On 2015 Sep 04, Casey M Bergman commented:

Orignally posted at PubPeer: https://pubpeer.com/publications/B37A9BD035CAEA3EEE79C48DDB0378

This paper provides a nice example of natural selection operating on a new gene duplicate at the Resistance to dieldrin (Rdl) locus in in D. melanogaster. Starting with an amino acid variant that is known to confer resistence to the insecticide dieldrin, the authors query whole genome sequences in the Drosophila Genetic Reference Panel (DGRP) [1] and find four strains that contain the resistance mutation. In three of the four strains, the resistance mutation is found unexpectedly in a heterozygous condition. The DGRP strains are highly inbred but are known to contain regions of residual heterozygosity scattered throughout the genome that could arise from incomplete inbreeding or segmental duplication.

By genotyping the offspring of "heterozygous" stocks, the authors find that 100% of progeny retain the heterozygous state, consistent with a segmental duplication. Analysis of the depth of sequence coverage in "heterozygous" strains confirms a 2-fold increase in the depth of coverage for a ~110 kb region containing Rdl and several other genes. The segmental duplication is flanked by two Roo transposable elements, suggesting it arose by ectopic recombination between repetitive sequences. The outcome of the duplication is that one duplicate contains the resitance allele and the other duplicate contains the susceptibility allele. Both copies are expressed. Transgenic analysis confirmed that the cause of the resistence allele is due to a single amino acid change in the Rdl gene only.

This paper shows how a newly arising allele with partial dominance can achieve a state of "permanant heterozygosis" by segmental duplication, conferring immediate adaptive advantage via the new allele while also retaining ancestral function via the old allele. This result nicely confirms a theoretical model developed by Spofford (1969) [2] (not cited in the current work) to explain selective forces that would govern the fixation of a new, polymorphic segmental gene duplicate. This work also shows that the "residual heterozygosity" in the DGRP lines may be more than just background noise from the vagaries of inbreeding, and may in fact point to many other functional segmental duplications in these lines.

[1] Mackay, Trudy FC, et al. "The Drosophila melanogaster genetic reference panel." Nature 482.7384 (2012): 173-178. http://www.nature.com/nature/journal/v482/n7384/full/nature10811.htm

[2] Spofford, Janice B. "Heterosis and the evolution of duplications." American Naturalist (1969): 407-432. http://www.jstor.org/stable/2458991

On 2013 Oct 31, John Cannell commented:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2014 Sep 26, George Ntoumenopoulos commented:

The ability to determine the need for and deliver physiotherapy for patients managed on VV-ECMO is challenging, both from the acute respiratory and rehabilitative perspective. The cannulae type, location (e.g. femoral and/or jugular veins) and stability of delivery of VV-ECMO may impact on the clinicians opinions (physiotherapy and medical staff) about physiotherapy care. Deep sedation levels during ECMO may limit the ability the deliver physiotherapy and we need to explore this further. The use of ultra-low tidal volume delivery during mechanical ventilation for lung protective ventilation may also mask the detection of problems such as secretion retention, with standard methods such as"sawtooth" patterning on expiratory flow waveforms. We need to further investigate the role of physiotherapy (both acute respiratory and rehabilitative) in this complex patient group.

On 2014 Oct 15, Gerd Heusch commented:

The skepticism of Dr. Berthelsen is well taken. However, contrary to his expectation, the results of our study with its primary endpoint troponin release have already been replicated, see Candilio L, 2015.

On 2014 Oct 13, Preben Berthelsen commented:

This study was registered with ClinicalTrials (NCT01406678). According to the registration, the authors planned to include 500 patients in a randomised study of remote ischaemic preconditioning (RIP) in isoflurane anaesthetised patients. No interim statistical analyses were planned. The study was, however, stopped early after the inclusion of 162 patients in the RIP-group and 167 patients in the group where no RIP was used. (Only 121 and 137 patients could be included in a per protocol analysis).

It is an indispensable rule not to break the randomisation code before all patients have completed the investigation. The authors have not followed this imperative. They have kept running track of the results as can be seen from the admission on page 598 of this paper “After use in some patients, however, we became aware of apparent interference of propofol with remote ischaemic preconditioning and discontinued its use in the remainder of the study”. So at least theoretically, it has been possible for the authors to analyse the data continuously and therefore stop the investigation – when a statistical difference was demonstrated – and before the stipulated number of patients had been reached.

A re-calculation of the power of the study using the 17% difference in 72h-troponin AUCs and a SD of 200 (standardised difference 0.27) shows that there is less than a 50% chance that another study would replicate the authors’ findings. It is my opinion that the result of this investigation must be viewed with some scepticism. Preben G. Berthelsen MD. Charlottenlund, Denmark.

