On 2016 Mar 31, Lydia Maniatis commented:

Todd, Egan & Kallie (2015) provide more demonstrations of violations of the "darker-is-deeper" rule. They note that the rule is already falsified by literature: "the evidence is overwhelming that observers' judgments of 3D shape from shading do not conform to the predictions of [the rule]" (Of course - the possible falsifying cases are infinite in number).

They also note that the rule was falsified by Langer and Bulthoff (2000), who concluded from their observations that "the perception of shape from shading must be based on a process that is more sophisticated that a simple darker-is-deeper heuristic." (Todd et al, 2015).

I wondered whether Chen and Tyler (2015) had cited Langer & Bulthoff (2000) in this paper. Indeed, they do, but the implication is the opposite of the description cited above:

"Langer & Bülthoff (2000) showed that the observer can indeed discriminate between “hills” and “valleys” on a surface with this “dark-is-deep” rule" (Chen and Tyler, 2015). This description conveys a very different and misleading (I would prefer to describe it as dishonest) impression of the results of the cited reference, papering over complications and contradictions to smooth the way to a preferred but non-viable argument.

On 2015 Nov 27, Lydia Maniatis commented:

I just want to expand a little on my previous comment, with regard to the direction of the light source. As I said, the implied direction of the light is front-parallel to the corrugations, but my suggestion that the shadowing is consistent with attenuation due to distance along the normal seems inadequate. I want to add that the putative peaks of the corrugations would tend to reflect light from all directions, not only light coming along the normal, and that the troughs would receive less light because, from some directions, it would be blocked by the protruding sections. (I realise now that this is the point the author was trying to make with the north-south wall example. He is correct but again, that does not mean that the light is not directional, only that it comes from a number of directions. I think that my assumption of fronto-parallel lighting may be more consistent with the patterns than his assumption that the directions form the surface of a half-sphere (the sky), but they might be equivalent.)

On 2015 Nov 27, Lydia Maniatis commented:

In his comment above, CCC says that “The issue of diffuse illumination is a rather minor aspect of the paper...” However, based on the abstract, confirming the “diffuse illumination assumption" is presented as central goal: “These results validate the idea that human observers can use the diffuse illumination assumption to perceived depth from luminance gradients alone without making an assumption of light direction.”

In the authors' stimuli, light does, in fact, have an implied direction – the light source is fronto-parallel to the (apparent) corrugations, such that its intensity is attenuated with distance in the direction along the normal.

As a general fact, the greater weighting of what we could call pictorial cues over binocular disparity in the perception of depth did not require more corroboration. It is evident in the fact that very convincing pictorial depth effects are easy to achieve, despite the zero disparity. Also, with regard to the presence of positive (rather than zero) disparity cues, a relevant reference that was not included is Pizlo, Zygmunt, Yunfeng Li, and Robert M. Steinman. "Binocular disparity only comes into play when everything else fails; a finding with broader implications than one might suppose." Spatial Vision 21.6 (2008): 495-508. (Update 12/2: I apologise, the authors do cite this paper).

In their abstract Langer and Buelthoff (2000) conclude that “overall performance in the depth-discrimination task was superior to that predicted by a dark-means-deep model. This implies that humans use a more accurate model than dark-means-deep to perceive shape-from-shading under diffuse lighting.”

On 2015 Nov 23, Chien-Chung Chen commented:

First of all, it should be noted that this paper is about cue combination, not shape-from-shading per se. The issue of diffuse illumination is a rather minor aspect of the paper, to which most of the discussion in the thread is quite irrelevant.

The commenter’s argument that no shadows are generated under diffuse illumination is a serious misunderstanding of the properties of diffuse illumination. Although, the light comes from every direction, it does not reach everywhere. For instance, suppose one erects a wall running from north to south on an otherwise flat surface. Immediately to the west of the wall, all the light from the east would be blocked by the wall. Thus, this location would receive less light and in turn appear darker than it would be without the wall, effectively forming a diffuse shadow. In short, the light received at a position on a surface depends on the extent of the sky it is exposed to. On an uneven surface, a position in a “valley” would “see” less of the sky than a position on a “hill” and thus would appear darker. This is why under diffuse illumination, darker implies deeper. Notice that, deeper means deeper than the surrounding surface, not from the source of light as the commenter erroneously states.

The difference between the “light-from-above” but with “darker-is-deeper” assumptions is one of the range of scenes to which they apply. It is indeed an empirical issue whether an interpretation that applies to many scenes is a true default or is cued by some aspect of the scene. Our claim is that the directional lighting assumption that is so common in computer graphics is typically cued by the discrepancy between symmetric contour information and asymmetric shading information. (In the case of Ramachandran’s disks, the contour information is circularly symmetric.) This particular discrepancy is a narrow subset of all possible arrangements of contours and shading in images. If this discrepancy is removed, either by making the shading (circularly) symmetric or by making the contours asymmetric, the prediction is that the visual system will not maintain the “light-from-above” assumption but will default to the “darker-is-deeper” assumption. The “darker-is-deeper” assumption is considered the more basic default because it can apply to images with any degree of symmetry or asymmetry. Of all possible visual scenes generated at random, only a small subset will contain symmetries and an even smaller subset will have contour/shading discrepancies consistent with any kind of oblique lighting direction. It is only in the presence of such discrepancies that a default for the lighting direction could come into play.

Finally, it is true that is difficult to separate “darker-is-deeper” and “darker-is-darker-colored” from one instance. However, such heuristics do not depend on one instance but the statistics of nature scenes. In a complex scene, one does not just use one visual cue. “Good shape” is not a necessary condition for darker-is-deeper rule as the complex random stimuli used by Langer & Buelthoff (2000) showed.

On 2015 Sep 26, Lydia Maniatis commented:

You say in the article that under diffuse illumination "light comes from every direction." In that case, there should be no shadows; a uniformly-colored surface should appear uniformly-colored in the image and unevenness (bumps, etc) will not be evident. The fact that you postulate a “darker-deeper” rule indicates that illumination is directional – “deeper” being equivalent to “farther from the source.”

When you say that diffuse illumination is “the default assumption when there is not strong enough evidence to support other interpretations of the scene,” how is this different from my saying that “light from the top right” is the default assumption when there is not strong enough evidence to support other interpretations of the scene?

The information available to the visual system is simply a luminance pattern, which could derive from an infinite number of reflectance/illumination combinations at each point. A “diffuse illumination” assumption – or any illumination assumption – cannot drive a solution to this problem, because it actually cannot decide between the possibilities: “darker is deeper” and “darker-is-darker-colored.” The drivers of the solution are good shape assumptions, combined with the assumption that good shapes have uniform reflectance. So if we have a good shape, (e.g. the Ramachandran bumps) then we assume that the luminance differences are illumination-based, and the illumination assumption follows.

(I would like to add that my earlier comment was deleted by moderators b/c it was a duplication, not b/c it was inappropriate!)

On 2015 Sep 25, Chien-Chung Chen commented:

The commenter seemed to confuse the concept of “default” with “predominate”. The default assumption is like a null hypothesis in statistics. The visual system uses the default assumption when there is not strong enough evidence to support other interpretations of the scene. The commenter seemed to take it that the default assumption meant that the observer would always make this assumption about the scene over other possible interpretations (thus, her statement that we “could not explain why we often perceive illumination to be directional”). With this clarification, it should be clear that in our paper, there is no logical contradiction in our claim that diffused illumination is a visual system default.

On 2015 Aug 18, Lydia Maniatis commented:

The authors claim to their results a. "validate the idea that human observers can use the diffuse illumination assumption to perceive depth from luminance gradients alone without making an assumption of light direction and b. confirm that "observers can recover shape from shading under diffuse illumination on the basis of the “dark-is-deep” rule."

There is a logical problem with the idea that the visual system has a "diffuse illumination assumption," on the basis of which it applies a dark is deep rule.

In viewing a scene, we perceive both the quality of the illumination (including direction) and the albedo of surfaces. Both of these qualities are constructed by the visual system, which must resolve the reflectance/illumination ambiguity. There is no default assumption as to either the reflectance of a particular patch nor the quality of the illumination. Both are inferred on the basis of structural constraints.

For example, in the case of the Ramachandran bumps demonstration, the fundamental choice is between seeing concave or convex circles with a homogeneous reflectance, or seeing part moon shapes of varying reflectances (some dark, some light, some in-between). In the Chen/Tyler stimuli, structural assumptions as to the "best" shape consistent with the stimulus produce a percept consistent with a homogeneously coloured surface under diffuse illumination. If the diffuse illumination assumption came first, we could not explain why we see the corrugations in the obviously flat picture, and we could not explain why we often perceive illumination to be directional. In addition, we can create cartoonish versions of corrugations, in which the deeper dark region is not signalled by a gradient, and still produce a similar impression of dark is deep. If structural assumptions don't favour it, however, dark will not be deep, it will just be dark.

In order to defend an a priori diffuse illumination assumption, the authors would need to explain why the assumption is made only sometimes (i.e. only when structural constraints demand it. )

I would add that their stimuli are not really consistent with diffuse illumination conditions, in the sense that both the upper and lower edges are straight, and thus we must be seeing the corrugated surfaces through a rectangular aperture. The concave sections of the surface would be at some distance from the edge of this aperture and I assume the luminance profile would indicate this in some way, with shadowing, or black holes in the gaps, something.

When it comes to 3D perception, shading follows shape, not vice versa.

On 2015 Aug 19, Seyed Moayed Alavian commented:

Dear Author, I would like to emphasize the role of supervision on barbers and more efforts for increasing the people awareness . Yours Prof Alavian

On 2015 Dec 09, Donald Forsdyke commented:

PURINE LOADING AS A THERMAL ADAPTATION The proteins of thermophiles are generally more heat-stable than the corresponding proteins from mesophiles. This must be reflected in either, or both, of two major amino acid variables – composition and order. In the past the notion that amino acid composition might be reflective of the pressure in thermophiles to retain purine-rich codons (3) has been disparaged by Zeldovich et al. (6). In this elegant new paper (5), Venev and Zeldovich (2015) agree that the “multiple factors” not accounted for in their modelling “include the influence of the genetic code and guanine-cytosine (GC) content of the genomes on amino acid frequencies.” However, there is puzzlement that the “theory and simulations predict a strong increase of leucine content in the thermostable proteins, whereas it is only minimally increased in experimental data.” Perhaps it is of relevance that leucine is on the top left quadrant of the standard presentation of the genic code, its codons being extremely poor in purines.

My response to Zeldovich et al. (6) in 2007, and my follow-up references in 2012 (1, 4), are set out below. One of the coauthors of the 2007 paper has recently further contributed to this topic (2).

2007 Response

This paper draws conclusions tending to oppose those of myself and coworkers (cited). A "key question" is held to be: "Which factor - amino acid or nucleotide composition - is primary in thermal adaptation and which is derivative?" Previous evidence is considered "anecdotal." Now there is evidence for "an exact and conclusive" relationship, based on an "exhaustive study" that provides a "complete picture." A set of amino acids - IVYWREL - correlates well with growth temperature. It is noted:

"Signatures of thermal adaptation in protein sequences can be due to the specific biases in nucleotide sequences and vice versa. ... One has to explore whether a specific composition of nucleotide (amino acid) sequences shapes the content of amino acid (nucleotide) ones, or thermal adaptation of proteins and DNA (at the level of sequence compositions) are independent processes."

In other words, are primary adaptations at the nucleic acid level driving changes at the protein level, or vice- versa? To what extent are the two processes independent? Their conclusion:

"Resolving the old-standing controversy, we determined that the variation in nucleotide composition (increase of purine-load, or A + G content with temperature) is largely a consequence of thermal adaptation of proteins."

Thus, the superficial reader of the paper, while noting the purine-richness of some of the codons corresponding to the IVYWREL amino acids, will conclude that the "independent processes" alternative has been excluded. Reading the paper (e.g. Figure 7) one can question the validity of this conclusion. Many of the IVYWREL amino acids have purine-poor alternative codons (especially IYLV, which at best can only change one purine unit in their codons). One of the IVYWREL amino acids has relatively purine-rich alternative codons (R, which at best can change two purine units). Two (EW) are always purine-rich, and there are no alternatives.

Displaying more EW's as the temperature got hotter would satisfy a need both for more purines and for more tryptophan and glutamate, so here there is no discrimination as to whether one "shapes" the organism’s content of the other. Displaying more IYLVs gives only minimal flexibility in accommodating a purine-need. Most flexibility is provided by R codons.

The authors do not give statistics for the differences between the slopes of Figs. 7a (unshuffled codons) and 7b (shuffled codons), but they appear real, presumably reflecting the choice biologically of purine-rich codons, a choice the organisms might not have to make if there were no independent purine-loading pressure. Thus, the authors note, but only in parenthesis, that the slopes "are somewhat different suggesting that codon bias may be partly responsible for the overall purine composition of DNA."

2012 Response

As a follow up, it can be noted that Dehouck et al. (2008) report that relationship between a protein's thermostability and the optimum growth temperature of the organism containing it, is not so close as previously thought (1). Furthermore, Liu et al. (2012) now conclude from a study of xylanase purine-rich coding sequences that "The codons relating to enzyme thermal property are selected by thermophilic force at [the] nucleotide level," not at the protein level (4).

1.Dehouck Y, Folch B, Rooman M (2008) Revisiting the correlation between proteins' thermoresistance and organisms' thermophilicity. Protein Engineering, Design and Selection 21:275-278.Dehouck Y, 2008

2.Goncearenco A, Berezofsky IN (2014) The fundamental tradeoff in genomes and proteomes of prokaryotes established by the genetic code, codon entropy, and the physics of nucleic acids and proteins. Biology Direct 9:29 Goncearenco A, 2014

3.Lambros RJ, Mortimer JR, Forsdyke DR (2003) Optimum growth temperature and the base composition of open reading frames in prokaryotes. Extremophiles 7:443–450.Lambros RJ, 2003

4.Liu L, Wang L, Zhang Z, Wang S, Chen H (2012) Effect of codon message on xylanase thermal activity. J. Biol. Chem. 287:27183-27188 Liu L, 2012

5.Venev SV, Zeldovich KB (2015) Massive parallel sampling of lattice proteins reveals foundations of thermal adaptation. J. Chem. Phys. 143: 055101Venev SV, 2015

6.Zeldovich KB, Berezofsky IN, Shakhnovich EI (2007) Protein and DNA sequence determinants of thermophilic adaptation. PLOS Comput. Biol. 3(1), e5.Zeldovich KB, 2007

On 2015 Sep 23, Klaas Vandepoele commented:

Datasets described in the paper are available as BED files via http://bioinformatics.intec.ugent.be/blsspeller/ :

BLS files (contain phylogenetic conserved genomic regions given certain cutoffs, in bed file format): osa/zma_C90F20B15.bed

Conserved known motif files (conserved known motif files, from CisBP database v1.02): osa/zmaconservedknownmotifsBLS15/95.bed

DNase hypersensitive sites (regions of open chromatin, in bed file format): DNase1hypersensitivesites_osa.bed

On 2015 Aug 09, Andrzej Miskiewicz commented:

The clinical study fall in line with the previously published genetic research of Zhang L. and Wong D.T. However in the presented manuscript the authors focus their research on the inflammation mechanism as a possible molecular mechanism common for the mentioned conditions.

On 2015 Aug 12, Donald Forsdyke commented:

WINGE PROPOSED HYBRID STERILITY CURED BY WHOLE-GENOME DUPLICATION

That hybrid sterility would be 'cured' by whole genome duplication was suggested by 'the father of yeast genetics' [1], Őjvind Winge [2], and has been extensively discussed in modern texts on speciation [3] and evolutionary biology [4].

He would doubtless have been delighted that his favorite organism (apart from dogs) had formed the basis of the elegant study by Marcet-Houben and Gabaldon that provides a welcome endorsement of his viewpoint.

[1] Szybalski W (2001) My road to Őjvind Winge, the father of yeast genetics. Genetics 158:1–6.

[2] Winge Ő (1917) The chromosomes. Their numbers and general importance. Comptes Rendus des Travaux du Laboratoire Carlsberg. 13:131–275. see Webpage.

[3] Forsdyke DR (2001) The Origin of Species Revisited. Montreal: McGill-Queen’s University Press, pp. 72–79.

[4] Forsdyke DR (2011) Evolutionary Bioinformatics. 2nd edition. New York: Springer, pp. 184–186.

On 2015 Aug 27, Trevor Marshall commented:

I see that Figure 2 of this paper closely resembles the in-silico image of D-Binding Protein which I presented (inter alia) at the Autoimmunity Congress in 2008, in Porto. I wonder if it was attributed properly? The caption I see on the journal's website does not seem to give any citation at all. Additionally, the text seems to be describing my image as the VDR, whereas this image is actually of the D-Binding Protein, not the VDR, despite the caption: "Figura 2 di 2. Il recettore della vitamina D (VDR) [D Binding Protein Complexed with 1,25-D]"

On 2017 Jul 10, Randi Pechacek commented:

Elisabeth Bik discussed this paper in a microBEnet blog about microbial biofilms in water meters.

On 2015 Nov 24, Salzman Lab Journal Club commented:

This thought-provoking paper describes a potential mechanism for cryptic exon splicing due to TDP-43 proteinopathy. It is interesting to consider the evolutionary history of the sequence surrounding these cryptic exons and the origins of TDP-43 binding. The authors show that GU-repeats, the consensus motif for TDP-43, often flank these cryptically spliced exons. Is it that these GU repeats originally promoted the splicing of these cryptic exons and TDP-43 evolved the ability to bind GU repeats to restrict this process or did the GU-repeats arise later to repress the splicing by recruiting TDP-43, leading to repression? Additionally, we would be interested in seeing the effect of other engineered TDP-43 mutants and truncations on cryptically spliced exons, specifically, a mutant lacking the C-terminus and other splicing repressor domains and the full length TDP-43 rescue as simple controls.

