers (e.g. in their reviews of flu vaccines) frequently identify blatant weaknesses in available studies which, apparently, are beyond of the reach of the McHarm instrument… On the contrary, Maglione et al. expressed frequent reservations about the “the strength of evidence” suggesting potential safety issues, but none about the power of the reviewed studies to effectively grasp evidence of vaccine hazards… Likewise, the authors are clearly not concerned with the tremendous tendency of vaccine studies (pre- or post-marketing) to underreport adverse events: experience suggests that physicians have a worrying reluctance to accept that vaccines might have adverse effects and, besides their indisputable tendency to brush aside any such suggestions from their patients, they do not hesitate to present as reassuring that “immunizations are up to date” when confronted with an unexplained disease, without even considering that these immunizations could have triggered the disease in question… Another illustration of the same bias: when reassuring, VAERS data are unchallenged, whereas the shortcomings of the system are immediately pointed out each time they may suggest a safety problem…

Maglione et al.’s review is regrettably silent on two crucial issues of the continuous extension of immunizations against trivial diseases:

i) maybe acceptable for one vaccination against a severe disease, the autoimmune risk related to the administration of foreign material increases arithmetically when vaccinations are multiplied beyond any sound limit;

ii) extended immunizations greatly alters the natural ecology of a number of infectious diseases (e.g. measles), a situation the assessment of which would be far more complex than the basic inventory of straightforward side-effects which, as mentioned above, is already severely defective from a methodological point of view: no reason to believe that vaccine promoters are better in complex assessments than in trivial ones…

As is easy to document, the never-ending extension of immunizations against anything is based upon the dramatization of anecdotic stories, sometimes tragic but fairly rare or even exceptional at a community scale. Yet, experience of drug assessment suggests that below frequencies of, at best, 1-2% of exposed patients, clinical trials fail to identify drug side-effects with a minimum of reliability (the statistical power of postmarketing surveillance being even lower by far). In a country like the USA, this detection threshold is consistent with a shadow area on iatrogenic risk of about 40,000-80,000 persons per vaccine for each vaccinated class of age: it should be obvious that risk-taking of such a size is simply disproportionate to the potential benefits of reducing the morbidity of trivial diseases (even taking into account the natural tendency of vaccine promoters to exaggerate the efficacy of immunizations…). The stubborn obfuscation of this evident arithmetical imbalance by health professionals or governmental agencies suggests that there is something rotten in the kingdom of immunization…

Marc GIRARD, MSc, MD

On 2014 Aug 04, Paul Brookes commented:

This paper addresses a very important point... namely, one of the proposed mechanisms by which ischemic preconditioning (IPC) is thought to bring about cardioprotection. It has been hypothesized that increased leakiness of the mitochondrial membrane to protons may lead to a lowering of ROS generation. We provided some evidence to support increased H+ leak in IPC in 2006 Nadtochiy SM, 2006. Unfortunately, it's not immediately clear to me exactly how the authors measured H+ leak in this study, and also whether their ROS measurements were performed correctly.

First proton leak: The normal way this is determined is to make simultaneous measurements of membrane potential and respiration in a single chamber equipped with an oxygen electrode and another electrode sensitive to a lipophilic cation such as TPP+. The authors are to be commended on their choice to use a TPP+ electrode, which instantly makes this paper more quantitative than others using qualitative fluorescent probes such as TMRE. They measured membrane potential (as reported in Figure 3) using succinate as a substrate for complex II, with rotenone to inhibit complex I. All good so far. However, normally oligomycin is added to inhibit the ATP synthase as a potential source of leak. In addition nigericin is added to equilibrate K+/H+ and thus ensure the potential is a measure of protonmotive force. As such, the potential measured here was not in a true state 4 condition which is typically required for quantitation of proton leak.

After describing membrane potential measurements, the methods section describes respiration and H+ leak methodology. It appears oligomycin was added this time (good!), but then something very odd happens... it is stated that "H+ leak was measured as the state 2 respiration rate required to maintain membrane potential at -150mV". Nowhere is it stated how that value of 150mV was arrived at. Normally, when doing these measurements, you set the mitochondria in state 4 (succinate, rotenone, oligomycin, nigericin), and then titrate the activity of the respiratory chain with an inhibitor such as malonate. Step-wise titration then gives you a series of membrane potential and respiration traces, a curve, from which the leak rate at any given potential can be read off. The question is, if such curves were made, how were they made (no mention of malonate anywhere), and why aren't they shown in the paper? The scary alternative explanation may be that they "measured" leak by imposing the 150mV membrane potential by titrating in uncoupler. This is incorrect - you can't add something that changes the leak as a way of measuring the leak. The other odd thing is that the average baseline membrane potential in the IR group barely above 150mV, so some replicates in that group must have had a potential value below 150mV to begin with. How did they authors get those mito's UP to 150mV for the leak measurement? Overall, it would be a whole lot better if they just showed the full leak curves.

What about ROS? The problems here are two-fold. First it appears that SOD was not added to the incubations to scavenge any stray superoxide and turn it into H2O2 for the assay to pick up. Second, the calibration of the assay was performed incorrectly. It is stated in the methods that the signal was calibrated by "adding known concentrations of H2O2 to buffer solution containing horseradish peroxidase and Amplex-Red" The problem is, it is necessary to calibrate in the presence of mitochondria, so the signal you measure with the calibrating H2O2 is under the same condition as the measurement itself. The way this is commonly done is to just add a bolus of H2O2 at the end of each run. This has the advantage of making every run internally calibrated, which cuts down on noise (this is a very noisy assay). The outcome here is a bit odd... the values of H2O2 generation are in the range of 10-40 nM/min/mg protein. Ignoring the odd units (rates of things should be expressed in moles not Molar), let's assume they meant to write nmols/min/mg - that would put their rates about 1500-fold greater than typical for these conditions (e.g.Chen Q, 2003).