On 2014 May 21, Amanda Capes-Davis commented:

INT 407 is a widely used cell line. However, many scientists are unaware that it is NOT an intestinal cell line. INT 407 is cross-contaminated by HeLa, which comes from cervical carcinoma. For a list of known cross-contaminated cell lines, see http://iclac.org/databases/cross-contaminations/.

On 2013 Oct 26, Lorene M Nelson commented:

This article provides an excellent summary of the state of knowledge regarding the role of pesticides in the etiology of Parkinson’s disease, and nicely highlights the challenges faced by environmental epidemiologists in identifying specific pesticide chemicals that are associated with PD. I agree with Dr. Kamel that it has been difficult to make substantial progress in identifying specific pesticide chemicals that increase PD risk except in unique circumstances, such as the two recent high-quality cohort studies that she cites. Another notable example is the retrospective (case-control) study conducted in central California by Dr. Ritz (UCLA investigator) whose group has produced findings implicating workplace and ambient pesticide exposure in PD [Wang A, 2011; also cited by Dr. Kamel], well-water consumption Gatto NM, 2009, specific pesticide combinations Costello S, 2009, and gene-pesticide exposure interactions Manthripragada AD, 2010.

On 2014 Nov 17, Raphael Levy commented:

This article belongs to the “stripy nanoparticles” series. The evidence behind the structure and special properties of these nanoparticles has been challenged by Cesbron Y, 2012. The publication in 2012 of Cesbron Y, 2012 took three years and has been followed by post-publication peer review of the various existing and new stripy articles on my blog, PubPeer, etc.

A detailed analysis of this body of work is published today in PloS One by Stirling et al; from the abstract: “through a combination of an exhaustive re-analysis of the original data with new experimental measurements of a simple control sample comprising entirely unfunctionalised particles, we conclusively show that all of the STM evidence for striped nanoparticles published to date can instead be explained by a combination of well-known instrumental artefacts, strong observer bias, and/or improper data acquisition/analysis protocols.”

On 2014 Jun 30, Karel Koberna commented:

The method described in the article of Cappella et al. was patented by us in 2012 and at the end of the same year published in great detail in our article “Atomic Scissors: A New Method of Tracking the 5-Bromo-2′-Deoxyuridine-Labeled DNA In Situ” PLoS (7(12): e52584). This fact is not evident as the paper of Cappella et al. contains incorrect reference to our original paper. We asked editor of Cytometry A to correct this error so we believe that it will be corrected soon.

On 2013 Dec 02, Kenneth N Litwak commented:

This study, by Ruff et al, attempted to demonstrate negative effects of excess sugar consumption on mouse survival, competitive ability, and reproduction, as a means of demonstrating the utility of Organismal Performance Assays (OPAs) in quantifying toxicities. However, there are multiple methodological issues in the article affecting its value.<br> 1. In the discussion, the authors state that the diet was “analogous to human subjects consuming a healthy diet plus the equivalent of ~3 cans of soda per day”. This statement does not correctly reflect the study diet vs. control diet, as the two diets were isocaloric. Adding 3 cans of soda to the diet would add additional calories to a diet, not replace a source of carbohydrates.<br> 2. The authors correctly noted that this experiment would have ideally maintained the different groups on their original diets for duration of the study; however, all mice were given the F/G diet once they entered the OPA enclosures at 26 weeks of age and for the next 7 – 8 months. This change in diet is a major confounding point of the entire experiment. It might be reasonably assumed that initial outcomes in the OPA enclosures would be due to different diets, but the authors do not provide any data to support this premise, for initial or long-term outcomes.<br> 3. Mouse pups were only counted every six week to determine reproductive success (Methods). The long duration between counts would likely result in an under-count of reproductive success, as cannibalism and early pup death would be difficult to determine. Further, the duration between counts was sufficient to allow multiple litters to be born to founder mice and to F1 mice.<br> 4. The confounding effects of the OPA enclosures and their locations (Supplementary Figure S3) are potentially significant. Each enclosure was immediately adjacent to others. However, some enclosures were only bounded by two enclosures, while some were bounded on three sides by other enclosures. Stress associated with territorial boundaries has been repeatedly documented in mice. Due to the small size of this study, the potential space interaction could have had significant effects on the outcomes.