On 2015 Sep 25, David Keller commented:

More observational evidence suggesting cardiovascular benefits associated with testosterone replacement

This observational study found that TRT (testosterone replacement therapy) dosing resulting in normalization of serum testosterone levels was associated with greater benefits than were lower doses. Association of an intervention with a benefit in an observational study does not prove causation and cannot be used to prove the safety or efficacy of the intervention. Observational studies are inherently affected by confounding variables which can only be eliminated by conducting a randomized controlled trial of the intervention.

Indeed, the authors conclude that "adequately powered, prospective, well-designed trials with a long-term follow-up will be needed to reach a conclusive agreement regarding the effect of TRT on CV risk."

We certainly have more than enough results from observational studies at this time to justify the expense of a large prospective randomized trial of testosterone replacement therapy. Given the possibility that the cardiovascular benefits associated with TRT may be caused by TRT, it is a major disservice to ageing men to delay the definitive randomized trial any longer.

On 2016 Jan 25, Noa Krawczyk commented:

Versão em Português do artigo disponível [Portuguese version available at]: https://figshare.com/s/f82b748e5f4133e4af2c

"A interação entre comportamentos de consumo de droga, contextos e acesso aos cuidados de saúde: Um estudo qualitativo explorando atitudes e experiências de usuários de crack no Rio de Janeiro e São Paulo, Brasil"

Resumo:

Antecedentes: Apesar da crescente atenção em torno do uso de crack no Brasil, pouco se sabe sobre as histórias dos usuários, seus padrões de consumo, e a interação de hábitos de consumo de droga, contextos e acesso/barreiras ao(s) cuidados de saúde. Estudos qualitativos raramente comparam os achados de pessoas que usam crack a partir de diferentes contextos. Este estudo tem como objetivo explorar os insights de usuários regulares de crack em duas grandes cidades brasileiras e examinar como fatores sociais e contextuais, incluindo o estigma e a marginalização, influenciam o uso inicial e diversos problemas de saúde e sociais.

Métodos: Entrevistas em profundidade e grupos focais foram realizados com 38 adultos usuários de crack recrutados em bairros pobres do Rio de Janeiro e São Paulo. As entrevistas e grupos focais foram gravadas em áudio e transcritas na íntegra. Procedeuse à análise qualitativa e os conteúdos foram organizados e analisados por temas recorrentes relevantes para os interesses do estudo.

Resultados: Para os participantes do estudo de ambas as cidades, o uso frequente de crack desempenha um papel central na vida diária e leva a uma série de consequências físicas, psicológicas e sociais. Os interesses comuns entre os usuários incluem o uso excessivo de crack, o engajamento em comportamentos de risco, a utilização pouco frequente de serviços de saúde, a marginalização e a dificuldade em reduzir o consumo de drogas.

Conclusões: As condições desfavoráveis em que muitos usuários de crack crescem e vivem podem perpetuar os comportamentos de risco, e o estigma marginaliza ainda mais os usuários dos serviços de saúde e de recuperação necessários. A redução do estigma e do discurso moralizante relacionado ao uso de drogas, especialmente entre os profissionais de saúde e policiais, pode incentivar os usuários a procurar atendimento necessário. Novas alternativas de cuidado para usuários marginalizados, baseados em redução de danos estão sendo desenvolvidas em algumas localidades no Brasil e outros países, e deveriam ser adaptadas e expandidas para populações vulneráveis.

On 2015 Sep 05, Andrew R Kniss commented:

It is certainly plausible that herbicides (glyphosate or other) might have some direct effect on earthworms. However, due to the design flaws, these effects cannot be evaluated from this particular study. The problems with this paper boil down to two main points:

1) One of the herbicides applied in the study was "Roundup Speed" which contains the herbicide pelargonic acid in addition to glyphosate, so it is impossible to conclude anything about the direct effects of glyphosate. 2) More importantly, the researchers didn't include a control treatment where they killed the plants without herbicides. All of the effects on earthworms and nutrients observed in this study could simply be due to killing the plants. It is perfectly plausible the exact same effects would be observed if the plants were clipped or pulled out of the pots.

In addition, the glyphosate rate used in this study is far greater than would be used in field applications of this product. I calculated the amount of glyphosate applied to the pots (adding up the three applications they made) and converted it into the amount of glyphosate per unit area. It turns out the amount of glyphosate applied to each pot is equivalent to a field rate of 12,680 grams per hectare. A typical application rate in a field of glyphosate-resistant crops would be somewhere between 800 to 1,300 grams per hectare. So the amount of glyphosate they applied is about an order of magnitude too high to be relevant to most field situations.

Blog post with more detail: http://weedcontrolfreaks.com/2015/09/dead-plants-are-probably-bad-for-earthworms/

On 2015 Oct 16, Andrea Messori commented:

How to manage the price of the newest expensive agents approved for HCV therapy? Pharmaceutical firms do not adopt the same policies across different countries and different regions

by Andrea Messori, PharmD, Sabrina Trippoli, PharmD, Claudio Marinai, Pharm D

HTA Unit, Tuscany Region, ESTAR, Regional Health Service, 50100 Firenze, Italy

In managing the price of the newest expensive agents approved for HCV therapy, Scripts (the largest pharmacy benefit manager in the US) was successful in fostering the competition between Abbvie and Gilead. In particular, Abbvie accepted an exchange of more prescriptions for price rebates, and so Scripts decided to cover Viekira Pak for the majority of HCV patients and restricted the coverage of Gilead products only under certain exceptions (1).

While Wilensky (1) emphasizes that this strategy based on competition in prices can be an effective way of reducing the cost of these expensive treatments, other experiences in this field have not been successful and therefore deserve to be mentioned. In May 2015, the Tuscany region of Italy undertook a competitive tender scheme aimed at the prescriptions for 18,000 HCV patients of our region (those without cirrhosis) (2) in which both Abbvie and Gilead were expected to participate. In fact, at national level the majority of reimbursed treatments for HCV (currently restricted essentially to patients with cirrhosis) are those based on the products of Gilead and Abbvie. Surprisingly enough, Gilead did not participate in this tender while Abbvie offered their product with no substantial price rebate thus leading to the failure of the Tuscan experience in fostering more prescriptions for price rebates. This is probably because of the reluctance of these two pharmaceutical firms to accept local agreements in which the nominal price of their products is explicitly reduced.

On the other hand, at national level both Gilead and Abbvie have accepted a price-volume reimbursement agreement with our national Medicines Agency (AIFA) according to which the drug prices are progressively subjected to very substantial price rebates (up to 80%) as the number of treated patients increases (3-5). One reason why the two companies have accepted this national agreement is that the agreement has been kept confidential, and so the nominal values of these discounted prices have remained unknown and have not become a reference price for other jurisdictions.

In conclusion, to manage sustainability in this field, payers of Western countries and national health systems have different tools of procurement and reimbursement at their disposal, but choosing the best strategy is a difficult task because pharmaceutical firms do not adopt the same policies across different countries and different regions.

References

1. Wilensky G. A New Focus on Prescription Drug Spending. JAMA 2015;314(5):440-441.

2. Brunetto MR, De Luca A, Messori A, Zignego AL. Reducing the price of new hepatitis C drugs in the Tuscany region of Italy. BMJ 2015;350:h3363 doi: 10.1136/bmj.h3363 (Published 24 June 2015), available at http://bmj.com/cgi/content/full/bmj.h3363?ijkey=xYS3zhzXoox8A8t&keytype=ref

3. Messori A. Newest treatments for hepatitis C: how can we manage sustainability? Clin Infect Dis. 2015 Aug pii: civ667. [Epub ahead of print], preprint available at http://www.osservatorioinnovazione.net/papers/cid2015pricing.pdf

4. Messori A. Managing the high cost of innovative drugs: anti-cancer agents vs direct-acting antivirals for hepatitis C (Comment posted 2 April 2015), Ann Intern Med 2015, available at http://annals.org/article.aspx?articleid=2212249#tab

5. Quotidiano Sanità Website. “Epatite C. Pani (Aifa) al Senato: Con accordi Aifa risparmi per 2,5 miliardi in due anni. Ma c’è troppo divario tra le Regioni”. http://www.quotidianosanita.it/governo-e-parlamento/articolo.php?articolo_id=30276 , accessed 30 July 2015

On 2016 Nov 22, Lydia Maniatis commented:

his article exhibits at least four common pathologies of the vision science literature.

First, the authors have adopted what, after Graham, I refer to as the “transparent brain hypothesis.”

Graham (1992), notes that, at a time when neuroscientists thought V1 was pretty much all there was to the visual cortex, many psychophysical experimental results perfectly matched the early descriptions of V1 neural characteristics.

Unfortunately, later evidence showed not only that there were many other hierarchically later levels of processing (V2, etc) but that even the descriptions of V1 receptive fields were overly simplistic.

How then, Graham asks, can we explain the mountains of lovely psychophysical results? Her reply is that, under certain conditions, the brain becomes “transparent” and experience directly reflects V1 activity. Teller (1984) had already described this attitude as the “nothing mucks it up proviso,” which she didn’t think was sound. Here, Kwon et al seem to believe that the use of short line segments in their stimuli causes them to directly tap into the V1 layer. (Discussions of both Teller (1984) and Graham (1992) can be found on PubPeer).

Proponents of this frankly bizarre view need, at the least, to meet the burden of explaining what happens at all the other levels of visual processing, with respect to their phenomenon of interest - bearing in mind that the same V1 receptive field activity that mediates observers' experience of their stimuli underlies, and thus is consistent with, and thus must explain, every aspect of their visual experience.

Second, and relatedly, the authors treat perception as a detection problem, rather than as an indirect, inferential process. They say: “Before providing details of our model, we will summarize some relevant characteristics of the topographical map of area V1, which make it uniquely suited for detecting closed curves on a retinal image.”

The reference to closed curves on a retinal image is unfortunate, since the stimulation of the retina is point stimulation, and curves are not a property of the proximal stimulus, but of the percept. Figure-ground segmentation is not, as I'm sure the authors are well aware, achieved by directly reading off retinal activity, in a process of detection. Any description of “contour detection” that can be developed with respect to the known properties of V1 receptive fields will be too simplistic; as a result, it will be trivial to construct any number of falsifying cases requiring a much broader - both geometrically and theoretically - perspective.

Actually, we don’t even have to try to find a falsifying case, because the authors do it for us: “But how about extracting overlapping and intersecting curves, for example, two elongated ellipses intersecting at 4 points? One of the anonymous Reviewers raised this question. The model, in its present form, does not guarantee that such [overlapping] individual ellipses can be extracted: the ambiguities at X intersections will usually not be resolved correctly…” The literature is filled with ad hoc models of this type, i.e. models that “in their present form” are inadequate to their stated purpose.

In the article highlights, the authors call theirs “the first principled explanation of the center of gravity tendency;” but one could argue that it isn’t very principled to describe a failed hypothesis as having explained anything. Perhaps the model can be fixed so as to handle a couple of overlapping ellipses, but it will almost certainly fail again soon thereafter. Sequential ad hoc fixes probably won’t result in an adequately limber model.

Finally, as is often the case with psychophysical experiments, the number of observers is very small (three), one of whom is an author. We’re told, for good measure, that one of the observers was naïve to the purpose of the experiment. Is this naivete important? If so, then what about the other two observers? If not, then why mention it?

I think what the authors refer to as "contour fragments" can also be considered "contours," i.e. they're homogeneously colored figures with a (closed) contour. That is, the "contour fragment" / "contour" dichotomy is a false one.

On 2015 Sep 21, Toni Mueller commented:

Please note regarding the subcellular fractionation protocol to obtain a synapse-enriched fraction (SYN) as indicated in the Methods and Figure 1: subsequent experiments in our lab have indicated that it is not necessary to combine P1 and P2 prior to triton solubilization. While combining these pellets enhanced the protein yield of the SYN fraction and the triton-insoluble other heavy/intermediate membrane fraction to facilitate western blot sample preparation, the SYN fraction generated by triton solubilization of only P2 appears to have less contamination by nuclear proteins. The steps performed in calculating GABA(A) receptor subunit expression and ratios do account for this potential confound (subunit expression is normalized to an inhibitory synapse marker-gephyrin, a loading control- VCP, and subunit expression in total homogenate), but other researchers and labs measuring proteins with both nuclear and synaptic expression patterns should be aware of this limitation in the fractionation method.

On 2016 Dec 01, Graham Walker commented:

There's an interactive version of the Revised Myeloma International Staging System at MDCalc (and one for the original Myeloma International Staging System as well). Both have supplemental content on how to use them clinically, next steps, and info about the creators.

On 2015 Oct 06, KEVIN BLACK commented:

A point of clarification for Ballard et al's Table 1: Nichols et al 2013 do report a death in the placebo group (1 of 9 subjects allocated to placebo, vs 0 of 15 allocated to olanzapine; see Figure 1 and Results text). We would be glad to share additional subject-specific information about adverse events.

On 2016 Aug 23, Ben Goldacre commented:

This trial has the wrong trial registry ID associated with it on PubMed: both in the XML on PubMed, and in the originating journal article. The ID given is NCT015553305. We believe the correct ID, which we have found by hand searching, is NCT01553305.

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

Many thanks,

Jessica Fleminger, Ben Goldacre*

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

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

On 2015 Aug 12, Friedrich Thinnes commented:

Solanezumab: a therapeutic breakthrough including theoretical gain.

The exciting paper of Siemers et al. [1] on slowing down the progress of Alzheimer´s by Solanezumab antibodies, from my point of view, not only represent a therapeutic breakthrough. The effects observed also broaden the understanding of the pathogenesis of Alzheimer´s Disease, this by pointing to induced neuronal cell death as basic in AD.

Accordingly, I propose plasmalemmal VDAC-1 (Swiss Prot P21796) to work as a receptor of amyloid Aß mono- or oligomers.

In line, it has been shown that docking of those Aß forms to cell surfaces result in an opening of cell membrane-standing VDAC-1, a process finally ending in neuronal cell death.

In consequence, whenever critical brain regions and their redundant structures are affected this way neuronal loss must be expected. In contrary, to capture Aß by adequate mAbs should minimize Aß toxicity, in other words slow down AD progress.

The voltage dependent anion channel (VDAC) is an archaic channel and thus suggested to be involved in housekeeping functions. The channel is well established in the outer mitochondrial membrane, here playing its role in the intrinsic apoptotic pathway. It is thus of proven relevance for Alzheimer´s Dementia [2].

First data on an extra-mitochondrial came up in 1989 by showing that human lymphocytes carry a heavy load of the molecule in their plasmalemma. Those data, meanwhile, found manifold support by several laboratories using different approaches; for review see [3] and www.futhin.de.

After studies focussed on the regulatory volume decrease (RVD) of HeLa or murine respiratory epithelial cells, respectively, had shown that cell membrane-integrated type-1 VDAC is part of the cell volume regulatory system of mammalian cells [4,5] data came up indicating that plasmalemmal VDAC-1 plays its role in apoptosis.

In a first effort it was elaborated that opening of VDAC-1 in the plasma membrane precedes the activation of caspases in neuronal apoptosis, induced by staurosporine. In other words, the authors documented that keeping type-1 porin in the plasmalemma of neurons closed by different specific antibodies abolishes the apoptotic volume decrease (AVD) of the cells [6].

Next, studies on the toxic effect of amyloid Aß peptides on septal (SN56) and hippocampal (HT22) neurons corroborated that blocking VDAC in cell membranes means preventing an apoptotic development of cells, and additionally demonstrated that VDAC-1 and the estrogen receptor α (mERα) co-localize and interact in cell membrane caveolae, mERα working towards neuroprotection. The topographic relationship of the molecules was further specified demonstrating that both are integrated in caveolar lipid rafts [7].

To notice: plasmalemmal VDAC-1 carries a GxxxG motif cell outside, amyloid Aß40/42 includes several of them in series. However, GxxxG motifs are established aggregation and membrane perturbation motifs.

Given this background recent data on an enhancement of BACE1 expression of hypometabolic neurons [8] made me ask if amyloid Aß, cut from ubiquitous APP by ß-secretase BACE1 and γ-secretase, may occasionally induce neuronal cell death via opening ubiquitous VDAC-1 in cell membranes of critical brain regions - a proposal including a general model for an induction of cell death [9].

The authors, remembering cerebral hypometabolism and amyloid accumulation as prevailing neuropathological characteristics of Alzheimer's disease, had tried to define effects of neuronal hypoactivity on amyloid plaque pathogenesis in the Tg2576 transgenic mouse model of Alzheimer's disease. They found that unilateral naris-occlusion resulted in an elevation of the ß-secretase BACE1 in neuronal terminals of deprived bulb and piriform cortex in young adult mice [8].

Conclusion: taking for granted that 1) amyloid Aß mono- and/or oligomers dock to cell membrane-standing type-1 VDAC via GxxxG motifs, 2) the docking reactions result in plasmalemmal VDAC-1 channel opening followed by cell death, and 3) Solanezumab antibodies neutralize Aß oligomers by scavenging, a revised version of the amyloid cascade hypothesis of Alzheimers´s pathogenesis comes up.

Accordingly, familial as well as sporadic Alzheimer's disease rests on a putative form of extrinsic cell death via opening cell membrane-standing VDAC-1 (= receptor), and is boosted by excessive amyloid Aß (= regulating agonist) production via processing of the amyloid precursor protein (APP) of weakening cells.

The synopsis of a series of solid data from several laboratories thus helps to further understand the pathogenesis of either form of AD.