So, TL/DR - good ideas, but more info' is needed on the proton leak method, and the ROS numbers are just wild. Also, lest anyone think I'm attacking this work because I happen to be one of the people who originally proposed proton leak --> lower ROS --> protection in IR injury, that's not the case. In-fact, my lab' is now pursuing a completely different downstream signaling mechanism, as a mediator of the protective effects of mitochondrial uncoupling, so if the results of this paper are true, my life just got a whole lot easier! I just want to be sure before I start citing it.

On 2014 Sep 09, Vahid Rakhshan commented:

Reply to the author’s response: Statistical errors in a recent article: Sella Turcica in patients with Type 1 diabetes

1. The authors kindly stated in the authors’ reply that it was clearly written in the 4th paragraph of Methods that how the subjects were matched: "This was clearly defined in the 4th paragraph of Material and Methods section. In addition, the need to match the groups according to bone age, not the chronological age was discussed briefly in Discussion (paragraph 3, page 183) with supporting references." (A) I re-checked the 4th paragraph and there was no indication of criteria for matching. The authors had indeed talked about grouping the patients according to the bone age, in the 4th paragraph ("that were equally distributed into subgroups according to bone age and sex."), but that was not something about "matching" the patients. (B) Moreover, there was no mention of the chronological age in that 4th paragraph of the Methods section. However, this factor is used for correlating the two groups, according to the Results and Discussion. (C) Furthermore, in the same Methods sentence that had stated that bone age was used for grouping ("that were equally distributed into subgroups according to bone age and sex."), sex was mentioned as a grouping factor as well. So if that quoted sentence implies that bone age was a matching factor, should we consider sex as a matching criterion as well? The above-quoted sentence from the 4th paragraph of the Methods is actually the criteria for dividing the sample into subgroups. However, the authors are referring to it (in the authors’ reply) as something about matching. (D) Besides, I had noticed the comparisons and correlations of groups regarding bone ages, in the pages 182 and 183, but since those factors were not pre-defined as criteria for matching, those comparisons and the authors’ accompanying correlations could not indicate a matching between the groups to the reader.

2. I appreciate the authors’ point #2. They kindly clarified the authors’ method.

3. The authors have stated in the authors’ reply that they had tested both the whole sample and each subgroup regarding the normality... First that the authors’ article stated n > 50 for the normality test. However, the authors’ subgroups were much smaller than this. So n > 50 could not relate to subgroups of n = 19. Therefore, either the statement "n > 50" was incorrect, or they had not tested for normality in the subgroups. Moreover, normality should not be tested in the whole sample. It is completely incorrect. Besides, they did not state why they had used two different normality tests?

4. There is nothing wrong with doing a pairwise comparison, and I did not say pairwise comparisons are bad, in my original letter. However doing a pairwise comparison without an ANOVA is statistical malpractice (when the ANOVA is necessary) and bad. It might have the following problems: (1) The absence of ANOVA disallows to assess the interactions. (2) Without an ANOVA design first, we do not know if any post hoc (or pairwise) comparisons are necessary or not, in the first place. If the ANOVA is non-significant, then performing the pairwise comparisons is simply moot (if we disregard the family-wise error introduced by the unnecessary statistical analyses). So this was why I was emphasizing on performing an ANOVA before doing the pairwise comparisons.

5. The authors kindly stated " In our manuscript, "matched" means "constructing two groups whose data would be comparable with each other"." ... "The data of these groups are independent from each other and the presence of one group cannot alter the data of the other. " (A) Please note that "matched" has its own globally accepted definition that is to select groups with similarities in certain aspects. So using a personal definition for this word is not a healthy practice. (B) Moreover, the statement "constructing two groups whose data would be comparable with each other" implies (by the word "comparable") that there were actually similarities between the two groups, and thus some sort of matching had been actually performed. This again reveals that the two groups were not independent. (C) Furthermore, the Results and the Discussion (pages 182 and 183) clearly indicate that the two groups were correlated with each other according to the chronological and bone age: Page 182: "For both groups, the chronologic and bone ages of the subjects were correlated to each other" Page 183: "The control group, on the other hand, was created from skeletal Class I patients with no systemic diseases and whose chronologic and bone ages were correlated." (D) This evidence completely suffices to invalidate the use of ANY independent-samples test. Correlated groups CANNOT be independent. How "the presence of one group cannot alter the data of the other" when the authors had themselves selected the control group in a way that they were correlated to diabetic patients in terms of chronological and bone age? The control group was already altered in a way that it could be correlated to the diabetic patients.

6. The authors have kindly re-stated that they had used the Bonferroni correction method. I had already seen the authors’ similar statement in the authors’ paper, but had not found any evidence supporting the authors’ statement. Please note that the Bonferroni correction is simply dividing the alpha by the number of pairwise comparisons in each family of tests. So we would expect to see alphas below 0.05 (for example 0.013 etc. depending on the number of pairwise comparisons). In that case, if the alpha had been corrected using the Bonferroni method to something like 0.008, and the P value had become P = 0.04, the P was still Non-Significant. However, throughout the text, and in the tables, only P values above the alpha = 0.05 had been considered as non-significant. This indicated that no Bonferroni had been used to adjust the alpha. (the alpha had not been adjusted in the first place).

7. I much appreciate the authors’ correction and clarification.