On 2017 Jul 08, David Keller commented:

Why Physicians Favored Lipitor Over Simvastatin

In their commentary on generic drug use, Alldredge and Kayser state the following:

"In 2011, fewer prescriptions for generic simvastatin were written than for Lipitor (atorvastatin calcium; Pfizer Inc) despite a significant cost differential and no apparent clinical differences in efficacy and safety." [1]

That statement is an unfair criticism of physicians and conflicts with the expressed views of the Food and Drug Administration (FDA). On June 8, 2011, the FDA issued a safety warning concerning simvastatin [2] in which physicians were advised to discontinue the use of simvastatin, 80 mg (except in patients who had already been taking it safely for over 1 year).

For patients taking amlodipine, the FDA warned against prescribing more than 20 mg of simvastatin, and for patients taking diltiazem or verapamil, the dose of simvastatin was limited to 10 mg. These safety warnings were based on reports of adverse events including serious myopathy caused by simvastatin, 80 mg, and by lower doses of simvastatin when combined with widely prescribed calcium channel blockers.

As a result, physicians were left with a maximum safe simvastatin dose of 40 mg, which lowers low-density lipoprotein cholesterol level by approximately 40%, whereas atorvastatin, 80 mg, lowers low-density lipoprotein cholesterol level by approximately 55%.[3] The FDA has not deemed it necessary to issue similar safety restrictions on the use of atorvastatin, which may explain why physicians favored Lipitor over generic simvastatin in 2011.

References

1: Alldredge BK, Kayser SR. Bending the curve toward increased use of generic drugs. JAMA Intern Med. 2013;173(3):233-234. PubMed Article

2: FDA Drug Safety Communication: new restrictions, contraindications, and dose limitations for Zocor (simvastatin) to reduce the risk of muscle injury. www.fda.gov/Drugs/DrugSafety/ucm256581.htm. Accessed February 21, 2013.

3: Law MR, Wald NJ, Rudnicka AR. Quantifying effect of statins on low density lipoprotein cholesterol, ischaemic heart disease, and stroke: systematic review and meta-analysis. BMJ. 2003;326(7404):1423. PubMed Article

Author's note: the format of the above letter is scrambled and difficult to read, as published online at the following URL:

https://jamanetwork.com/journals/jamainternalmedicine/fullarticle/1726968

Due to the difficulty of getting the published version corrected, I have posted the corrected version above.

On 2014 Jan 15, Satoko Hattori commented:

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

On 2013 Nov 14, Gareth Highnam commented:

Awesome study, are cancerous areas changed to a uniform density across ethnicities? Or does density of cancerous areas also have an ethnic component? All factors for an optimized detection model, I suppose. [CoI - RH is my favourite published uncle :) ]

On 2013 Nov 20, Jamie Horder commented:

It would be wrong to say that reverse coded items are rarely not unconfusing!

On 2013 Nov 17, James C Coyne commented:

Formal Correction: This article has been formally corrected to address the following errors.

As a result of an error by PLOS ONE, an incorrect version of the article was typeset. Below is a link to a version of the article based on the correct manuscript.

http://www.plosone.org/annotation/listThread.action?root=71757

On 2013 Nov 05, Pedro Mendes commented:

This yeast metabolic reconstruction improves on the work described in Herrgård MJ, 2008, Dobson PD, 2010, and Heavner BD, 2012. The data set is available at http://yeast.sf.net/

On 2017 Jun 10, Casey Greene commented:

We used this method in our own preprint. Our preprint is available on bioRxiv: https://doi.org/10.1101/147645

For the convenience of the research community, we have made our implementation available as an open source python package. It is currently maintained on GitHub (https://github.com/kathyxchen/crosstalk-correction) and distributed via PyPI (pip install crosstalk-correction). We hope that others will also find this useful.

On 2014 Jul 12, Jorge H Ramírez commented:

The overall quality of this randomized clinical trial is poor:

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

• The study evaluates surrogate instead of definitive outcomes.

• No description of allocation concealment mechanisms.

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

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

• No appropriate description of blindings in the study.

• No intention-to-treat analysis

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

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

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

A recent publication entitled "Solifenacin/tamsulosin FDC squeezes out competitors."(4) supports that this combination is cost-effective only based in the pharmacoeconomic analysis of this study (Neptune trial. van Kerrebroeck and colleagues. Eur Urol 2013).

I have the following open-question for anyone reading this comment:

What would happen with pharmacoeconomic evaluations involving solifenacin/tamsulosin in the treatment of lower urinary tract symptoms after considering the risk of severe hypotension and the results of unpublished studies?

Finally, I personally think that the title "Solifenacin/tamsulosin FDC squeezes out competitors." resembles more a pharmaceutical marketing campaign (a misleading one) than a serious paper reporting the results of a pharmacoeconomic analysis.