Phenotypically mild at the beginning, increasing brain function disturbances evidenced by worsening stages of the disease point to a progressive process on the somatic level. First occasional or just a few cells being affected, over time a burden of cell deaths accumulates finally ending in Alzheimer dementia whenever critical brain regions and their redundant structures are affected. In line, to block free amyloid by antibodies, allows slowing down AD. Finally, t he model presented allows to formally explain the reverse relationship of AD and cancer [10].

References

[1] Siemers ER, Sundella KL, Carlson C, Michael Case, Sethuraman G, Liu-Seifert H, Dowsett SA, Pontecorvo MJ, Dean RA, Demattos R. Phase 3 solanezumab trials: Secondary outcomes in mild Alzheimer’s disease patients Alzheimer’s & Dementia 2015; epub ahead: 1-11.

[2] Demetrius LA, Magistretti PJ, Pellerin L. Alzheimer's disease: the amyloid hypothesis and the Inverse Warburg effect. Front Physiol. 2015 Jan 14;5:522. doi: 10.3389/fphys.2014.00522. eCollection 2014.

[3] Thinnes FP.Phosphorylation, nitrosation and plasminogen K3 modulation make VDAC-1 lucid as part of the extrinsic apoptotic pathway-Resulting thesis: Native VDAC-1 indispensible for finalisation of its 3D structure. Biochim Biophys Acta. 2015; 1848:1410-1416. doi: 10.1016/j.bbamem.2015.02.031. Epub 2015 Mar 11. Review. PMID: 25771449

[4] Thinnes FP, Hellmann KP, Hellmann T, Merker R, Brockhaus-Pruchniewicz U, Schwarzer C, Walter G, Götz H, Hilschmann N. Studies on human porin XXII: cell membrane integrated human porin channels are involved in regulatory volume decrease (RVD) of HeLa cells. Mol Genet Metab. 2000; 69:331-337.

[5] Okada SF, O'Neal WK, Huang P, Nicholas RA, Ostrowski LE, Craigen WJ, Lazarowski ER, Boucher RC. Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume regulation in murine cells. J Gen Physiol. 2004; 124:513-526. Epub 2004 Oct 11.

[6] Elinder F, Akanda N, Tofighi R, Shimizu S, Tsujimoto Y, Orrenius S, Ceccatelli S. Opening of plasma membrane voltage-dependent anion channels (VDAC) precedes caspase activation in neuronal apoptosis induced by toxic stimuli. Cell Death Differ. 2005; 12:1134-1140. PMID: 15861186 Free Article

[7] Marin R, Ramírez C, Morales A, González M, Alonso R, Díaz M. Modulation of Abeta-induced neurotoxicity by estrogen receptor alpha and other associated proteins in lipid rafts, Steroids 2008; 73:992–996.

[8] Zhang X-M, Xiong K, Cai Y, Cai H, Luo XG, Feng JC, Clough RW, Patrylo PR, Struble RG, Yan XX. Functional deprivation promotes amyloid plaque pathogenesis in Tg2576 mouse olfactory bulb and piriform cortex, Eur. J. Neurosci. 2010; 31: 710–721.

[9] Thinnes FP. Amyloid Aß, cut from APP by ß-secretase BACE1 and γ-secretase, induces apoptosis via opening type-1 porin/VDAC in cell membranes of hypometabolic cells-A basic model for the induction of apoptosis!? Mol Genet Metab. 2010; 101:301-303. doi: 10.1016/j.ymgme.2010.07.007. Epub 2010 Jul 15. No abstract available.

[10] Thinnes FP. Alzheimer disease controls cancer - concerning the apoptogenic interaction of cell membrane-standing type-1 VDAC and amyloid peptides via GxxxG motifs. Mol Genet Metab. 2012; 106:502-503. doi: 10.1016/j.ymgme.2012.06.004. Epub 2012 Jun 15. No abstract available.

On 2015 Sep 27, Ivan Oransky commented:

The first author of this paper is appealing the retraction: http://retractionwatch.com/2015/09/22/author-appeals-retraction-after-co-authors-dispute-nature-comm-paper/

On 2015 Sep 15, David Keller commented:

Proposal for a clinical trial to test the safety of a widely-used radionuclide scan

A recent letter to JAMA Internal Medicine [1], asked whether substantia nigra (SN) neurons weakened by Parkinson disease (PD) may be more sensitive to the adverse effects of ionizing radiation than are healthy mature neurons. Dosimetry safety studies assume that neurons are relatively resistant to damage from ionizing radiation. Radiation safety is, instead, calculated based on exposure of such tissues as the thyroid and the lining of the bladder. If SN neurons in PD are significantly more radiosensitive than healthy neurons, then PD patients might suffer progression of PD caused by the level of ionizing radiation exposure caused by certain diagnostic scans.

In a widely-used clinical diagnostic brain imaging procedure known as the "DaT scan", a radiopharmaceutical tracer marketed as "DaTscan" (Ioflupane I-123) is injected intravenously, crosses into the brain and binds to dopamine transporters. The tracer emits gamma radiation, thereby allowing for imaging which can help distinguish Parkinsonism from other causes of similar symptoms. According to Table 1 of the DaTscan product information, the highest concentration of injected activity occurs in the striatum, close to the substantia nigra [2]. At the recommended adult dose of DaTscan, the striatum is exposed to 185 MBq x 230 microGray/MBq = 42550 microGray = 42.55 mSv = 4.25 Rad of gamma radiation (1 Sv = 1 Gray = 100 Rad). The nearby SN receives approximately the same exposure, although the exact figure is not specified in the DaTscan product information.

How damaging is a gamma exposure of about 42.5 mSv to SN neurons already weakened by PD? For comparison, a head CT exposes the entire brain to about 2 mSV uniformly; so the radiation exposure to the striatum caused by a dose of DaTscan is the same as it would receive from 21 brain CT scans [3]. The SN and other nearby basal ganglia presumably receive about the same exposure, although the DaTscan product insert does not specify this important information.

The clinical effect of this radiation dose on PD patients may be found by conducting an observational study of patients who have been ordered to get a DaTscan by their neurologist. Each patient would be given a thorough UPDRS exam (a detailed PD-focused neurologic exam) prior to being scanned, and at appropriate intervals after scanning. The overall rate of UPDRS score deterioration in the study subjects should be compared with that of matched PD patients who have not undergone scanning. Any significant worsening of UPDRS scores in the intervention group, compared to the control group, would presumably be an adverse effect of the DaTscan radiotracer, and should be investigated further.

With the increasing use of DaT scans, PD patients should be informed whether their clinical condition, as measured by the UPDRS, will be expected to worsen as a result of these scans, and if so, approximately how much.

I emailed the above observations to the Commissioner of the FDA recently, and received a reply which failed to address my radiation safety concerns regarding the FDA-approved radiopharmaceutical tracer marketed by General Electric as DaTscan.[4]

The Code of Federal Regulations Title 21, Section 601.35 (Evaluation of safety of diagnostic radiopharmaceuticals) mandates evaluation of "changes in the physiologic or biochemical function of the target and nontarget tissues". The effect of 42.5 mSv of gamma radiation concentrated on the already diseased neurons in the substantia nigra of patients with Parkinson's disease has not been determined, as is required under the above-cited Federal regulation.

I urge neurologists and their patients with PD to consider the high concentration of gamma radiation caused by DaTscan and ask, before injecting this tracer, "is this scan really necessary, and how will it substantively alter clinical management?".

References

1: Keller DL. Non-neurologists and the Dopamine Transporter Scan. JAMA Intern Med. 2015 Aug 1;175(8):1418. doi: 10.1001/jamainternmed.2015.2497. PubMed PMID: 26236969.

2: DaTscan drug prescribing information, visited on 9/16/2015:<br> http://us.datscan.com/wp-content/uploads/2014/06/prescribing-information.pdf

3: M.I.T. online guide to radiation exposure, accessed on 9/20/2015 at:<br> http://news.mit.edu/2011/explained-radioactivity-0328

4: Email received from FDA pharmacist identified only by the initials "H.P.", 10/15/2015.

On 2016 Jun 16, Stefano Biffo commented:

Lipid metabolic kinase, ACC1? It is not a carboxylase?

On 2016 May 11, Jonathan Heald commented:

Posted on behalf of the American Academy of Sleep Medicine.

Consensus Conference Panel., 2015

On 2016 May 05, Jonathan Heald commented:

None

On 2016 May 17, Annika Hoyer commented:

With great interest we noticed this paper by Nikoloulopoulos. The author proposes an approach for the meta-analysis of diagnostic accuracy studies modelling random effects while using copulas. In his work, he compares his model to the copula approach presented by Kuss et al. [1], referred to henceforth as KHS model. We appreciate a lot Nikoloulopoulos referring to our work, but we feel there are some open questions.

The author shows in the appendix that the association parameter from the copula is estimated with large biases from the KHS model, and this is what we also saw in our simulation study. However, the association parameter is not the parameter of main interest which are the overall sensitivities and specificities. They were estimated well in the KHS model, and we considered the copula parameter more as a nuisance parameter. This was also pointed out by Nikoloulopoulos in his paper. As a consequence, we are thus surprised that the bad performance in terms of the association parameter led the author to the verdict that the KHS method is 'inefficient' and 'flawed' and should no longer be used. We do not agree here, because our simulation as well as your theoretical results do clearly show that the KHS estimates the parameters of actual interest very well. Just aside, we saw compromised results for the association parameter also for the GLMM model in our simulation.

Nikoloulopoulos also wrote that the KHS approximation can only be used if the 'number of observations in the respective study group of healthy and diseased probands is the same for each study'. This claim is done at least 3 times in the article. But, unfortunately, there is no proof or reference or at least an example which supports this statement. Without a mathematical proof, we think there could be a misunderstanding in the model. In our model, we assume beta-binomial distributions for the true positives and the true negatives of the i-th study. They were linked using a copula. This happens on the individual study level because we wanted to account for different study sizes. For estimating the meta-analytic parameters of interest we assume that the shape and scale parameters of the beta-binomial distributions as well as the copula parameter are the same across studies, so that the expectation values of the marginal distributions can be treated as the meta-analytic sensitivities and specificities. Of course, it is true that we used equal sample sizes in our simulation [1], however, we see no theoretical reason why different sample sizes should not work. In a recently accepted follow up paper on trivariate copulas [2] we used differing sample sizes in the simulation and we also saw a superior performance of the KHS model as compared to the GLMM. In a follow-up paper of Nikoloulopoulos [3], he repeats this issue with equal group sizes, but, unfortunately, did not answer our question [4,5] with respect to that point.

As the main advantage of the KHS over the GLMM model we see its robustness. Our SAS NLMIXED code for the copula models converged better than PQL estimation (SAS PROC GLIMMIX) and much better that Gauss-Hermite-Quadrature estimation for the GLMM model (SAS PROC NLMIXED). This was true for the original bivariate KHS model, but also for the recent trivariate update. This is certainly to be expected because fitting the KHS model reduces essentially to the fit of a bivariate distribution, but without the complicated computations or approximations for the random effects as it is required for the GLMM and the model of Nikoloulopoulos given here. Numerical problems are also frequently observed if one uses the already existing methods for copula models with non-normal random effects from Liu and Yu [6]. It would be thus very interesting to learn how the authors’ model performs in terms of robustness.

Annika Hoyer, Oliver Kuss

References

[1] Kuss O, Hoyer A, Solms A. Meta-analysis for diagnostic accuracy studies: A new statistical model using beta-binomial distributions and bivariate copulas. Statistics in Medicine 2014; 33(1):17-30. DOI: 10.1002/sim.5909

[2] Hoyer A, Kuss O. Statistical methods for meta-analysis of diagnostic tests accounting for prevalence - A new model using trivariate copulas. Statistics in Medicine 2015; 34(11):1912-24. DOI: 10.1002/sim.6463

[3] Nikoloulopoulos AK. A vine copula mixed effect model for trivariate meta-analysis of diagnostic test accuracy studies accounting for disease prevalence. Statistical Methods in Medical Research 2015 11 Aug; Epub ahead of print

[4] Hoyer A, Kuss O. Comment on 'A vine copula mixed effect model for trivariate meta-analysis of diagnostic test accuracy studies accounting for disease prevalence' by Aristidis K Nikoloulopoulos. Statistical Methods in Medical Research 2016; 25(2):985-7. DOI: 10.1177/0962280216640628

[5] Nikoloulopoulos AK. Comment on 'A vine copula mixed effect model for trivariate meta-analysis of diagnostic test accuracy studies accounting for disease prevalence'. Statistical Methods in Medical Research 2016; 25(2):988-91. DOI: 10.1177/0962280216630190

[6] Liu L, Yu Z. A likelihood reformulation method in non-normal random effects models. Statistics in Medicine 2008; 27(16):3105-3124.

On 2015 Aug 14, David Mage commented:

The authors have done an excellent job in reviewing the effect of fetal sex on prenatal development. However, they seem to reach an incongruous conclusion above that "male fetuses exposed to prenatal adversities are more highly impaired than those of female fetuses." Given that the numbers of X and Y sperm in the race to conception must be identical if Dad is XY, the human primary gender distribution at the instant of conception must be 0.5 XY and 0.5 XX. However, given the nominal 5% excess male live birth rate, there must be an excess of female fetal loss during pregnancy, between the moment of conception and moment of exit from the birth canal. Conceptus and fetal loss in the first trimester can occur even before Mom knows she is pregnant, and later without fetal recovery for gender identification. Even if there is a male excess of observed fetal loss in the third trimester, from spontaneous abortion or stillbirth, it cannot be greater than the prior female fetal loss. The authors also do not appear to consider as valid Naeye et al. (1971)'s page 905 concluding explanation for the male infant disadvantage: "The biologic difference must originate in the genetic difference between the sexes and those genetic differences are the consequences of the disparity in the number of the X chromosomes." Indeed, Mage and Donner, Scandinavian Journal of Forensic Science, 2015;21(1) doi:10:1515/sjfs-2015-0001 show that an X-linked gene in Hardy-Weinberg Equilibrium with a dominant allele protective against respiratory failure with frequency p = 1/3 and non-protective recessive allele with frequency q = 1 - p = 2/3, can explain the 50% male excess rate of infant death from respiratory failures,such as SIDS, and the 25% male excess rate of ALL infant mortality up to their 5th birthday.

David Mage, PhD (WHO retired)

On 2015 Aug 20, Lydia Maniatis commented:

The author says that “If... [slant] judgments were based on scaling contrast or scaling gradients, then surfaces viewed under orthographic projection should all appear fronto-parallel. In order to evaluate these predictions, it is useful to consider the image of a planar surface under orthographic projection in Figure 3D.”

This is not a fair test. Figure 3D was constructed on the basis of a set of upright rectangles. Under orthographic projection, we still have upright rectangles, and the visual system treats projections shaped like rectangles as fronto-parallel, regardless of their source. If the rectangles had been tilted (resulting in parallelogram-shaped projections), or if we were dealing with circles instead of rectangles (producing elliptical projections), then the orthographic projection would not appear fronto-parallel.

The failure to take shape into account is typical of many studies on slant (e.g Ivanov et al 2014; Saunders and Chen 2015). But shape, whether collected in a “texture” or individually, is dispositive in slant perception and it needs to be explicitly considered, or else results will be inconsistent and uninterpretable.

The idea that foreshortening could even be a potential cue to slant is logically untenable, as I explain in a comment on Ivanov et al (2014).

I would also note that in the perspective projections in Figure 3, edges and objects are visually grouped to produce oblique lines, more so for the larger slants. It is known that obliques tend to be perceived as receding (e.g. Deregowski and Parker 1992). This presents a confound very difficult to disentangle from other suggestions.

On 2016 Dec 18, Laura Williams commented:

Our online journal club discussed this paper on October 6, 2015. https://www.youtube.com/watch?v=bbfzFcRgmaM

On 2015 Sep 22, Kenneth Witwer commented:

Dr. Dweep's response is appreciated but does not engage our suggestions or explain why the miRWalk validated target module was:

1) extensively erroneous (1.0, as we pointed out I believe in 2011),

2) greatly expanded and again erroneous (2.0 at the start of 2015, apparently due to a database error, and as we shared with the authors earlier this year),

3) and finally what appears to be a mirror of miRTarBase, at least according to our results.

Instability and a lack of clear information on versions and methods causes confusion, especially when scientists take these data at face value. I have reviewed submitted manuscripts that used miRWalk "validated" results as input for further experiments or analysis, even when these results were, unbeknownst to the authors, completely erroneous.

Dr. Dweep suggests that our analysis was deficient because of the terms we used for our 50 selected genes. This would indeed be important had we attempted to gauge the comprehensiveness of miRWalk or other databases (we expressly did not), or if we had used one set of terms for our miRWalk query, and another set for querying all the other databases (we did not, unless forced to do so by an interface). Which gene terms we used in our comparison of databases, then, is irrelevant.

Dr. Dweep's second point is that our analysis focused only on a small portion of the miRWalk database, the validated target module. Should we have ignored perceived problems with such modules, simply because the file sizes for these data are smaller than the sum of all predictive or correlative information on miRNAs in any given database, or on the internet, or whatever other set of information we might consider?

Finally, Dr. Dweep refers to supplemental methods that explain, albeit in vague terms that do not allow reproduction of the results, how validated targets are gathered using text searches and four databases. This does not explain why the results we downloaded from miRWalk2.0 in the period of April-August 2015 were exactly the same as found in another database, miRTarBase (last updated two years ago), down to the sorting of the hits, nor does it explain the drastic fluctuations in numbers of validated hits over the years, almost all of which we examined were erroneous. Thus, a miRWalk validated target module user in January, 2015, would have received a completely different set of results compared with the same query a year earlier or six months later. As we have suggested to Dr. Dweep, one might simply to link to miRTarBase in cases where miRWalk2.0 does not provide additional information, and provide more extensive versioning information or even an automated email update to users when mistakes are found or major changes implemented.

We agree that validated target databases have potential uses and hope that our findings are somehow helpful, as a cautionary note even if they are not used to improve databases.

On 2015 Sep 22, Harsh Dweep commented:

We congratulate Dr. Witwer for the publication of his results in Drug Development Research journal. This publication is based on analyses of only one of the many key features of miRWalk2.0 comparing it with 50 genes by considering GeneCard terminology. Here, we would like to mention that the gene synonymous information is neither comprehensively maintained by Genecard nor MeSH (and other databases). For example, a total of 14, 22 and 23 synonyms are documented in NCBI-gene, MeSH and GeneCards, respectively, for the Clusterin (CLU) gene. Only 5 synonyms are common between GeneCards (as used by Witwer et al based on) and MeSH. However, the information of PubMed is relying on the terms stored in MeSH. By considering only GeneCards for evaluation (text-mining), a large amount of information on synonyms as well as their related articles can be missed. In addition, an alias of a gene can be commonly found in other genes, for example, VH alias is common for 36 different genes. These comments reflect only part of the problems related to text mining.

Moreover, Witwer addresses only 0.008% of the information contained in miRWalk2.0.

Additionally, it is clearly mentioned in the supplementary information of miRWalk2.0 article (page 11) that “information on miRNA interactions associated with pathways, diseases, OMIM disorders, HPOs, organs, cell lines and proteins involved in miRNA processing, is extracted by an automated text-mining search in the titles and abstracts of PubMed articles. In a next step, this information was combined with experimentally verified miRNA-target interactions downloaded from four databases”.

On 2015 Sep 03, Kenneth Witwer commented:

We recently published a small comparison of several validated miRNA-target databases (Lee YJ, 2015)--that is, catalogs of miRNA-target interactions with published experimental evidence. A "validated target" module is one part of miRWalk2.0, so this database and its predecessor (1.0) were included in our study. We queried 50 genes at different times. 82 miRNA-target interactions were returned by miRWalk1.0, 5468 by miRWalk2.0 in January, 2015, and 91 by miRWalk2.0 in May, June, and August, 2015, with only 5 from the original 82. As of August, 2015, the final set of 91 interactions was identical to that returned by miRTarBase (Hsu SD, 2014, Hsu SD, 2011), down to the sort order. Although miRTarBase is cited as one of numerous sources of information for miRWalk output, it was not clear from the methods that it would be the only source for the genes we queried. Experimental validation databases have the potential to provide useful information, but in light of the stability and accuracy issues we seem to have observed over time, users and reviewers are encouraged to 1) consult multiple sources of information (we found Diana TarBase to be among the most comprehensive and best-curated of those we tested, Vlachos IS, 2015); 2) at the same time be aware that different databases may rely entirely or to some extent on other databases; and 3) check the strength of interaction evidence in the primary literature.

On 2017 Oct 12, Sander Houten commented:

This paper focuses on the role of KLF14 in insulin signaling via the PI3K/Akt pathway. The authors study mouse models of obesity and the Hepa 1-6 cell line, a derivative of a mouse hepatoma. The authors show that Klf14 mRNA and KLF14 protein expression is decreased in liver, adipose and muscle of C57BL/6 mice on high fat diet and db/db mice when compared to control animals. In subsequent experiments the authors use ectopic KLF14 expression in Hepa1-6 cells and show that KLF14 stimulates insulin signaling via the classical PI3K/Akt pathway (Yang M, 2015). I would like to point out that there is little evidence to support the hypothesis that KLF14 plays an important role in adult mouse liver biology. We found no evidence for expression of KLF14 in adult mouse liver as we were unable to amplify Klf14 cDNA, did not find Klf14 mapped reads in liver RNA sequencing data and found no specific signal upon immunoblotting (Argmann CA, 2017). Our data on the absence of Klf14 expression in liver are consistent with previously published work by others (Parker-Katiraee L, 2007) and publicly available data sources. We also investigated the physiological functions of KLF14 by studying a whole body KO mouse model and focused on the metabolic role of this transcription factor in mice on chow and high fat diet. Our results indicate that KLF14 does not play a role in the development of diet-induced insulin resistance in male C57BL/6 mice (Argmann CA, 2017).

On 2017 Jul 13, Randi Pechacek commented:

Elisabeth Bik described this paper in a microBEnet blog post.

On 2015 Nov 04, Milos Filipovic commented:

Dirty dancing of NO/RSNO and H2S

In this report by Cortese-Krott et al, 2015, the existence of SSNO− as a product of the anaerobic and aerobic reaction of H2S with NO or RSNOs, was claimed based on MS experiments. The authors failed to prove HSNO using a demo MS instrument, although HSNO was prepared by the acidification of nitrite, pulse radiolysis and trans-nitrosation and characterized by MS, IR (14N/15N labelling) and 15N-NMR (Filipovic at al, 2012), and even reported by Cortese-Krott et al, 2014. HSNO has also been recently detected by 15N NMR as a product of the reaction of PNP+SSNO- with sulphide (Wedmann et al, 2015), again going against the claim of Cortese-Krott et al, 2015 that “SSNO- mix” is stable in excess of sulphide.

The authors use LTQ OrbiTrap to concentrate reactive ions (as demonstrated by the absence of any MS signal in the first 1.5 min of continuous injection of the reaction mixture) all of which can intercombine in the ion trap (Figure 3C). They never show the control spectra, actual intensities of the signals, nor do they show the spectrum with broad m/z. It is puzzling that the signal of the reactant, SNAP, reaches its maximum almost at the same time as SSNO− and all other reaction products. While SNAP peak slowly decays in agreement with Uv/Vis experiments, the reaction products remain at maximum intensity (although most of them have questionable S/N ratio, Figure 3C). Contrary, HS2O3− starts to disappear, suggesting that it was present even before reaction started.

The authors refer to the formation of persulfide NONOate ([ONN(OH)S2]−). This species should have m/z 124.9479. While the Figure S6A in indeed has the title: “High resolution mass spectrum of [SS(NO)NOH] −“, the actual figure shows the spectrum of the species with m/z 141.9579, which authors assign to H4O2N3S2−. Strangely, in the same reaction mixture, identical mass peak is assigned to another species, HO5(14)N(15)NS−, (Figure 3B, right panel). 3 peaks are present in m/z ~143 in Figure S6A in. As this is the only MS spectrum shown in high resolution, one can calculate the mass of those unassigned peaks and observe that none of them show up in Figure 3B which is recorded under the same conditions. The isotopic pattern of SULFI/NO (Figure 3B (left panel)) is inconsistent with what should be expected for this species. In the Figure 3A (right panel) there is a huge unassigned background peak at m/z ~94.9252, which does not appear in the Figure 3A (left panel). It is unclear whether the reported peaks are smaller or bigger than actual background noise of the instrument.

In the unexplainable absence of 15N NMR and IR characterisation of SSNO−, which by authors’ claim is “stable” and “abundant”, and correct isotopic patterns for reported species, the authors should have performed experiments with pure 14N and 15N labelled SNAP to independently demonstrate the isotopic distribution of each species and their corresponding m/z shifts.

The authors use maXis Impact instrument to show that they cannot detect HSNO in the reaction of RSNO and H2S. The injection of buffer alone creates the signal intensities of ~1.5x107 (the upper detection limit of this instrument) that makes the background noise stronger than the actual signal of few milimolar GSNO (Figure S8C). The authors also send a message that due to the presence of DMSO and acetonitrile in the tubing no one should try to detect anything at m/z range 50-70. The authors could have cleaned the instrument instead, used new tubing for every measurement or used stainless steel tubing to solve this problem as it is done in the laboratories with MS experience. Furthermore, the ionization conditions which “break” DSMO (BDE ~ 50 kcal/mol, Blank et al, 1997) into CH3SO/CH3SO+ are inappropriate for RSNO/HSNO detection (BDE ~30 kcal/mol, Wedmann et al, 2015). Results look like as they were produced in a limited amount of time and on a very dirty demo instrument and should not have been used for publication.

To prove that 412 nm species is NO dependent the authors trap NO by cPTIO (Figure S4F), ignoring the fact that nitronyl nitroxides readily reacts with H2S and therefore no conclusion can be drawn from this experiment (Wedmann et al, 2013).

The authors also use water soluble triphenylphosphine (TXPTS) to trap nitroxyl from their “SSNO- mix” ignoring the fact that triphenylphosphines are good trapping agents for sulfane sulphur (by mixing PNP+SSNO- with triphenylphosphine Seel et al, formed SNO-), and that S-nitrosothiols react/decompose in the presence of triphenylphosphines in general and TXPTS in particular (Bechtold et al, 2010), so nothing can be concluded from those experiments either.

In conclusion, the data presented in this study ask for more critical and in-depth re-evaluation.

Cortese-Krott MM, et al. (2015) Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl. Proc Natl Acad Sci USA 112(34):E4651-60.

Filipovic MR, et al. (2012) Chemical characterization of the smallest S-nitrosothiol, HSNO; cellular cross-talk of H2S and S-nitrosothiols. J Am Chem Soc 134(29): 12016-27.

Cortese-Krott MM, et al. (2014) Nitrosopersulfide (SSNO(-)) accounts for sustained NO bioactivity of S-nitrosothiols following reaction with sulfide. Redox Biol 2:234-44.

Blank DA, North SW, Stranges D, Suits AG, Lee YT (1997) Unraveling the dissociation of dimethyl sulfoxide following absorption at 193 nm. J Chem Phys 106(2):539-550.

Wedmann R, et al. (2015) Does Perthionitrite (SSNO(-)) Account for Sustained Bioactivity of NO? A (Bio)chemical Characterization. Inorg Chem 54(19):9367-9380.

Wedmann R, et al. (2013) Working with “H2S”: facts and apparent artefacts. Nitric Oxide 41:85-96. Seel F, et al. (1985) PNP-Perthionitrit und PNP-Monothionitrit. Z Naturforsch 40b:1607–1617.

Bechtold E, et al. (2010) Water-soluble triarylphosphines as biomarkers for protein S-nitrosation. ACS Chemical Biology 5(4):405-414.

On 2015 Nov 04, Ivana Ivanovic-Burmazovic commented:

No evidence for SSNO- in “SSNO- mix”

The results and conclusions published by Cortese-Krott et al. 2015 (Proc Natl Acad Sci USA 2015, 112(34):E4651-60) in the manuscript entitled “Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl” urgently call for comments from the chemical point of view, because they include a number of chemical misconceptions.

The main conclusion of this work, as stated in its title, is that SSNO- (according to the authors a “S/N-hybrid species”) is one of three key bioactive reaction products of the reaction of H2S with NO or S-nitrosothiols (RSNOs). Based on authors’ claims, SSNO- can be obtained in high yields in aqueous solutions at pH 7.4 (even in the presence of oxygen) and it is stable for hours. However, everything known about the chemical/spectroscopic properties of SSNO- contradicts authors’ conclusions. We are afraid that the biological community will be confused by these results, and that researchers without inorganic chemistry background will use such undefined reaction mixtures of NO and H2S, as well as of RSNO and H2S, as a source of SSNO- that in reality is not present under given conditions at all. To help clarifying confusion in the field we bring important facts about SSNO- chemistry below.

In general, a history of the identification of S-S bond-containing compounds in solutions was rich in contradicting conclusions (Seel F, et al. 1977), and “some of those who dared to tackle this challenging task were victims of delusions because such species that were optically (or even by other methods) observed in non-aqueous solutions could not easily be established as defined substances” (Seel F and Wagner M, 1985). This is a translated quotation from Seel F, Wagner M 1985, who first synthesized SSNO- under exclusion of water and dioxygen (Seel F, et al. 1985). However, although citing work of Seel and Wagner, Cortese-Krott et al, 2015 do not mention that i) SSNO- solutions are sensitive to oxygen and water (Seel F and Wagner M 1985) and ii) even in very alkaline aqueous solutions only 10 % of SSNO- was obtained from NO and S2-, although even that was questionable, as SSNO- could not be confirmed by 15N-NMR in aqueous solutions (Seel F and Wagner M, 1988). This contradicts that SSNO- is stable for hours at pH = 7.4, especially in high concentrations in “SSNO--enriched mixtures” (“SSNO- mix”) (Cortese-Krott et al, 2015). Related to the claimed determination of the high SSNO- yield in “SSNO- mix”, in SI (page 9) Cortese-Krott et al, 2015, state: “The (theoretical) maximal yield of SSNO- under these conditions is 1 mM, corresponding to the concentration of added nitrosothiol (please refer to Fig. S9 for experimental determination of reaction yield).” However, Fig. S9 deals with MS of dimethylsulfoxide, and nowhere in SI the clamed experimental results confirming a high SSNO- yield could be found. Instead there is some confusing statement that their putative SSNO- contains two sulfur atoms based on an observation of “two times as much sulfide as sulfane sulfur” (Cortese-Krott MM, et al. 2015). (Even more general, since authors do not provide stoichiometry of the considered reactions, they cannot provide any quantification.)

In agreement with the original work of Seel and Wagner, we demonstrated SSNO- inherent instability by preparing pure crystals of PNP+SSNO- and characterizing its properties by 15N-NMR, IR, EPR, MS, X-ray analysis, electrochemical and computational methods (Wedmann R, et al. 2015). For example, when ca. 10% water was added to an acetone solution of a pure SSNO- salt (Wedmann R, et al. 2015), it decomposed within ca. 100 s. Cortese-Krott et al. report that SSNO- does not react with thiols, H2S and cyanide (Cortese-Krott MM, et al. 2015). However, solutions of a pure SSNO- salt, which Cortese-Krott et al. never used, quickly decompose in the presence of thiols, H2S (Wedmann R, et al. 2015) and cyanide. These authors state that SSNO- is resistant to the biological reductants (Cortese-Krott MM, et al. 2015). However, SSNO- is reduced at a physiological potential of −0.2 V vs. NHE (Wedmann R, et al. 2015). Being unstable at pH = 7.4, in the presence of thiols and biological reducing agents, SSNO- cannot exist under physiological conditions in any relevant concentration. They also report that HSSNO is more stable than HSNO, because HSSNO supposedly has increased electron density on the proximal sulfur (which is a statement for which they do not provide any experimental support) and therefore does not easily react with HS- and positive metal centers (which is contradictio in adjecto) (Cortese-Krott MM, et al. 2015). The facts are quite different: i) the proximal-S has a +0.24 charge (Wedmann R, et al. 2015), ii) the S-N bonds in HSSNO and SSNO- (calculated BDE 16.0 and 22.1 kcal/mol, respectively; B3LYP/aug-cc-pv5z, in the presence of solvent/water) are weaker than those in HSNO and SNO− (BDE 27.74 and 36.21 kcal/mol, respectively), which makes (H)SSNO more prone to homolysis than (H)SNO, and iii) SSNO- reacts with metal centers (as evidenced by the reaction with [Fe3+(TPP)]) (Wedmann R, et al. 2015). Cortese-Krott et all. quote that (H)SNO is (only) stable at 12 K (Cortese-Krott MM, et al. 2015), but the PNP+SNO- crystals have been isolated at room temperature (Seel F, et al. 1985). Furthermore, Cortese-Krott et al. have previously observed alone that (H)SNO forms at room temperature from a 1:1 mixture of RSNO and sulfide in water (pH = 7.4) at even higher yield than their “SSNO-“ (Cortese-Krott MM, et al. 2014).

Thus, it is highly problematic to make further conclusions about the physiological effects and reactivity of the product mixtures with undefined chemical composition. To obtain valid (bio)chemical conclusions, use of pure compounds instead of undefined reaction mixtures is recommended. We are willing to provide pure SSNO- and SS15NO- salts to interested researchers.

References:

Cortese-Krott MM, et al. (2015) Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl. Proc Natl Acad Sci USA 112(34):E4651-60.

Seel F, Guttler, H-J, Simon G, Wieckowski A (1977) Colored sulfur species in EPD-solvents. Pure Appl. Chem. 49:45-54.

Seel F, Wagner M (1985) The reaction of polysulfides with nitrogen monoxide in non-acqueous solvents - nitrosodisulfides. Z Naturforsch 40b:762–764, and refernces therein.

Seel F, et al. (1985) PNP-Perthionitrit und PNP-Monothionitrit. Z Naturforsch 40b:1607–1617.

Seel F, Wagner M (1988) Reaction of sulfides with nitrogen monoxide in aqueous solution. Z Anorg Allg Chem 558(3):189–192.

Wedmann R, et al. (2015) Does Perthionitrite (SSNO(-)) Account for Sustained Bioactivity of NO? A (Bio)chemical Characterization. Inorg Chem 54(19):9367-9380.

Cortese-Krott MM, et al. (2014) Nitrosopersulfide (SSNO(-)) accounts for sustained NO bioactivity of S-nitrosothiols following reaction with sulfide. Redox Biol 2:234-44.

On 2016 May 20, Preben Berthelsen commented:

A grant application is not a clinical trial registration. The content of such an application is not a blueprint of the research but merely an indication of what the researchers contemplate. What counts scientifically is the pre-trial registration of the study with ClinicalTrials. To finalise the discussion on the question of the aim of the study, I have copy pasted below the Primary Outcome Measure from the ClinicalTrials registration (NCT01680744).

Primary Outcome Measures: • Renal Function [ Time Frame: 12 hours of mild hypothermia ] [ Designated as safety issue: No ] The primary outcome measures are renal function as determined by creatinine and cystatin c between declaration of neurological death and organ recovery in each of the two treatment groups. Delta creatinine and terminal creatinine are important predictors of graft quality and function, as demonstrated in the present data (HRSA study and Region 5 DMG/DGF study), and will be compared between the control and treatment group.