References

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

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

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

4. Nazir, J. Solifenacin/tamsulosin FDC squeezes out competitors." PharmacoEconomics & Outcomes News 706 (2014): 10-5.

On 2013 Nov 01, Gerard Ridgway commented:

This is an updated version of Weiner MW, 2012, with additions highlighted in the PDF version. Somewhat confusingly, the abstract still refers to "results as of February 2011", but the masthead on the PDF clarifies that it was "Updated April, 18, 2013". To give a rough idea of the scale of the update, the number of cited references has increased from 225 to 349. Li Shen has been added as an author.

On 2013 Nov 19, Ilene Mizrachi commented:

The genome assembly is available in GenBank. The assembly was recently updated to version 2, APWK02000000 and is retrievable at http://www.ncbi.nlm.nih.gov/nuccore/APWK00000000

On 2013 Nov 19, Gustavo Costa commented:

The Genbank Acession APWK01000000 for the contigs, mentioned in the paper, is unavailable. Maybe the submitting authors should inform Genbank to release the sequences publicly.

On 2014 Dec 07, Harri Hemila commented:

Rerksuppaphol S, 2013 reports a highly significant baseline imbalance on age

Rerksuppaphol S, 2013 randomized 100 children to the zinc and placebo groups using blocks of two. According to their Table 1, average age was 10.0 yr (SD 0.5 yr) in the zinc group, but 11.4 yr (SD 0.8 yr) in the placebo group; the authors calculated P=0.0001 for the difference in baseline age. In their paper, Rerksuppaphol S, 2013 do not comment on their calculation of highly significant imbalance on age.

As the most interesting finding, Rerksuppaphol S, 2013 report that coughs and runny noses were shorter in the zinc group, with P=0.01. The median “duration for cough” was 1.0 days in the zinc group, and 6.0 days in the placebo group. The total duration of the trial was 3 months and it seems obvious that many participants had more than 1 cold episode per 3 months. However, the Methods and Results do not describe whether the “duration of cough” means duration per episode or duration per person. Thus the outcomes are not well described. Given the reported baseline imbalance and the lack of transparency in the outcomes, it is difficult to trust the reporting on coughs and runny noses.

Rerksuppaphol S, 2013 refer to the Cochrane review on zinc for the common cold by Marshall I, 2000 (ref. 19), which was withdrawn several years ago in Marshall I, 2007. Thereafter a new Cochrane review was written by Singh M, 2011 which is also cited (ref. 25), without any mention that the latter replaced the old withdrawn Cochrane review (2000), which was also cited (ie ref. 19). Such a presentation of previous literature on zinc and colds gives an impression of sloppiness.

Rerksuppaphol S, 2013 has been cited in JAMA by Das RR, 2014. Therefore the validity of this study is a relevant issue.

On 2017 Nov 29, Liz Dooley commented:

This Cochrane Review has been superseded. A new author team will publish a new protocol 'Vitamin C for pneumonia'. This information has been provided by the Cochrane Acute Respiratory Infections Group.

On 2014 May 14, Martine Crasnier-Mednansky commented:

The feedback strategy established by Doucette CD, 2011 relates to data obtained under conditions of 'extreme' catabolite repression elicited by nitrogen limitation. A direct inhibitory effect of α-ketoglutarate on Enzyme I inhibits phosphorylation of the components of the phosphotransferase system (PTS), including phosphorylation of EnzymeIIAGlc which activates adenylate cyclase. Therefore, cAMP-mediated catabolite repression occurs in these conditions. Another not-yet propounded effect of an increase in α-ketoglutarate is an enhanced inducer exclusion, which further hinders uptake of carbon sources other than PTS substrates. Under nitrogen-limited conditions, β-galactosidase synthesis in a wild-type strain growing on glucose is most likely affected by inducer exclusion. Thus, nitrogen-limited growth may not 'rely completely' on cAMP-mediated gene regulation as transcriptional regulation by cAMP and inducer exclusion both interfere with β-galactosidase synthesis Crasnier-Mednansky M, 2008. By not taking into account inducer exclusion the authors undermine the power of 'quantitative physiology'. Within the same frame of thought, using pts strains grown on lactose to measure β-galactosidase activity is physiologically irrelevant.

In sharp contrast with the present data, Daniel J, 1986 reported that α-ketoglutarate (or pyruvate indirectly by accumulation of α-ketoglutarate) caused repression of the lac operon - but not oxaloacetate. In addition repression did not occur in crr strains (lacking Enzyme IIAGlc) in support of Doucette CD, 2011.

On 2013 Oct 31, John Cannell commented:

The authors found markers oxidative stress is present in autism spectrum disorder (ASD). I wonder if the authors are aware that the genes for the antioxidants superoxide dismutase and thioredoxin reductase are directly up-regulated by the secosteroid 1,25 di-hydroxy vitamin D3 (calcitriol). I believe both genes also harbor a vitamin D response element.