Ethical Problem. If the authors planned - as the thorny lifeline thrown by Dr. Greenwald (HRSA) seems to suggest - to study recipient graft function all along, the kidney recipients should have been informed that they took part in a randomized clinical trial and they should have given their consent before being enrolled in the investigation. This did not happen.

Preben G. Berthelsen, M.D. Charlottenlund, Denmark.

On 2016 Apr 25, Preben Berthelsen commented:

None

On 2016 Mar 09, Melissa Greenwald commented:

The United States Health Resources and Services Administration (HRSA) is the federal agency that awarded the grant funding for this research proposal. Grant awards were based on ranking after applications were reviewed by an external Technical Review Committee. “Delayed graft function” (DGF) was clearly stated as one of the goals of this research study as noted in the original grant application submitted in March 2011: “The goals of the this intervention are to demonstrate that TH [Therapeutic Hypothermia]: 1) better preserves deceased donor renal function while awaiting organ recovery when compared to normothermia; 2) increases the number of suitable organs for transplantation; and 3) improves recipient renal function after transplantation as measured by a reduction in DGF [Delayed Graft Function] and SGF [Slow Graft Function].” The grant application listed “Initial graft function” one of four variables to be measured for assessment of the first of two specific objectives of this research study. This is further specified in the Methods section of the grant application as: “The primary outcome measure will be number of patients in each group showing DGF/ SGF.” The parameters for information about research grants that is included and displayed on the Clinical Trials.gov website is under oversight of the U.S. National Institutes of Health.

Melissa Greenwald MD, Acting Director, Division of Transplantation, Health Resources and Services Administration, Rockville, Maryland, USA

On 2016 Jan 04, Preben Berthelsen commented:

According to the ClinicalTrials registration (NCT01680744) for this study, the primary outcome measure was renal function as determined by changes in creatinine and cystatin c between declaration of neurological death and organ recovery in donors randomised to normothermia or hypothermia. No secondary outcome measures were stipulated.

When Niemann et al report the results of their investigation, the primary outcome measure has been radically altered from changes in donor renal function to delayed graft function in the kidney recipients. This change in end-point - mirabile dictu – resulted in a positive outcome of the authors’ intervention.

In my view, the paper does not present evidence for a benefit of induced hypothermia in brain death donors prior to kidney transplantation. As the paper falsely suggests such a benefit the only safe option is a retraction of the paper - either by the authors or by the New Engl J Med where the review process seems to have been substantially substandard.

P.G.Berthelsen, M.D. Charlottenlund, Denmark

On 2015 Dec 04, Claus U Niemann commented:

Thank you for reviewing the study. I agree that the ethics and logistics of this study were exceedingly complex. This is further complicated by the complete lack of regulatory oversight for this type of research. Some of our efforts to alleviate this lack of oversight are described in the supplement. Two specific comments: 1.) Recipient covariates were included. Table S4 in the supplementary appendix provide important recipient variables that are known to be associated with renal allograft function. In fact, these variables are validated in several studies and are provided to the transplant community by the Scientific Registry of Transplant Recipients (SRTR),http://www.srtr.org/ 2.) Creatinine levels and GFR ( last determination prior organ recovery) appeared be lower in the hypothermia group. However, we are unsure of the effect of hypothermia on creatinine production itself and therefore cannot state with certainty that hypothermia actually resulted in an improvement. Nevertheless, the last creatinine prior organ recovery has been demonstrated to be a significant predictor of delayed graft function in the recipient.

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

This study was discussed on Sep 22nd and 23rd in the open online nephrology journal club, #NephJC, on twitter. Introductory comments are available at the NephJC website.

The discussion was quite detailed, with about 40 participants, including nephrologists, fellows and residents.

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

The highlights of the tweetchat were:

• The authors were to commended for designing and conducting this trial, using a relatively safe and low risk intervention, which may have potentially important implications for care of potential donors and outcomes in transplant recipients.

• A considerable discussion occurred around the ethics of doing a clinical trial without needing to obtaining consents from the recipients, which admittedly would have been logistically challenging.

• Though the results on delayed graft function were dramatic, as also were biologically plausible with a greater benefit observed in extended criteria donor organs, most discussants thought the results needed replication and also would like to see benefit in longer term clinical outcomes. Some key issues also brought up included the lack of covariates (especially related to recipient characteristics) and the pre-transplant improvement in kidney function seen in the intervention arm.

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

On 2017 Jul 04, Randi Pechacek commented:

Holly Ganz mentioned this article on microBEnet while discussing the spread of antibiotic resistance to wildlife populations.

On 2017 Jan 16, Martine Crasnier-Mednansky commented:

Growth of Escherichia coli on excess glucose under aerobic conditions is NOT diauxic, and the acetate switch is NOT "classically described as a diauxie". It is therefore extraordinary that the authors are now "showing diauxic behavior does not occur under such conditions". A diauxie in the presence of BOTH glucose and acetate was reported by Kao KC, 2005.

On 2017 Mar 19, Harri Hemila commented:

A manuscript version is available at HDL.

On 2015 Sep 01, Zhiping Pang commented:

We appreciate that Dr. Rinaman acknowledges that our conclusions are consistent with previous studies (Alhadeff et al., 2012; Dickson et al., 2012; Dossat et al., 2011; Skibicka, 2013), but strongly disagree with her surmise that our study is flawed based on the specificity of the mouse line used to manipulate GLP-1 neurons. We apologize that, primarily due to space limitations, we did not cite all the papers from Dr. Rinaman and colleagues. However, we argue that the specificity of the mouse line may be not as clearly doubted as Dr. Rinaman states, nor do we believe that our conclusions depend on that specificity alone. Based on the totality of our experimental work, we believe that our conclusions, as stated in the paper, are sound. As with all published scientific work, we provide experimental evidence for a particular hypothesis that is logical and plausible, but do not claim that our model provides a definitive answer to the question — in this study, how central GLP-1 regulates feeding. Thus, we feel that the comment from Dr. Rinaman et al. is much more apodictic and definitive than the phrasing of our paper’s conclusions.

We would like to respond to the specific concerns raised by Dr. Rinaman and her co-authors in the comment posted on PubMed Commons.

Dr. Rinaman et al. suggested that we claimed that Phox2b is GLP-1 specific:

We did not state, explicitly or implicitly, that endogenous expression of Phox2b and GLP-1 are 100% overlapping. We acknowledged that it is possible that not all GLP-1 expressing neurons express the phox-2b-cre transgene and that other types of neurons may express Phox-2b-cre as well. The purpose of utilizing the Phox2b-Cre transgenic line was to assess whether a defined group of central GLP-1 neurons was involved in regulating food intake. Our experimental results provide evidence that GLP-1 neurons likely participate in the regulation of food intake, although they do not exclude the involvement of non-GLP-1 Phox2b-Cre expressing neurons. Specifically, our data support a specific role of GLP-1 neurons in the regulation of food intake behavior in the following ways: a) the anorexic effects induced by the activation of Phox2b-Cre expressing neurons are blocked by the GLP-1R specific blocker Exendin-9 (also discussed below); b) retrograde-labeled NTS-VTA projecting neurons are positive for GLP-1; c) Cre-activated expression of EYFP colocalizes with GLP-1 in brain sections detected by a commercially available antibody (Peninsula Laboratories T-4363) (Zheng and Rinaman, 2015; Zheng et al., 2015); d) injection of CNO at the VTA in DREADD-expressing animals leads to suppressed food intake after 5 hours. In ongoing, unpublished studies, we have expressed Cre-dependent channelrhodopsin in NTS neurons of Phox2b-Cre transgenic mice to express channelrhodopsin in Phox2b-Cre positive NTS neurons, and found that neuronal activation by optical stimulation of the NTS nerve terminals is blocked by Exendin-9. This presents additional evidence to support that GLP-1R (GLP-1 receptor) is expressed in Phox2b-cre expressing cells. Taken together, these findings, along with reports from Scott et al (Scott et al., 2011) and the collection of studies cited throughout our manuscript, lead us to propose evidence for the involvement of GLP-1 signaling in the VTA in the regulation of feeding behavior.

Additionally, the transgenic mice used in this study were created based on the Bacteria Artificial Chromosome (BAC) technology. Unlike in the case of gene knockins generated by homologous recombination, in the case of transgenics, including BAC transgenics (Heintz, 2001), the introduced foreign gene is randomly inserted into the genome (Beil et al., 2012) and the expression of the transgene is influenced by epigenetic factors and genetic background (Chan et al., 2012). Therefore, the expression of the transgene does not always faithfully mimic endogenous gene expression. Indeed, the three Phox2b-Cre transgenic lines generated in Dr. Elmquist’s laboratory exhibit different expression patterns (Scott et al., 2011). Given these considerations, we did not conclude that Phox2b-Cre was only expressed in GLP-1 neurons or vice-versa. As described in the paper, the Phox2b-Crehese animals were employed as a tool to interrogate the function of a group of GLP-1 expressing neurons in regulating food intake behavior.

Due to size limitation, full response please refer to (please copy the hyperlink address): https://www.dropbox.com/s/0n129f2ugn3tgjz/Response.pdf?dl=0

On 2015 Aug 24, Linda Rinaman commented:

Phox2b is Not Specifically Expressed by Hindbrain GLP-1 Neurons

M.R. Hayes, University of Pennsylvania, Philadelphia, PA, USA; L. Rinaman, University of Pittsburgh, Pittsburgh, PA, USA; K.P. Skibicka, Sahlgrenska Academy at the University of Gothenburg, Sweden; S.Trapp, University College London, London, U.K.; D.L. Williams, Florida State University, Tallahassee, FL, USA

The first part of this study sought to extend published work [1-7] supporting a role for central GLP-1 signaling in suppressing palatable food intake. For this purpose, the authors virally expressed DREADDs within the caudal medulla of transgenic Cre-driver line mice, followed by chemogenetic DREADD activation to increase or decrease the activity of transfected neurons. Unfortunately, their experimental design depends on a Phox2b-Cre mouse model [8] that is non-specific for GLP-1 neurons.

Phox2b is expressed by a diverse set of autonomic-related neurons distributed throughout the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (DMV), and other regions [9-13], including catecholaminergic and HSD-2-positive neurons that innervate mesolimbic, hypothalamic, and other central targets [14-16]. The present study includes a supplementary figure (S1) purporting to show co-localization of GLP-1 immunolabeling with mCherry reporter expression, but the depicted coronal section is well rostral to the established location of GLP-1 neurons in rats and mice [17-19].  Thus, the GLP-1 immunolabeling is non-specific, and the authors present no credible evidence that GLP-1 neurons express virally-encoded DREADDs.

It is not surprising that food intake was suppressed in mice in which Phox2b-expressing AP, DMV, and NTS neurons were transfected to express hM3Dq. CNO activation of these neurons should disrupt physiological functions and activate stress-sensitive GLP-1 neurons [19-21] whether or not they express DREADDs. However, other than food intake, no physiological or behavioral measures were performed.  The authors report that the hypophagic effect of CNO was specific to the high-fat diet, with no effect on chow intake, but their experimental design and results are insufficient to support this claim.  Further, i.p. injection of Exendin-9 was reported to block the hypophagic effect of CNO (Figure 1F). The basis for this effect is difficult to understand, because the utilized i.p. dose of Exendin-9 is well below the established threshold for antagonizing GLP-1 receptors in the periphery, let alone within the brain [22,23].  In addition, stereotaxic injections were used to deliver a GLP-1 receptor agonist or CNO into the ventral midbrain of mice just before measuring their food intake (Figure 2), with no consideration of how acute surgery, presumably in anesthetized mice, might affect subsequent feeding behavior.   

In summary, we believe that the present report is seriously flawed.  Although the authors' conclusions are consistent with previous work in rats, their report fails to demonstrate a specific role for endogenous central GLP-1 signaling in the control of palatable food intake in mice.

REFERENCES

1. Dossat, A.M., et al., J Neurosci, 2011. 31: 14453.

2. Alhadeff, A.L., L.E. Rupprecht, and M.R. Hayes, Endocrinol, 2012. 153: 647.

3. Mietlicki-Baase, E.G., et al., Am J Physiol Endocrinol Metab, 2013. 305: E1367.

4. Dickson, S.L., et al., J Neurosci, 2012. 32: 4812.

5. Skibicka, K.P., Front Neurosci, 2013. 7: 181.

6. Richard, J.E., et al., Plos One, 2015. 10(3).

7. Hayes, M.R., L. Bradley, and H.J. Grill, Endocrinol, 2009. 150: 2654.

8. Scott, M.M., et al., J Clin Invest, 2011. 12: 2413.

9. Kang, B.J., et al., J Comp Neurol, 2007. 503: 627.

10. Lazarenko, R.M., et al., J Comp Neurol, 2009. 517: 69.

11. Geerling, J.C., P.C. Chimenti, and A.D. Loewy, Brain Res, 2008. 1226: 82.

12. Mastitskaya, S., et al., Cardiovasc Res, 2012. 95: 487.

13. Brunet, J.F. and A. Pattyn, Curr Opin Genet Dev, 2002. 12: 435.

14. Geerling, J.C. and A.D. Loewy, J Comp Neurol, 2006. 497: 223.

15. Delfs, J.M., et al., Brain Res, 1998. 806: 127.

16. Mejias-Aponte, C.A., C. Drouin, and G. Aston-Jones, J Neurosci, 2009. 29: 3613.

17. Llewellyn-Smith, I.J., et al., Neurosci, 2011. 180: 111.

18. Vrang, N., et al., Brain Res, 2007. 1149: 118.

19. Rinaman, L., Am J Physiol, 1999. 277: R582.

20. Maniscalco, J.W., A.D. Kreisler, and L. Rinaman, Front Neurosci, 2013. 6: 199.

21. Maniscalco, J.W., et al., J Neurosci, 2015. 35: 10701.

22. Williams, D.L., D.G. Baskin, and M.W. Schwartz, Endocrinol, 2009. 150: 1680.

23. Kanoski, S.E., et al., Endocrinol, 2011. 152: 3103.

On 2016 Dec 17, John Tucker commented:

The authors of this highly publicized petition raise the issue of the untenable rate of increase in the price of oncology drugs. They point out that the average cost of new cancer drugs at launch has increased by 5-fold to 10-fold over the last 15 years, and express concern regarding the effects of these drug costs on patient's financial well-being, treatment decisions, and the financial stability of the healthcare delivery systems. They propose a variety of solutions, including price controls, re-importation, reforms to the patent system, and encouraging professional groups to incorporate the cost of medical interventions into guideline development decisions.

Certainly we can all agree that the cost of healthcare cannot be allowed to perpetually outstrip the rate of economic growth. But ultimately, controlling healthcare spending requires a data-driven examination of what is driving costs, and not just politically expedient finger pointing at other contributors.

Per CMS (https://www.cms.gov/Research-Statistics-Data-and-Systems/Statistics-Trends-and-Reports/NationalHealthExpendData/Downloads/highlights.pdf), US healthcare spending increases from 2014 to 2015 included:

• A $53B increase in hospitalization costs, from $947B to $1000B
• A $37B increase in physician and clinic fees, from $597B to $635B
• A $26B increase in drug costs, from $298B to $324B.

Further, a recent IMS study (available at http://www.imshealth.com/en/thought-leadership/quintilesims-institute/reports/global-oncology-trend-report-2014) has shown that for many cancer drugs, including bevacizumb, cetuximab, pertuzumab, rituximab, and trastuzumab among others, the price paid by patients to hospitals exceeds the average wholesale price of the drug by more than 100%. This is in spite of the increasing number of hospitals that pay far below AWP for these drugs due to 340b discounts.

In order to control healthcare costs, it will be necessary to put all cost sources on the table, not just those of pharmaceuticals. This will include the very high drug administration fees pay to hospitals, and in the long run, the mid-six figure to seven figure salaries of many signatories of the Mayo Petition on Drug Prices.

On 2015 Dec 24, Vatsalya Vatsalya commented:

NIAAA Director's Statement for the Record on NIAAA FY 2015 Budget Request, Senate Subcommittee on Labor-HHS-Education Appropriations (Context of Varenicline in the 4th paragraph of NIAAA Research Section): http://niaaa.nih.gov/about-niaaa/our-funding/congressional-testimony/directors-statement-record-fy-2015-budget

On 2015 Dec 11, Vatsalya Vatsalya commented:

The NIAAA director's report for September 2015 council meeting included varenicline clinical trial manuscript as one of the research highlights:

http://www.niaaa.nih.gov/about-niaaa/our-work/advisory-council/directors-reports-council/niaaa-directors-report-institute-10#research-highlights

On 2015 Dec 03, Robert M Flight commented:

I have some concerns about the procedure of propagation to GO child terms mentioned as part of the procedure for generating the mutation-GO profile, as this results in new gene-GO annotations that do not exist in the original gene-GO annotation. A github repo with the results of my investigation of this issue is available, along with copies of the paper, supplementary data, all R and octave code used, and my correspondence with Sael.

The authors state that this propagation is necessary for the function of the ONMF procedure, but do not provide any evidence that this is so, and as part of the overall analysis actually perform propagation back to parent terms to determine significant GO terms. I wonder if both sets of propagation might have been avoided by using a GOSlim or another GO subset.

On 2015 Nov 06, Kenneth Witwer commented:

The figures in this article appear to have been previously published by another group (Tian Q, 2012) in the context of a different miRNA, miR-550a. The miRNA sequence in Figure 2 is also the sequence of miR-550a, not miR-1246 as labeled.

On date unavailable, commented:

None

On 2015 Aug 05, Roger H Reeves commented:

Response to Benoit Bruneau (Bruneau’s comments in italics)

Bruneau: The paper by Polk et al purports to demonstrate a genetic interaction between Tbx5 and Ts65Dn. I have a number of comments and questions related to the data used to reach this conclusion. First, the reduced amount of Tbx5 in the Ts65Dn is interesting, but puzzling is the almost complete absence in the Ts65Dn;Tbx5+/- embryos: based on previous investigations, this level of Tbx5 mRNA (almost zero) should result in extremely severe defects in heart formation, which are not observed.