Peehl DM, Shinghal R, Nonn L, Seto E, Krishnan AV, Brooks JD, Feldman D. Molecular activity of 1,25‐dihydroxyvitamin D3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis. J. Steroid Biochem Mol. Biol 2004;92:131–141. PMID:15555907>

Calcitriol also directly up-regulates glutathione reductase and increases glutathione levels.

Jain SK, et alo. Vitamin D upregulates glutamate cysteine ligase and glutathione reductase, and GSH formation, and decreases ROS and MCP-1 and IL-8 secretion in high-glucose exposed U937 monocytes. Biochem Biophys Res Commun. 2013 Jul 19;437(1):7-11. doi: 10.1016/j.bbrc.2013.06.004. Jain SK, 2013

Also, supplemental vitamin D significantly reduces oxidative stress in humans.

Nikooyeh B, et al. Daily intake of vitamin D- or calcium-vitamin D-fortified Persian yogurt drink (doogh) attenuates diabetes-induced oxidative stress: evidence for antioxidative properties of vitamin D.J Hum Nutr Diet. 2013 Jul 5. doi: 10.1111/jhn.12142. Nikooyeh B, 2014

Asemi Z, et al. Vitamin D supplementation affects serum high-sensitivity C-reactive protein, insulin resistance, and biomarkers of oxidative stress in pregnant women. J Nutr. 2013 Sep;143(9):1432-8. doi: 10.3945/jn.113.177550. Asemi Z, 2013

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

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

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

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

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

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

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

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

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

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

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

70% of American toddlers do not take the Ameri

On 2014 May 07, Matthias Girndt commented:

The full title of this article in German is: "Paresthesia and muscle cramps after dialysis: can they be prevented?"

On 2016 Nov 20, Morten Oksvold commented:

An investigation committee at Wayne State University (WSU) recommends that 42 articles from Fazlul Sarkar to be retracted (report finished August 31, 2015). This article represents one of them.

This information was published by Retraction Watch (November 17, 2016) and you can find a link to the full report here:

http://retractionwatch.com/2016/11/17/details-of-investigative-report-into-sarkar-released-by-aclu/

This article should therefore no longer be cited.

On 2013 Oct 31, John Cannell commented:

The authors found markers oxidative stress is present in autism spectrum disorder (ASD). I wonder if the authors are aware that the genes for the antioxidants superoxide dismutase and thioredoxin reductase are directly up-regulated by the secosteroid 1,25 di-hydroxy vitamin D3 (calcitriol). I believe both genes also harbor a vitamin D response element.

Peehl DM, Shinghal R, Nonn L, Seto E, Krishnan AV, Brooks JD, Feldman D. Molecular activity of 1,25‐dihydroxyvitamin D3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis. J. Steroid Biochem Mol. Biol 2004;92:131–141. PMID:15555907>

Calcitriol also directly up-regulates glutathione reductase and increases glutathione levels.

Jain SK, et alo. Vitamin D upregulates glutamate cysteine ligase and glutathione reductase, and GSH formation, and decreases ROS and MCP-1 and IL-8 secretion in high-glucose exposed U937 monocytes. Biochem Biophys Res Commun. 2013 Jul 19;437(1):7-11. doi: 10.1016/j.bbrc.2013.06.004. Jain SK, 2013

Also, supplemental vitamin D significantly reduces oxidative stress in humans.

Nikooyeh B, et al. Daily intake of vitamin D- or calcium-vitamin D-fortified Persian yogurt drink (doogh) attenuates diabetes-induced oxidative stress: evidence for antioxidative properties of vitamin D.J Hum Nutr Diet. 2013 Jul 5. doi: 10.1111/jhn.12142. Nikooyeh B, 2014

Asemi Z, et al. Vitamin D supplementation affects serum high-sensitivity C-reactive protein, insulin resistance, and biomarkers of oxidative stress in pregnant women. J Nutr. 2013 Sep;143(9):1432-8. doi: 10.3945/jn.113.177550. Asemi Z, 2013

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

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

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

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

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

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

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

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

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

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

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

70% of American toddlers do not take the Ameri

On 2014 Jan 03, Ian Lyons commented:

In this paper, Park and Brannon showed that training adult subjects on an approximate, nonsymbolic arithmetic task (adding and subtracting estimates of the number of dots in various dot arrays) led to improvement on a symbolic arithmetic task (i.e., using Indo-Arabic numerals). The authors suggest this result points to a causal role for the ‘approximate number system’ (ANS) in more complex, symbolic math processing and may thus inform the development of interventions designed to improve mathematical competence in children and adults. We believe the authors’ work takes an important step forward in terms of understanding the building blocks of mathematical performance, and, using their work as a jumping board, we offer several points of reflection concerning (1) the nature of the ANS, and (2) what it means to train performance on a task versus the process it is meant to measure.