Response: We show a qPCR analysis at a single developmental time point, the E12.5 heart. Although continuous low Tbx5 levels beginning earlier in heart development would result in severe heart defects as Bruneau suggests, the ramifications of diminished expression at E12.5 are unclear. Perhaps the increased lethality in Ts65Dn;Tbx5+/- mice is related to this observation. In an unnecessary ad homonym attack, Bruneau intimates that we are incapable of using qPCR appropriately (“This brings into question the quantitation of the mRNA levels”). Instead, it is Bruneau’s supposition that our observation at E12.5 could only be accurate if severe heart malformations were observed which is incorrect.

Bruneau: Data are presented only for 2 of the 4 genotypes that would be necessary to derive any conclusion [about genetic association]. In table 3, WT and Ts65Dn genotypes are not present. In Figure 2, only the compound heterozygote Ts65Dn;Tbx5+/- and WT are shown.

Response: The expected frequencies for these defects in wt and Ts65Dn mice have been published multiple times by us and others and three relevant studies are cited [1-3] showing that in a sample of this size, we would expect to see <1 ASD, <1 VSD, 0 AVSD and 0 OA in the same genetic background as this study. That is, we would expect to observe few or no defects and the same for wt. Perhaps we could have made a more concrete reference to the cited studies with regard to this specific point; however, neither we nor the reviewers found our presentation to be problematic. Even for those who missed the reference, we disagree with the statement that there is “no evidence [in Table 3] for genetic interaction.” The combination of genotypes unequivocally changes the phenotype; what forces not involving genetics might be responsible?

Bruneau: The same genotypes are missing from Fig 4.

Reponse: Fig. 4 is a histological representation. Since the defects don’t occur and such controls in the same genetic background have already been published, we choose to cite them rather than reproduce them here.

Bruneau: The in situ hybridization in Fig 4 suggests a reduction in Pitx2 expression in Ts65Dn;Tbx5+/- hearts; despite a very weak signal, this may be true, but this in no way indicates that the mice have atrial isomerism nor that the left-right pathway is involved in the defects shown.

Response: Here Bruneau overstates our conclusions about left-right patterning in order to criticize them (straw man argument). We show that OA incidence increases in Tbx5;Ts65Dn mice (Tbl. 3) – Bruneau tacitly agrees. We show that Pitx2 expression is lower in LA of Ts65Dn – Bruneau agrees with this as well. We discuss our findings in the context of the literature on OA where one finds frequent references to the relationship between OA and left-right development of the heart (see 12 references to the relationship between OA, Pitx2 and left-right signaling, 2nd paragraph of the Discussion). We point out the established link between altered patterns of Pitx2 expression, left-right isomerism and OA as a justification for doing this experiment in Fig 4 with the results shown. However, we do not state that OA is due to atrial isomerism, nor do we state that any of these mice have atrial isomerism. Bruneau has misstated our conclusion.

Bruneau: The atria of the mutant mice (in Fig 4) clearly have their normal morphology

Response: We note that it is not possible to correctly deduce complex pathological relationships from a single histological image. Regardless, it is irrelevant, as we do not assert that left-right isomerism is observed; that is a straw man of Bruneau’s invention.

Bruneau: I look forward to reading the authors' responses to these issues.

Response: We expect differences of opinion in interpretation by experts as a useful and necessary part of the scientific enterprise. Here, however, Bruneau has offered a superficial and needlessly aggressive critique replete with mischaracterization of our stated conclusions. We trust that objective readers interested in the complex phenomena resulting in heart defects in Down syndrome will consider our data for its intrinsic value and not mischaracterize our qualified speculations in the Discussion as conclusions.

Signed: Roger Reeves, Renita Polk, Peter Gergics, Ivan Modkowitz and Sally Camper

1. Moore CS (2006) Postnatal lethality and cardiac anomalies in the Ts65Dn Down syndrome mouse model. Mamm Genome 17: 1005-1012.
2. Williams AD, Mjaatvedt CH, Moore CS (2008) Characterization of the cardiac phenotype in neonatal Ts65Dn mice. Dev Dyn 237: 426-435.
3. Li H, Cherry S, Klinedinst D, DeLeon V, Redig J, et al. (2012) Genetic modifiers predisposing to congenital heart disease in the sensitized Down syndrome population. Circ Cardiovasc Genet 5: 301-308.

On 2015 Jul 29, Benoit Bruneau commented:

The paper by Polk et al purports to demonstrate a genetic interaction between Tbx5 and Ts65Dn. I have a number of comments and questions related to the data used to reach this conclusion. First, the reduced amount of Tbx5 in the Ts65Dn is interesting, but puzzling is the almost complete absence in the Ts65Dn;Tbx5+/- embryos: based on previous investigations, this level of Tbx5 mRNA (almost zero) should result in extremely severe defects in heart formation, which are not observed. This brings into question the quantitation of the mRNA levels. This is very minor point compared to the presentation of the data: data are presented only for 2 of the 4 genotypes that would be necessary to derive any conclusion. In table 3, WT and Ts65Dn genotypes are not present. In Figure 2, only the compound heterozygote Ts65Dn;Tbx5+/- and WT are shown; how can one judge any genetic interaction if the individual genotypes (Ts65Dn and Tbx5+/-) are not shown? In certain genetic backgrounds we see such defects occasionally in Tbx5+/- neonates, therefore the conclusions proposed by the authors cannot be reached with the data provided. The same genotypes are missing from Fig 4. The in situ hybridization in Fig 4 suggests a reduction in Pitx2 expression in Ts65Dn;Tbx5+/- hearts; despite a very weak signal, this may be true, but this in no way indicates that the mice have atrial isomerism nor that the left-right pathway is involved in the defects shown. The atria of the mutant mice clearly have their normal morphology, and there is no evidence presented of isomerism (e.g. ectopic or missing sinoatrial node, abnormal venous valve connections), not are any left-right pathway components (upstream or downstream of Pitx2) explored. The authors' conclusions regarding overriding aorta as a product of defective LR asymmetry, especially that of the atria, is particularly puzzling, as these are opposite poles of the heart. Therefore the conclusions related to disruption of left-right pathways in this genotype is not at all supported. I look forward to reading the authors' responses to these issues.

On 2016 Oct 27, Andy Collings commented:

Jawdat Al-Bassam's comment on this article (https://elifesciences.org/content/4/e08811#comment-2953448767) is reproduced below:

Negative stain EM 3D-reconstruction and consequent interpretations are often ambiguous due to their low resolution and rely on biochemical data for substantiation as provided in our manuscript. During the past year, we have used different 3D-reconstruction strategies to re-analyze negative stain data and obtain structures. From this re-analysis, we observed some changes in the features of 3D-reconstructions in Figures 5 and 7 in a program-dependent manner, which could result in changes to the fitted models described in Figures 5 and 7. As such, we would like the community to be aware that there are possible ambiguities and alternative interpretations of the published reconstructions, which likely arose from complex heterogeneity due to different conformational states or deformations from the negative staining process. However, these potential reconstruction differences do not change the general conclusions made in the manuscript regarding the overall organization of the complexes and the sites of binding for tubulin and tubulin cofactor C at low resolution. In addition, we deposited our raw data several months ago (EMPAIR-10034, 10035) and welcome suggestions and input from the community. We are currently focused on high-resolution structural studies using cryo-electron microscopy that will allow us to determine the de novo structure for the Tubulin cofactors-D-E with Arl2 assembly in complex with Tubulin dimer and Tubulin cofactor C.

Jawdat Al-Bassam (jawdat@ucdavis.edu)

On 2016 Nov 09, NLM Trainees’ Data Science Journal Club commented:

This is a preliminary study examining the discoverability of and access to biomedical datasets generated by research funded by the U.S. National Institutes of Health (NIH). More specifically, it focuses on datasets not deposited in a known repository and considered “invisible.” Analysis of the NIH-funded journal articles shows that only 12% explicitly mention deposition of datasets in known repositories, leaving a surprising 88% that are invisible datasets. The authors suggest that approximately 200,000 to 235,000 invisible datasets are generated from NIH-funded research published in one year alone. The study identifies issues to improve discoverability and access to research data: definition of a dataset, determining which data should be preserved and archived, and better methods for calculating the number of datasets of interest.

Our group had the honor of having two of the authors in attendance – Betsy Humphreys and Lou Knecht – to provide personal insights into the study. Betsy pointed out one the article’s strength – the opportunity to share this surprising discovery that has potential practical benefits to the research community. The study has received a fair amount of positive feedback, and Betsy mentioned that Clifford Lynch from the Coalition for Networked Information (CNI) has distributed this paper and calling for more studies on this subject. She also recognized the study’s weakness of not holding up as a model of research methodology and lack of consensus among the annotators on the definition of a dataset.

There was a lively discussion from the group on what constitute “invisible” dataset – are links to scientist or institutional websites considered invisible and how to detect visibility with better JATS tagging. For clinical researchers, personal domain self-archiving is the norm and considered invisible. Everyone agreed that it would be a priority to define visible and invisible for future research. Another interesting part of the discussion focused on having dataset be “in context” i.e., datasets are meaningful only when considered along with the published paper. It was surprising to learn that a large part of the formal repository name mentioned in the full text did not turn out to be in the context of an actual deposit. We discussed that it would be helpful to have guidelines and a consistent way to label the information – database name, date of deposit, accession number – in the acknowledgement for easier discoverability and preservation. We were pleased to see this preliminary study published because it brought light to the large problem of invisible dataset, and look forward to seeing more research in this area.

On 2015 Jul 25, Irving I. Gottesman commented:

This experimental confirmation of a "rumor" about pain insensitivity in patients with schizophrenia is a welcome one. If (when) it is noticed by schizophrenia researchers, the finding could be extended to testing the first degree relatives of patients for such decreased pain sensitivity.

On 2016 Dec 21, Daniel Corcos commented:

It is surprising that in situ cancer cumulative incidence after 7 years in the intervention group is only twice that in the control group, as these cancers are usually detected by screening, and a 4-fold difference would be expected instead.

On 2016 Apr 18, Duke RNA Biology Journal Club commented:

This comment is the summary of a discussion from our journal club meeting.

General Impressions: An impressively thorough paper which uses a combination of cell biology, biochemistry and high-throughput sequencing approaches to first identify lncRNAs associated with repressed chromatin, determine what genes a model lncRNA regulates and, finally, identify a specific structural mechanism by which this occurs. This paper lays the groundwork for future identification and characterization of chromatin associated lncRNAs. One overarching criticism with this work is it reads as two separate papers - one identifying the biological role that MEG3 plays in transcription regulation and a second developing the triple helix method of regulation.

Specific Points:<br> A technique, ChRIP-seq, was used to determine the global lncRNA environment of repressive chromatin. The design of the RIP protocol using two different proteins associated with repressed chromatin greatly simplified the final analysis by narrowing the pool of lncRNA targets to 70 unique lncRNAs. However, it was interesting that, even though both RIPs used 4SU crosslinking, the enrichment seen for T-to-C (or A-to-G) conversions was within the EZH2 pulldown and not the H3K27me3 pulldown. What are the 440 RNAs that specifically interacted with H3K27me3 but not EZH2 and why don’t they show crosslinking to the protein over input levels? While this was an interesting puzzle, the comparison that was done between the EZH2 crosslinked enriched RNAs and the chromatin enriched RNAs to provide a list of only 70 lncRNAs, was a clever way of finding chromatin associated RNAs that specifically bound to the PRC2 complex.

From the list of 70 candidate lncRNAs to study, MEG3 was selected. Even though the focus is on one lncRNA, a similar characterization pipeline could be used for the other RNAs identified through this technique. The cross-links identified through the T-to-C transitions were used as a starting point to identify a clear binding site to the protein. Luckily for them, the 4SU labeling, which usually over-crosslinks to its targets, shows only two identifiable clusters of crosslinks. Starting with crosslinks in the more conserved exon 3, they identified 9 bases that abolish ~50% of binding to EZH2 in vitro and in vivo, however, this would also imply there are separate sites on MEG3 that facilitate the other 50% of the binding. Additionally, the non-conserved sequences in MEG3 could fine-tune the binding to various proteins, including EZH2, in different tissues or organisms. Neither of these points is discussed further in the article but would be interesting to delve into if the ChRIP-seq analysis could be modified and applied to tissue samples.

After identifying MEG3’s putative site of binding to EZH2, the group did the obvious experiment and made knockdowns of both EZH2 and MEG3 to determine which genes were affected by RNA-seq experiments. An interesting addition might have been to use complete knockdown cells and a “rescue” with the mutant MEG3 from Fig 2 to provide insight into what genes that binding site specifically affects. They determine that both knockdowns show overlap for the TGF-beta pathway. This is further validated by using an orthogonal assay called ChOP-seq.<br> To explore the intricacies of the MEG3:TGF-beta gene interaction, Mondal and co-workers looked for sequence motifs within the MEG3-binding regions of the genome and discovered a GA-rich sequence motif. Interestingly, they also found GA-rich sequence motifs in the genomic binding locations of rox2 and HOTAIR, two well studied lncRNA known to bind the genome. This information suggests that GA-rich motifs are important for lncRNA localization across the genome.

Several previous studies explored the possibility that lncRNA form triple-helix structures with their target genome binding site using a combination of computational and experimental techniques; Mondal and co-workers used the Triplexator software, which is based on the binding rules needed for triple helix formation, as well as RNAse digestion assays. The Triplexator software identified several regions in MEG3 with high probability of forming triple helices, and those with the highest probability were GA-rich sequences. The assays combined GA-rich dsDNA probes and a target GA-rich segment of MEG3. After incubation, these solutions were separately treated with RNase A and RNase H. RNase A does not degrade ssDNA or dsDNA while RNase H degrades only ssRNA. The solution was sensitive to RNase A digestion, but resisted RNase H digestion. This indicates that the RNA present is not single stranded; however, the authors assume that a triple-helix structure is forming. Because they are not directly observing the structure, it is potentially possible that the RNA is displacing one of the DNA strands and forming an RNA:DNA double-helix. Mondal and co-workers use an anti- triplex dA.2rU antibody for their in vivo assays to confirm the presence of triple-helices, but there is no indication that this antibody was purchased from a commercial source, nor do they explain the method of raising the antibody in-house.

While this information indicates a RNA:DNA triple-helix structure, direct observation is necessary to confirm. Numerous methods could be used to assess the triple-helix structure: X-ray crystallography, nuclear magnetic resonance (NMR), small-angle x-ray or neutron scattering (SAXS/SANS), cryoelectron-microscopy (cryo-EM). As initially stated, we believe this work would have benefitted from splitting into two separate papers: one exploring the genomic binding of MEG3 to the TGF-beta pathway genes, and the other exploring the potential role of triple-helix formation in lncRNA binding.

On 2015 Sep 10, Holly G Prigerson commented:

Data appear to confirm that in some (most?) cases, less chemo is more QoL

On 2015 Aug 20, Bill Cayley commented:

A good example of when "less" is "more" - for more examples, see: https://lessismoreebm.wordpress.com/

On 2017 Feb 05, Stuart RAY commented:

I appreciate Prof Pybus' response, and have no objection to this use of the "Simplot" name; I was just providing context.

On 2017 Feb 01, Oliver Pybus commented:

I named the method "Deep Simplot" in homage to the very popular Simplot program. I apologise to Prof Ray and his colleagues for (i) not seeking their permission to use this term and (ii) not citing the original Simplot paper. To avoid confusion I recommend that, in future, the method described by our paper is referred to as "deep divergence plotting". My thanks to Prof Ray for bringing this issue to my attention.

On 2017 Jan 31, Stuart RAY commented:

As the author of Simplot (Lole KS, 1999) I infer that the "Deep Simplot" method described was named after Simplot; it also shares characteristic display elements (as seen in figure 3b). Of course, the bootscanning approach that I implemented in Simplot was pioneered by Mika Salminen et al (Salminen MO, 1995, which is cited by Iles et al.).

On 2015 Aug 12, Víctor M. Baizabal-Aguirre commented:

The PLKO1-GSK3beta1 and PLKO1-GSK3beta2 vectors used in our work were successfully used in a previous report by Yoeli-Lerner et al. (2009). As in our study, these authors were also able to fully silence the GSK3beta isoform (PMID: 19258413, see Figures 1A and 3A). Most of the studies on GSK3-dependent regulation of beta-catenin have used unspecific inhibitors that affect both GSK3 isoforms. Therefore, the contribution of GSK3alpha and GSK3beta to the regulation of beta-catenin is an issue that remains open. In this regard, Yu et al. (2003) reported that specific gene silencing of GSK3alpha or beta by siRNA expression vectors induces the stabilization of beta-catenin in P19 mouse embryonic carcinoma cells (PMID: 12597911, see Figure 5). As to the effect of GSK3 silencing on beta-catenin, results published in 2009 by Mamaghani et al., demonstrated that GSK3alpha or beta inhibition by siRNA increased the stabilization of beta-catenin in pancreatic cancer cells (PMID: 19405981, see Figure 3A). In contrast, Ryu et al., (2012) reported that specific GSK3alpha inhibition by siRNA decreased beta-catenin levels in human gastric cancer cells (PMID: 22328534, see Figure 3E). These findings indicate that complete removal of GSK3alpha or GSK3beta, as in our work, affect the relative abundance of beta-catenin and that GSK3alpha and GSK3beta alter in different ways the stabilization of beta–catenin, depending on the type of cell.

On 2015 Aug 04, Jim Woodgett commented:

Figure 3 appears to be missing panel B (beta-catenin blot).

The RNAi knockdowns here are remarkably efficient and it looks from the legend as though only one siRNA was used (although in the methods 3 alpha sequences and 2 beta sequences are listed). Figure 5 shows essentially complete removal. Blowing up the figure reveals some image artifacts. Typically, even complete inhibition of either GSK-3alpha or GSK-3beta has no effect on beta-catenin as the other isoform compensates fully (Axin is present at far lower concentrations that either isoform of GSK-3 and is the limiting factor in beta-catenin phosphorylation).

On 2016 Oct 24, Sean Ekins commented:

A poster based on this work from ACS in 2015 http://www.slideshare.net/ekinssean/mobile-apps-for-transporter-drugdrug-interaction-prediction-a-tool-of-the-future-now

On 2015 Dec 21, Will Rowe commented:

SEAR is now available for use as an App on the BaseSpace platform (Illumina).

This iteration of SEAR has been updated, changes include:

• The use of the open source VSEARCH (instead of USEARCH).

• New result output.

Please try it at:

https://basespace.illumina.com/apps/2083081/SEAR-Antibiotic-Resistance

Finally, the SEAR source code (for the App, Docker container image and original code) is available on GitHub:

https://github.com/wpmr2/SEAR

On 2016 Dec 05, Anne Carpenter commented:

As senior author on this paper, I report an error on page 10: the words "after cell fixation with 16% paraformaldehyde" should be replaced by "after cell fixation with 3.2% formaldehyde".

On 2015 Jul 25, David Keller commented:

This just in: pioglitazone proved futile for slowing the progression of Parkinson's disease

A large double-blind placebo-controlled randomized phase II study has proved pioglitazone futile for slowing the progression of early Parkinson's disease [1]. Pioglitazone is the only widely-prescribed thiazolidinedione ("glitazone") since the FDA placed safety restrictions on the use rosiglitazone (Avandia).

Glitazones now join the ranks of disproved neuro-protectants, including creatine, co-enzyme Q10, vitamin E and minocycline. Back to the laboratory....

Reference:

1: NINDS Exploratory Trials in Parkinson Disease (NET-PD) FS-ZONE Investigators. Pioglitazone in early Parkinson's disease: a phase 2, multicentre, double-blind, randomised trial. Lancet Neurol. 2015 Aug;14(8):795-803. doi: 10.1016/S1474-4422(15)00144-1. Epub 2015 Jun 23. PubMed PMID: 26116315.

On 2015 Jul 23, David Keller commented:

Exenatide, a different kind of diabetes drug, also exhibits activity against Parkinson's disease

The type-2 diabetes drug exenatide is in the category of incretin mimetics, and is not a thiazolidinedione ("glitazone") like the drugs in this study by Brauer and colleagues. Exenatide lowers blood sugar by raising levels of endogenous insulin, among other effects. It also has exhibited symptomatic benefits in Parkinson's disease, in a prospective randomized interventional trial [1], with persistent motor and cognitive benefits which suggest possible disease modification. Exenatide can be used concomitantly with glitazones, and such use should be accounted for in this glitazone study, to avoid skewed results. For example, if the percentage of glitazone patients taking exenatide was higher than the percentage of placebo patients taking exenatide, then the apparent benefits of glitazone use may have been all or partly due to this imbalance in the use of exenatide. Was this possibility controlled for?

Reference:

1: Aviles-Olmos I, Dickson J, Kefalopoulou Z, Djamshidian A, Kahan J, Ell P, Whitton P, Wyse R, Isaacs T, Lees A, Limousin P, Foltynie T. Motor and cognitive advantages persist 12 months after exenatide exposure in Parkinson's disease. J Parkinsons Dis. 2014;4(3):337-44. doi: 10.3233/JPD-140364. PubMed PMID: 24662192.

On 2017 Jun 28, Raphael Levy commented:

A response from Chad Mirkin. Well, nearly: a section of William Briley's PhD which starts with: "Though the endosomal escape of SNA nanostructures such as the Nanoflare and stickyflare is evident based upon their ability to provide sequence-specific information regarding RNA levels and locations within cells, one researcher [That’s me!] has concluded that SNAs cannot escape from endosomes.[75] That researcher is ignoring the many papers now that use such architectures for sequence-specific cell-sorting experiments." More here

On 2015 Nov 17, Raphael Levy commented:

David Mason and myself submitted a letter to the Editor of PNAS regarding that article. It was however deemed not to "contribute significantly to the discussion of this paper" by the editorial board, and therefore publication was declined. I leave it to the readers of PubMed Commons to decide: the letter was published as a PrePrint on bioRxiv. Our article argues that Briley et al own data show that the Sticky-Flare remain in endosome where they get degraded by nucleases (and therefore cannot report on RNA level and localisation).

On 2015 Nov 17, Raphael Levy commented:

The PNAS article itself includes a sentence which could be interpreted as SmartFlare advertising: "As a result, the Nanoflare has grown into a powerful and prolific tool in biology and medical diagnostics, with ∼1,600 unique forms commercially available today (sold under the SmartFlare trade name)." Furthermore, there is a Sticky-Flare patent which was published around a month before the communication of the PNAS article.

On 2015 Sep 28, George McNamara commented:

I posted this on PubPeer, https://pubpeer.com/publications/25CC01C366B9593D1686A78B52461F#fb36935

The Briley et al 2015 paper is deficient in methods - what is the length of the new product? what are the design criteria for specificity? how are the spherical nucleic acids constructed? Is there a mechanism by which the flare gets kicked off the SNA? I suggest PNAS explicitly require self contained full methods and materials in manuscripts they accept. The details can be in the supplemental file, and can both provide full details and cite -- or even quote -- earlier work.

The Briley COI statement states: "The authors declare no conflict of interest.". The authors and their University previously commercialized NanoFlare/SmartFlare - is PNAS sure they have not submitted patent applications for Sticky-Flare and intent to make money from it = financial interest. I am fine with commercialization of products, but if this is an advertisement for a future product, the authors should be honest in their COI an PNAS should mark the paper as an advertisement.

Citation for commercialization: "NanoFlares have been very useful for researchers that operate in the arena of quantifying gene expression. AuraSense, Inc., a biotechnology company that licensed the NanoFlare technology from Northwestern University, and EMD-Millipore, another biotech company, have commercialized NanoFlares. There are now more than 1,700 commercial forms of NanoFlares sold under the SmartFlare name in more than 230 countries." http://www.northwestern.edu/newscenter/stories/2015/07/new-tool-for-investigating-rna-gone-awry.html#sthash.GwI4hbRx.dpuf

One of their patents is US8507200B2 https://patents.google.com/patent/US8507200B2/en?q=mirkin&q=nanoflare

On 2017 Mar 27, Christopher Southan commented:

There was tutorial on this theme at the 2017 International Conference on Trends for Scientific Information (ICIC) https://www.slideshare.net/Haxel/icic-2017-tutorial-digging-bioactive-chemistry-out-of-patents-using-open-resources/1

On 2016 Jan 25, Dimitrios Tzalis commented:

The ELF Public Compound Collection (PCC) is a very unique collection of compounds that are already synthesized and available for screening within ELF Screening Campaign. The goal of the paper was to compare the PCC with other compounds that are available for biological screening or that were actually screened. That is why, with a full awareness, we have analyzed the PCC against part of the PubChem collection called MLP of NIH, commercially available Maybridge and already tested compounds represented by ChEMBL. It might be interesting to collate the PCC and the 15 millions of patent-extracted compounds, in respect to broadly understood novelty, but we wanted to avoid the contamination of our comparison with theoretical compounds. In such a way the work is much more consistent. Nevertheless, thank you for your valuable comment and we can think of extending our novelty check also in respect to theoretical compounds in the future analysis since our collection is still growing. We believe that by focusing on exploring underrepresented chemical space of spiro-compounds and saturated, fused hetero rings we are offering a very competitive and unprecedented collection.

On 2015 Jul 22, Christopher Southan commented:

This is part of a special issue "From chemistry to biology database curation" http://www.sciencedirect.com/science/journal/17406749/14/supp/C. The PubMed ID series is 26194580-6.

On 2015 Aug 03, Maria A. Landin commented:

Gratulere med ny artikkel Rivan :)

On 2015 Jul 22, Ellen M Goudsmit commented:

As one of the main authors, I note that the PACE trial could not have used the London criteria for ME, as often claimed, as there should have been at least one difference between the groups, and this was not reported. Ergo, they did not use the criteria for ME correctly and the results from the trial can not be extrapolated to this population. The attitude of the authors towards ME and the scientists who specialise in this disease is disappointing.

On 2015 Oct 03, Seyed Moayed Alavian commented:

Dear All, I would like to declare that receiving the medical services may be an important risk factor for acquiring the HCV and HBV infections in Middle East Countries. Yours Alavian SM

On 2016 Jan 14, Noman Shahzad commented:

The article is the highest level of evidence available on the topic. Reading the article, I am interested in knowing more about those who developed hernia. Like group wise details of how many of them were symptomatic, how many required surgical repair. Was there any statistical difference in detection method used to diagnose hernia? Was there difference in Quality of Life of those who developed hernia in traditional vs small suture group? This information will help in understanding the impact of change in technique from patient perspective. Clustering leading to increased risk of alfa error is a frequent problem of multicentric trials, it will be informative if information could be provided if adjustment was made for clustering in statistical analysis.

On 2016 Jan 10, Noman Shahzad commented:

None

On 2015 Jul 24, Jay Kaufman commented:

Etsuji Suzuki of Okayama University, Japan contacted the authors to note that on page 3 of the article, the text states that that conditioning on CVD in Figure 1c opens four paths that bias the effect of obesity on mortality. However, the first of these paths that is listed in the text (obesity <- smoking -> CVD -> mortality) does not in fact contain a collider on the path, since no nodes are entered and exited through arrowheads. Therefore, the text should state that there are three such paths, not four. This error in the exposition of the problem changes neither the quantitative results nor the conclusions of the paper.

On 2015 Dec 11, Mark Johnston commented:

Interactive protocols from this article are freely available at protocols.io.

On 2015 Sep 29, George McNamara commented:

Why does this paper's authorship section state:

Conflict-of-interest disclosure: The authors declare no competing financial interests.

when one of the authors works for the company that supplied the CB-839 drug feature in the abstract and key points?

http://www.calithera.com/programs/cb-839/

Genetically mandated alterations in the fundamental metabolic pathways of tumors often cause a dramatic rise in the uptake of the nutrients glucose and glutamine. Removal of glutamine leads to a substantial reduction in cell growth or induces cell death in certain types of cancer cells, indicating that these cells are dependent on, or “addicted” to, glutamine. Normal cells do not show this pronounced dependence on glutamine. The enzyme glutaminase, which converts glutamine to glutamate, has been identified as a critical choke point in the utilization of glutamine by cancer cells. CB-839 is a potent, selective, reversible and orally bioavailable inhibitor of human glutaminase.

On 2015 Oct 24, Geriatric Medicine Journal Club commented:

This article was critically appraised at the October 2015 Geriatric Medicine Journal Club (follow #GeriMedJC on Twitter). One of the study authors was also present for the tweet chat discussion! The full discussion can be found at: http://gerimedjc.blogspot.com/2015/10/october-2015-gerimedjc.html?spref=tw This is a very interesting study which separates common beliefs about older people in general as different from older patients in health care settings. This will help inform policies and education strategies to improve care for older adults.

On 2015 Aug 16, Xiang Ming commented:

Thanks very much for Murtaugh’s careful review. According to your comment, we make following explanations. 1. The pancreas photos on the first row of Fig.5A showed that the surgical removal of tissues do not include intestine. After being photographed, the tissues were embedded in paraffin, so we could ensure that all sections assessed in this paper are from pancreas. 2. As you said “the treatment might have induced cancers of an intestine-like pathology”, we should explain that a great quantity of non-tumor components existed in pancreatic cancer including stromal cells and lymphocytes. The immunohistochemical analysis in Figure.5 was consistent with this pathological characteristic which was similar to intestine-like pathology. 3. In our study, the malignant cell could secrete a small amount of amylase (Fig.5A). This also prove that the treatment didn’t induce cancers of an intestine-like pathology. On the other hand, the Ki67 is mainly expressed in the malignant acinar cell nucleus, so high levels of Ki67 expression could support our conclusion that Reg3g could promote proliferation in acinar cells

On 2015 Jul 30, L Charles Murtaugh commented:

While this is an interesting study, and the role of REG genes in pancreatic cancer is arguably understudied, the conclusions of this paper are tempered by irregularities in the histological analysis. Only one study group developed pancreatic cancer in this study, namely the high-dose pReg3g + DMBA treatment, referred to as HA10R. Inspection of the histology data presented for the HA10R group (Fig. 5A) reveals that several of the most relevant images (H&E staining, Ki67 and cytokeratin-19), for this group specifically, appear to be taken from sections of the intestine rather than the pancreas. While, in principal, the treatment might have induced cancers of an intestine-like pathology, this would not explain the clear organization of proliferative crypts and non-dividing villi, apparent from Ki67 staining. This raises doubts about the quantitative analysis of these and other variables, in Fig. 5B, as well as about the overall conclusion that treated mice developed cancer of the pancreas.

On 2015 Dec 05, David Keller commented:

Patient-oriented result: response rate to magnetic stimulation was the same as to placebo

This study of repetitive transcranial magnetic stimulation (rTMS) was designed to test the hypothesis that rTMS would result in a "statistically significantly greater percentage of responders to treatment in an active rTMS group compared with a placebo rTMS group" [1]. A relatively new metric called the Tinnitus Functional Index (TFI) was used to measure response to treatment. The TFI rated 18 of the 32 subjects actively treated with rTMS as responders to treatment (56%), while only 7 of the 32 subjects treated with sham therapy were rated as responders (22%). These two rates differed significantly, which was pre-specified in the Objectives section as defining a successful outcome.

However, 7 of the 18 treated subjects rated as "responders to therapy" using the TFI scale nevertheless believed they had received sham therapy, implying that they did not perceive any treatment benefit beyond the placebo effect. When a subject states that his treatments seemed like sham therapy, providing only placebo-strength benefit, this is important information. Since it is a direct expression of the subject's assessment of the efficacy of rTMS therapy, it has more validity than a contrived metric like the TFI, from a patient-oriented perspective.

The data in e-Table 12 indicate that, of the 32 subjects who received active rTMS treatments, only 11 correctly guessed they had received active therapy at the end of the last treatment, which implies that only 11 out of 32 actively-treated subjects (about 34%) noted perceptible improvement in their tinnitus symptoms. Coincidentally, 11 of the 32 placebo-treated subjects (also 34%) guessed that they had received active rTMS therapy, which equals the placebo effect. Thus, active rTMS treatments had the same response rate as sham therapy, equal to the placebo effect of 34%.

Conclusion: rTMS is no more effective than placebo for treating tinnitus, when assessed by subjects after a full course of treatments, based on their perception of whether they received active or sham therapy. The advantage of this assessment is that it eliminates uncertainty about the accuracy and clinical relevance of the TFI metric, because the assessment of treatment benefit came directly from the subjects themselves.

Reference

1: Folmer RL, Theodoroff SM, Casiana L, Shi Y, Griest S, Vachhani J. Repetitive Transcranial Magnetic Stimulation Treatment for Chronic Tinnitus: A Randomized Clinical Trial. JAMA Otolaryngol Head Neck Surg. 2015 Aug;141(8):716-22. doi: 10.1001/jamaoto.2015.1219. PubMed PMID: 26181507.

On 2015 Nov 29, David Keller commented:

38% of the reported "responders to therapy" thought they had been randomized to placebo & reply by author

At the end of this study, only 11 of the 32 subjects who received active treatment for tinnitus guessed that they had received active treatment. The remaining 21 subjects who were actively treated guessed that they had received placebo (sham treatments).

18 of the 32 actively treated subjects were rated as "responders" to therapy by Folmer et al. Thus, 7 of the actively treated subjects, who were rated as "responders" to therapy, thought they had received sham treatments. Bottom line: 7 of the 18 tinnitus sufferers (38%) who were reported to be "responders to therapy" actually did not perceive any benefit.

Tinnitus is a subjective phenomenon. I contend that, by definition, a responder to tinnitus therapy cannot believe that he received sham therapy. If a subject thinks he was treated with sham therapy, he did not perceive any benefit, and he cannot be a reported to be a "responder to therapy". This is the essence of my criticism of this study, and it has not been addressed.

Addendum (12/4/2015): Yesterday, in reply to the above comment, Dr. Folmer issued the following statement (start of quotation):

In our study, participants were categorized as "responders" or “non-responders” to TMS treatment based solely on the change in their TFI score from baseline to post-TMS assessment – this is stated in the article.

The definition of "responders" or “non-responders” we used had nothing to do with

1. Whether or not study participants “perceived” any benefit if that was based on anything else but their TFI score
   

2. Study participants’ guesses that they received active or placebo rTMS  
   

These are separate issues. You can debate, discuss or disagree with them, but they remain separate issues and definitions as specified in the article.

--Robert L. Folmer, Ph.D. (end of quotation)

On 2015 Oct 05, Eiko Fried commented:

A recent study published in Nature by the CONVERGE consortium [1] identified two Single Nucleotide Polymorphisms (SNPs) for Major Depressive Disorder (MDD) that replicated across two samples of Han-Chinese women with recurrent depression. The report was accompanied by an editorial [2] that hailed the findings as biologically and diagnostically relevant, suggesting that large-scale exploratory genome-wide studies offer enticing prospects towards aiding diagnosis and the development of new drugs.