Park and Brannon’s crucial experimental condition trained participants using approximate, nonsymbolic addition. This differs from tasks used more commonly in the literature to measure individual differences in the ANS – adaptation and comparison – which involve simply distinguishing between two approximate quantities (e.g., in comparison tasks, one typically decides which of two arrays contains more dots). The difference in tasks is significant because previous attempts to train participants on just a nonsymbolic comparison task failed to show significant improvement in individuals’ symbolic math performance [Wilson et al., 2006 (http://www.ncbi.nlm.nih.gov/pubmed/16734906); DeWind & Brannon, 2012 (http://www.ncbi.nlm.nih.gov/pubmed/22529786)]. Why, then, does training on nonsymbolic arithmetic lead to improvement in symbolic arithmetic skills, but training on nonsymbolic comparison does not, even though both have been shown to correlate with symbolic arithmetic [e.g., Gilmore et al., 2010 (http://www.ncbi.nlm.nih.gov/pubmed/20347435); Halberda et al., 2012 (http://www.ncbi.nlm.nih.gov/pubmed/22733748)]? One possible conclusion is that it is not enough simply to tap the ANS; instead, accessing the ANS must be structured in a manner that more directly parallels the target skill – symbolic arithmetic. From a broader perspective, such a conclusion suggests that it is time to take a deeper look at what exactly we mean by an ‘approximate number system’, as Park and Brannon’s results may in fact point to an important division between approximate quantity representation and manipulation within the ANS. The view that the ANS is not a unitary construct is also leant support by the fact that performance on nonsymbolic quantity comparison and nonsymbolic arithmetic tasks are uncorrelated [Gilmore et al., 2011 (http://www.ncbi.nlm.nih.gov/pubmed/21846265)].

That nonsymbolic arithmetic (but not nonsymbolic comparison) training leads to improved symbolic arithmetic brings us to a second point: It is crucial to make a distinction between a task meant to measure or be an index of some underlying process, and the process itself. To cure a fever, one does not build a more precise thermometer; and by extension, if one demonstrated that using a more precise thermometer indeed failed to reduce one’s fever, it would be rather rash to conclude ambient bodily temperature is irrelevant to one’s health. A nonsymbolic number comparison task may act like a thermometer, where the underlying process it indexes is ANS acuity. Training on nonsymbolic comparison tasks does not improve math skills (Wilson et al., 2006; DeWind & Brannon, 2012), but this does not mean that the ANS is irrelevant for math. By training on nonsymbolic arithmetic instead of nonsymbolic comparison, Park and Brannon showed that one’s training regimen simply needs to tap the ANS in a way that better parallels the types of cognitive operations used in symbolic arithmetic.

One sees a similar distinction between tasks that index versus train an underlying process elsewhere in the numerical domain: when a person is asked to mark the location of a number on a number line, the linearity of their estimates predicts math achievement [Booth & Siegler, 2006 (http://www.ncbi.nlm.nih.gov/pubmed/16420128), 2008 (http://www.ncbi.nlm.nih.gov/pubmed/18717904)]; but rather than train on this task per se, researchers found success using a board game that trained children to linearize their visuo-spatial representations of symbolic numbers – i.e., the underlying process that was presumably being measured by the number line task. Training on the board game improved performance on both the numberline task as well as math achievement [Siegler & Ramani, 2009 (http://psycnet.apa.org/journals/edu/101/3/545/)].

Further, we believe that Park and Brannon’s own dataset provides yet another example illustrating the distinction between a task meant to measure or be an index of some underlying process, and the process itself. The authors show a correlation between numerical ordering ability and symbolic math ability, replicating our previous work [Lyons & Beilock, 2011 (http://www.ncbi.nlm.nih.gov/pubmed/21855058)]. Nevertheless, training on the ordering task did not lead to improvement in symbolic math beyond what was seen for vocabulary training. If one concludes from this result that understanding ordinality is irrelevant for developing math skills, one is in danger of mistaking a means of measurement for the thing being measured – much as one might have done with the dot comparison or number-line tasks discussed above.

In conclusion, Park and Brannon’s recent paper showing a causal relation between nonsymbolic and symbolic arithmetic, represents a step toward understanding the building blocks of complex arithmetic. Perhaps missed in the excitement, though, is that this work underscores the need for researchers – especially those interested in educational applications – to carefully consider what their tasks and paradigms truly mean with respect to the processes and representations they aim to investigate. Failing to do so risks conflating the means of measurement with what is being measured, and may in turn lead to recommendations for educators to train the wrong thing.