We disagree with the editorial’s interpretation (and most of the media coverage) of these CONVERGE results, which also contrast with the careful phrasing of the authors themselves. Although the two SNPs discovered in the comparatively homogenous CONVERGE sample did replicate in a similarly ascertained group, the editorial fails to mention that they did not in the more heterogeneous Psychiatric Genomics Consortium (PGC) data also examined by the authors. Moreover, in polygenic risk score analysis, the genetic signal in the PGC sample explained less than 0.1% of disease risk in the CONVERGE data, implying a fundamental lack of overlap in genetic risk signal across samples.

The laudable effort of the CONVERGE consortium to ensure genetically and phenotypically homogenous samples confirms the elusiveness of the genetics of MDD. Hailing the results as robust insights into the biology of depression detracts from the true scientific relevance of the study: genetic effects for MDD are, even in large homogenous samples, small and do not generalize.

Given the hitherto negative results of genetic MDD studies [4,5], slogging along on this current road of ever-larger samples and discovering at best small effects is not an alluring prospect, especially so considering that these effects are likely not specific to MDD [6]. Instead, we suggest revising complex psychiatric phenotypes such as MDD that were transferred unquestioningly from psychiatry to genetics. Incorporating recently proposed network models [7], symptom- rather than syndrome-level analyses [8], and the development of new instruments that tap variation along the entire continuum [9,10] (i.e., in both "cases" and "controls") offer promising ways forward.

References

• 1.Cai, N. et al. Sparse whole-genome sequencing identifies two loci for major depressive disorder. Nature 523, 588–91 (2015).

• 2.Ledford, H. First robust genetic links to depression emerge. Nature 523, 268–269 (2015).

• 3.Keener, A. B. Genetic Variants Linked to Depression. Sci. (2015).

• 4.Hek, K., Demirkan, A., Lahti, J. & Terracciano, A. A Genome-Wide Association Study of Depressive Symptoms. Biol. Psychiatry 73(7), 667–78 (2013).

• 5.Daly, J. et al. A mega-analysis of genome-wide association studies for major depressive disorder. Mol. Psychiatry 18, 497–511 (2013).

• 6.Kendler, K. S. ‘A gene for...’: the nature of gene action in psychiatric disorders. Am. J. Psychiatry 162, 1243–52 (2005).

• 7.Cramer, A. O. J., Kendler, K. S. & Borsboom, D. Where are the Genes? The Implications of a Network Perspective on Gene Hunting in Psychopathology. Eur. J. Pers. 286, 270–271 (2011).

• 8.Fried, E. I. & Nesse, R. M. Depression sum-scores don’t add up: why analyzing specific depression symptoms is essential. BMC Med. 13, 1–11 (2015).

• 9.Lee, S. H. & Wray, N. R. Novel genetic analysis for case-control genome-wide association studies: quantification of power and genomic prediction accuracy. PLoS One 8, e71494 (2013).

• 10.Van der Sluis, S., Posthuma, D., Nivard, M. G., Verhage, M. & Dolan, C. V. Power in GWAS: lifting the curse of the clinical cut-off. Mol. Psychiatry 18, 2–3 (2012).

Authors

• EI Fried, University of Leuven, Belgium

• S van der Sluis, VU Medical Center, Amsterdam, The Netherlands

• AOJ Cramer, University of Amsterdam, The Netherlands

PDF of this commentary (DOI: 10.13140/RG.2.1.3480.4963) available at: http://eiko-fried.com/wp-content/uploads/Nat_Correspondence_blog.pdf

On date unavailable, commented:

None

On 2015 Sep 23, Angelo Gaitas commented:

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

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

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

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

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

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

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

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

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

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

On 2015 Aug 29, Michael King commented:

Our recent commentary discusses this paper:

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

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

On 2015 Sep 27, Ivan Oransky commented:

The first author of this paper is appealing the retraction: http://retractionwatch.com/2015/09/22/author-appeals-retraction-after-co-authors-dispute-nature-comm-paper/

On 2016 Nov 02, Eiko Fried commented:

What the study actually found is this: none of the 171 serum proteins differed between MDD cases and controls, seeing that none of the 171 markers differed after controlling for multiple testing.

The authors do not report this most important core finding of their study in the abstract, and instead claim that 28 of the 171 markers differed between the two groups. I struggle to understand why the authors chose to report the uncorrected findings instead of the proper statistical results — the corrected findings – in the abstract.

On 2017 Jan 19, Jameson Voss commented:

This paper identifies an interesting association between calories in the national food supply per capita and average population body weight. It was recently recognized by an independent review as an important work in the field. The recognition is appropriate as the analysis was based on worldwide comprehensive data, stratification of countries by income, assessment of longitudinal changes, incorporation of a mathematical model of weight gain and other strengths. The association is plausible and builds on other work connecting the food supply and body weight.

Despite these strengths, readers should be cautious about how they interpret the title and discussion. First, the title uses a causal phrase “major driver” which was also echoed in the recent review (linked above). Readers should be aware this title refers to an ecologic association and avoid making causal inferences about outcomes at a population or individual level based on this type of observational study design. Secondly, readers might misinterpret the term “major” as though the authors found the association to be stronger than other obesity correlates, but the paper did not provide an empiric comparison with any other obesity correlate. Finally, the discussion about opportunities for future research could also be misunderstood. The authors argue against testing the association with a study that moves people to different food systems by claiming it would be impractical. That might discourage readers from pursuing probative designs like “packet randomized experiments” which can utilize quasi-random assignment of migration (e.g., international adoptions, foreign exchange students, military household moves, etc.). If done properly, these studies can eliminate confounding among individuals, but confounding at the location persists. When participants are assigned to locations with higher energy in the food supply, they would simultaneously face other local, regional, or national exposures. That is, all exposures found at a location are assigned together as a single “packet,” so national food supply could be confounded by other differences between places (e.g., infrastructure, hygiene, ambient light, pollution, temperature, etc.).

There are empiric methods of handling packet level confounding, but it can be eliminated with a design called cluster randomization. Within the United States, an industry affiliated organization has worked to lower national per capita food energy supply and this has coincided with worsening average waist circumference, but it cannot be known if these trends are caused by the lower energy available in the food supply (as discussed here). Instead, system interventions could be randomized to different systems or subsystems or implemented at randomized start times or randomized locations.

While ecologic studies have confounding at both the location and subject level, there is still possible utility for prediction if the confounding remains stable across periods. Interventions, on the other hand, work based on causation and can have unintended consequences. Thus, readers should continue to feel encouraged to investigate net harms and benefits of altering the food supply.

The views expressed in this comment are those of the author and do not necessarily reflect the official policy or position of the Air Force, the DoD, or the U.S. Government.

On 2016 Apr 26, Ivan Oransky commented:

A researcher tried to publish a letter on this study, but the journal said prior publication on PubPeer meant he couldn't: http://retractionwatch.com/2016/04/26/does-posting-on-pubpeer-count-as-prior-publication-journal-says-yes-rejects-letter-rebutting-campus-sexual-assault-study/

On 2017 Aug 02, Gregory Francis commented:

The journal Perspectives on Psychological Science used to have an on-line commenting system. They seem to have discontinued it and removed all past comments. In April 2013, I published a comment on this article. I reproduce it below.

Simonsohn's arguments are without merit

Simonsohn presents two arguments to discredit the publication bias analyses I have published over the past year. Neither of these arguments are convincing to me, but some people seem to be taking them seriously. I treat them in more depth in a recent paper (Francis, 2013), but it might be helpful to have a comment with the article itself.

Whether to ignore data that appears to be biased

I have argued that sets of experiments that appear to be biased should be treated as "non-scientific" and be replaced by new experiments. Such a recommendation is admittedly a cautious approach, but I feel that such caution is warranted for scientific investigations; especially when it is fairly easy to gather new data. For example, in my POPS article, to which Simonsohn's directs his reply, the publication bias analysis suggests that the aversiveness memory effects reported by Galak & Meyvis (2010) appear to be biased. My recommendation to ignore their data is not so very harsh, since it is quite easy for interested researchers to gather new (unbiased) data and determine the magnitude of the effect. There may be situations where gathering new data is more difficult and such situations might justify efforts to try to mitigate effects of bias. However, existing statistical methods are not very good at compensating for bias, and they do not attempt to compensate for other types of questionable research practices that can also trigger the bias analysis.

Simonsohn raises an interesting issue about the relation between statistical significance and practical significance. His example of a literature of 100 studies, all with p<.05 and power of 97%, highlights a particularly bad property of bias. As Simonsohn describes it, bias appears to be present (the bias test gives .97¹⁰⁰ = 0.0476) but small because, given the power values, one would expect just three non- significant findings out of 100 experiments. Thus, Simonsohn concludes the bias is small because it appears to be small relative to what is published. Such a view would be fine if there was no reason to suspect bias, but it is difficult to maintain this view given that the analysis suggests there is bias. The bias could be small, but there may have been another 100 non-significant findings that were suppressed, in which case the bias is quite large. There is no way for a reader to know the extent of the bias.

One of the fundamental goals of science is to reduce uncertainty in measurements, but the appearance of bias introduces uncertainty about the experimental results and conclusions. The responsibility for making a strong scientific argument rests with the authors; and if their results appear to be biased, then it is difficult to make such an argument.

Cherry picking

Simonsohn charges that my analyses are ironically biased with the very practice that is used to produce the biased experiment sets that I criticize. To this charge, I reply "guilty", but I have an explanation. There are different types of biases; and some biases misrepresent empirical data, while other biases are simple byproducts of scientific exploration. Understanding the differences between these types of bias is very important.

Biases that misrepresent the data

If a researcher runs 10 direct replication experiments and gets six experiments to reject the null hypothesis and four experiments to not reject the null, then bias is going to be introduced if only the six significant experiments are reported. (Whether such bias will be detected by the bias analysis depends on the estimated power of those experiments.) A meta-analysis across those six published experiments will almost surely overestimate the true effect size, because the experiments with samples having small effect sizes tend to not reject the null. When the experiments are connected by a theoretical link (in this case by the idea that the experiments are measuring the same effect size), this kind of bias misrepresents the true state of nature, which is clearly a problem for a scientific field.

Biases that do not misrepresent the data

If a researcher runs 10 experiments on unconnected topics (separate studies on afterimages, reading rates, Stroop reaction times, aversiveness ratings of memories, etc.) and gets six experiments to reject the null and four experiments to not reject the null, then a bias is introduced by publishing only the six significant experiments. However, this kind of bias is mostly benign because it does not misrepresent the published data. Suppose the study on afterimages finds a significant effect with a standardized effect size of 0.6. None of the other experiments tell us anything about the effect size of afterimages, so whether those experiments are published or not does not influence the scientific conclusions that are drawn about afterimages from this published study.

The publication bias analyses I have reported over the past year have the latter type of bias. Whether other papers have bias or not is irrelevant to the conclusion about bias for Galak & Meyvis (or any other paper). This is not to say that the bias analysis cannot make a Type I error and conclude bias when it does not exist. If we cannot tolerate making a Type I error, then we should not make decisions.

An important caveat is that the selective reporting of my analyses prohibits us from estimating the proportion of biased papers in the field. Trying to make such an estimate from the reported analyses changes the bias from being benign to one that misrepresents the data. Importantly, just because this particular interpretation is inappropriate does not mean that the individual analyses are inappropriate.

In conclusion, Simonsohn's arguments are without merit. His claim that ignoring the data is unwise leads to scientifically vacuous arguments; and his claim of cherry picking is true but its meaning is misunderstood.

Francis, G. (2013). Replication, statistical consistency, and publication bias. Journal of Mathematical Psychology. http://dx.doi.org/10.1016/j.jmp.2013.02.003

Conflict of Interest: None declared

On 2015 Jul 15, Peter Hajek commented:

The key question is what effect this has on cigarette sales. If it is reducing cigarette sales, it is a positive development.

On 2015 Jul 15, Holly Brothers commented:

This is really interesting - I'll be happy to get access to the article. It reminds me of the RadioLab episode highlighting Jasper Lawrence who used hookworms to battle his hyperimmune responses (allergies and asthma) http://www.radiolab.org/story/91691-sculptors-of-monumental-narrative/.

On 2015 Dec 17, Peng Chen commented:

I realize that you have work very hard to publish all these excellent papers! Good job! Well done! And, I love U :)

On 2015 Jul 22, BSH Cancer Screening, Help-Seeking and Prevention Journal Club commented:

The HBRC journal club read with interest this paper that uses an innovative qualitative method to examine the barriers to colorectal cancer (CRC) screening experienced by people from medically under-served areas in the US. The paper offers an account of how macro level factors, such as the socio-political context, combine with individual level factors to determine uptake of CRC screening.

The authors used a participatory research method and PhotoVoice technique to engage participants in the research project, which took place over several months and included training sessions, group meetings, and individual meetings. We felt that one of the strengths of this approach was that it allowed rapport to be built over time, and that participants could express themselves in ways other than by just verbalising their thoughts and feelings. The photos taken by participants served as the “jumping off point” for the group discussions, which we felt helped obtain a richer picture of what people have to go through in order to obtain screening. Using each participant’s photos as a catalyst for the discussions may have encouraged a more evenly distributed participation across participants, because each participant was expected to contribute to the meetings. Although many of the barriers mentioned in this paper focused on the costs of colonoscopy screening and may therefore not necessarily generalise to healthcare contexts that use different screening methods or are free at the point of delivery, such as in the UK, some of the opportunity costs mentioned by participants did resonate with the journal club and may be more widely applicable. For example, those undergoing flexible sigmoidoscopy screening in the UK may also be faced with the opportunity cost of having to take (potentially unpaid) time off work.

We felt the authors could have commented more on the possible limitations of their study. The chosen study method required a great deal of commitment and active participation from participants. Although the researchers note that the Photovoice method may address shortcomings of other qualitative work in this field, which “may simply generate ‘impression management discourses’”, we felt that the method does not necessarily circumvent these discourses, but may just elicit more “crafted” image discourses. In addition, the high level of commitment expected from participants in the current study may mean that only those who were most highly motivated and health conscious self-selected to participate, which may limit the generalisability of the study findings. The authors chose to focus on the obstacles experienced by those who had been screened, but those may not necessarily be the same as the obstacles faced by people who have not screened. The HBRC journal club would encourage further study in samples of participants who are not engaged with screening. We also felt that the results were presented in a way that suggested a broad consensus among participants and wondered whether there were any dissenting voices? Some of us felt that a greater emphasis could have been placed on the experience of facilitators to screening uptake, although some felt that this was adequately addressed by discussing the importance of social support around the time of screening. Finally, we felt that the paper formed a good illustration of some of the barriers and facilitators to CRC screening that we already know about, but some of us felt that it did not offer many new insights, and wondered whether the wealth of data collected in the study could have generated any novel insights? We recognise that journal requirements may only allow for a limited discussion of all findings in a study, and we would argue that online supplements could make a valuable contribution to convey the results of this type of study, for example, as a photo gallery with accompanying quotations.

In sum, the HBRC journal club really enjoyed reading this paper and feel that the Photovoice method is a great method for doing explorative research in certain groups, because of the high level of active involvement of participants in the research project, and the meaning and value added by their pictures which may complement verbal explanations of behaviour. This study shows that decisions about screening are not just up to the individual, but are made in a socio-political context that can help or hinder individuals to obtain screening, and has important implications for policy making in preventive healthcare.

Conflicts of interest. We report no conflict of interests and note that the comments produced by the group are collective and not the opinion of any one individual.

On 2015 Jul 27, Chuan-Wei Jang commented:

In the paper, we failed to specify the sources of some antibodies used in the additional experiments a reviewer requested after our initial submission. These antibodies are:

H3K4me2: Active Motif, 39141

CenpA: Abcam, ab33565

TRF1: Alpha Diagnostic International, TRF12-A

TRF2: Novus, NB110

Beta-Actin: Abcam, ab8826

On 2015 Jul 16, Chuan-Wei Jang commented:

The figures in the paper have been reduced in size and resolution in the print publication process, and thus some details are compromised in their final form. The original high resolution figures can be viewed or downloaded from here.

On 2017 Apr 12, Siegfried Hekimi commented:

We obtained very different results that suggest that CLK-1 functions exclusively in ubiquinone biosynthesis and not in the nucleus to affect the mtUPR. In a paper entitled: A single biochemical activity underlies the pleiotropy of the aging-related protein CLK-1. By Liu et al. http://www.nature.com/articles/s41598-017-00754-z?WT.feed_name=subjects_energy-metabolism

On 2016 Aug 24, Ben Goldacre commented:

This trial has the wrong trial registry ID associated with it on PubMed: both in the XML on PubMed, and in the originating journal article. The ID given is NCT0210086. We believe the correct ID, which we have found by hand searching, is NCT02100865.

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

Many thanks,

Jessica Fleminger, Ben Goldacre*

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

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

On 2016 Feb 29, Jim Johnson commented:

Thanks Dave. Actually there are 2 related bioRxiv pre-prints. One is from the SFP facility and reports on the male littermates from this study. The other is from a conventional facility (same facility as the 2012 Mehran et al paper reports on).

Hyper-variability in Circulating Insulin Levels and Physiological Outcomes to High Fat Feeding in Male Ins1-/-:Ins2+/- Mice in a Specific Pathogen-free Facility Nicole M Templeman, Arya Mehran, James Johnson bioRxiv doi: http://dx.doi.org/10.1101/031799

Hyper-variability in Circulating Insulin and Physiological Outcomes in Male High Fat-fed Ins1-/-:Ins2+/- Mice in a Conventional Facility Arya Mehran, Nicole M Templeman, Xiaoke Hu, James Johnson

On 2016 Feb 28, Dave Bridges commented:

A preprint was posted by this group describing similar experiments in male mice: http://dx.doi.org/http://dx.doi.org/10.1101/031799

On 2017 Jul 13, Randi Pechacek commented:

Elisabeth Bik wrote a blog describing this paper on microBEnet in a discussion about human microbiomes at sea.