Signed, Ian Lyons and Sian Beilock

On 2017 Oct 10, L David Sibley commented:

We appreciate the posting of the comment by Dr. Meissner and also the primary data provided by the Kursula group (Kumpula et al., Kumpula EP, 2017). Although we agree that there are differences in the conclusions, we feel that these stem primarily from differences in methodology and biological substrates. As such, it would be premature from this new study to make sweeping statements about the polymerization behavior “apicomplexan” actins.

First, we note that our study used Toxoplasma gondii actin (TgACT1) Skillman KM, 2013, while Kursula group studied Plasmodium actin, PfACT1 more specifically. We agree that the two actins are somewhat similar, and so it would be surprising if they had an intrinsically different mechanism of polymerization. However, our previous studies have noted differences between these substrates and found that polymerization of PfACT1 is more efficient than that of TgACT1 as shown by light scattering and microscopy Skillman KM, 2011.

Second, Kumpula et al., suggest that sedimentation may not be a reliable method for monitoring polymerization kinetics of apicomplexan actins due to the formation of small oliogmeric species that do not fully sediment at 100,000g or even 450,000g. We are well aware of this limitation, and indeed it is also shown in our paper by several methods Gershon D, 1990. We considered the formation of heterogenous small oligomers in the experiments we performed, including in the simulations and determination of rate constants. Importantly all of our findings show that a simple isodesmic model can fit the data for assembly and turnover. These findings further predict that if there is a cooperative component, it is exceedingly small relative to conventional actins. Moreover, our conclusion that TgACT1 polymerizes by an isodesmic mechanism was not based solely on sedimentation but was supported by light scattering assays, which demonstrate a lack of a lag period associated with a normal critical concentration (Cc) Skillman KM, 2013.

Third, Kumpula et al., used a modified pyrene assay to monitor the “kinetics’ of filament formation. We agree that the pyrene assay is normally well suited for studying kinetics, but may not be ideal for monitoring thermodynamics associated with the intrinsic polymerization mechanism. Kumpula et al., report that at low concentrations of PfACT they detect evidence for a Cc, based on plotting the fluorescence signal vs. actin concentration. However, under these conditions where pyrene labeling was done at sub-stochiometric levels, the lack of a signal below a particular concentration may be due to lack of sensitivity, rather than an actual Cc. It would be important to determine whether such a Cc could also be detected by intrinsic tryptophan quenching, which may be more sensitive. We would also like to point out that unlike these two assays that attempt to monitor the Cc at very low concentration of protein, the estimate of Cc from sedimentation assays is based on behavior across a wide range of concentrations and thus it is more robust to small fluctuations, variability in protein levels, or assay differences.

Finally, we note than both our study and that of Kumpula used recombinant actin produced in insect cells. Hence, the previous suggestion that apicomplexan actins cannot be functionally expressed heterologously Olshina MA, 2016 is incorrect and not the reason for the differences described in polymerization behavior. Rather, it is clear that recombinant actins produced in an appropriate manner exhibit many interesting functional properties. Further studies may reveal to what extent these features are shared vs. unique among divergent actins.

On 2017 Sep 23, Markus Meissner commented:

A recent report from the Kursula group demonstrated that the sedimentation assays used in Skillmann et al., 2013 are unlikely to obtain reliable results for apicomplexan actins. In this study the Kursula group (see: https://www.nature.com/articles/s41598-017-11330-w) used a protocol based on pyrene labelling and demonstrated that polymerisation of apicomplexan (Plasmodium) actin is occurring in a cooperative manner, meaning it requires a critical concentration. Surprisingly, the Cc is very similar to canonical rabbit actin. Furthermore, it was shown that shorter filament lengths result from higher depolymerisation rate. In conclusion, it appears that -like all other known eukaryotic actins- apicomplexan actin polymerises in a cooperative manner, requiring a nucleation reaction (see Kumpula et al., 2017).

On 2016 Jul 06, David C. Norris commented:

In addition to the images above, an animation cited in the article may also be of interest: http://hemonc.org/docs/CMLhistory.avi

On 2013 Nov 18, vaigundaragavendran jegadeesan commented:

The review provides us with an in-depth overview of the multifarious mechanisms that underpin the pathophysiology of PCIBP. In particular, the pictorial representations are very informative and explain the critical steps more clearly. A nice read to concatenate PCIBP mechanisms to those underlying various other pain conditions to eventually form a string of plausible therapeutic approaches.

Highly recommended.

On 2013 Oct 28, John Cannell commented:

Didn't Finland start supplementing their children with vitamin D in 2003? I think increased vitamin D levels account for the reduction in type 1 diabetes in Finland.

On 2016 Apr 12, Oliver Kuss commented:

This is a clearly written paper on proportional hazards model for interval-censored data. I especially liked the authors sharing their SAS code for the piecewise constant model with which I was able to recalculate the results for the breast cancer data set as given by Lindsey/Ryan.

However, there are also two things that concern me a bit:

(1) The SAS code seems to be a only slightly shortened version of the %recurr macro of Lu/Liu (2008). It would have been fair by Gong/Fang to point to this source.

(2) The estimated hazard ratios for the breast cancer data set (table 6) do not coincide with the previous literature. For example, Lindsey/Ryan report 0.930 (0.287) as the hazard ratio from the piecewise constant model and I found the same results. However, the authors report 0.392 ...

Lindsey JC, Ryan LM. Tutorial in biostatistics methods for interval-censored data. Stat Med. 1998 Jan 30;17(2):219-38.

Lu L, Liu C. Analysis of Correlated Recurrent and Terminal Events Data in SAS®. www.lexjansen.com/nesug/nesug08/sa/sa16.pdf

On 2014 Nov 17, Raphael Levy commented:

I discuss the interpretation of the energy profiles shown in this paper (fig 4C) on my blog. I compare with two other articles addressing the same problem: Li Y, 2012 and Gkeka et al. One of the author of Gkeka et al, Lev Sarkisov (Edinburgh), left a detailed and helpful comment on the blog.

Beyond the theoretical work, detailed analysis of the stripy nanoparticle experimental evidence is published today in PloS One by Stirling et al; from the abstract: “through a combination of an exhaustive re-analysis of the original data with new experimental measurements of a simple control sample comprising entirely unfunctionalised particles, we conclusively show that all of the STM evidence for striped nanoparticles published to date can instead be explained by a combination of well-known instrumental artefacts, strong observer bias, and/or improper data acquisition/analysis protocols.”

On 2013 Nov 16, M Mangan commented:

Oh, sure, now they changed the title so nobody will understand the issue. The original title was "Environmental stress and transposon transcription in the mammalian brian."

On 2013 Oct 31, M Mangan commented:

So does this affect only Brians? All Brians?

On 2013 Dec 09, John Cannell commented:

The authors report that immune system dysregulation is common in autism spectrum disorder (ASD). They discuss a number of nutritional relationships to the immune system. If it is dietary, the question is why does this immune dysregulation occur and what has caused such dysregulation to skyrocket in recent decades?

Vitamin D deficiency produces very similar immune dysregulation to what the authors reported.

Prietl B, 2013

Kamen DL, 2010

Yang CY, 2013

Baeke F, 2010

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

Gong ZL, 2014

Meguid NA, 2010

Mostafa GA, 2012

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

DeLuca GC, 2013

Eyles DW, 2013

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

Cannell JJ, 2010

Cannell JJ, 2008

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

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

Kočovská E, 2012

As the authors point out, immune dysregulation is common in ASD. The question is why now and what is causing it?

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2013 Oct 29, John Cannell commented:

I congratulate the authors on an important work but it shows how little communication is occurring among autism scientists. Each scientist seems to be immersed in his or her own research interest but oblivious to the larger body of autism research.

The vitamin D theory of autism (vitamin D deficiency is the environmental risk factor for this highly heritable disorder) is entirely consistent with the authors work, as they briefly discuss in their paper. However, vitamin D's effect on the immune system is pervasive and robust and deserves much more attention as the factor implicated in both autism and immunity.

Schwalfenberg GK A review of the critical role of vitamin D in the functioning of the immune system and the clinical implications of vitamin D deficiency.Mol Nutr Food Res. 2011 Jan;55(1):96-108. doi: 10.1002/mnfr.201000174. Epub 2010 Sep 7. Review. Schwalfenberg GK, 2011

Three recent studies, using community controls, have found 25(OH)D levels are significantly lower in children with autism. Two of the studies below (Mostafa et al and Gong et al) also found autism severity, as rated on standard autism rating scales, is inversely correlated with 25(OH)D levels. Mostafa et al found an R value of -.86 for the association of serum 25(OH)D with autism severity.

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

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

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

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

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

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

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

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

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

70% of American toddlers do not take the American Pediatric Association's recommended vitamin D supplement of 400 IU/day.

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

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

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

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

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2014 Jul 19, Chris Carter commented:

HSV-1/host interactome

On 2013 Nov 04, AARON HSUEH commented:

This paper neglected earlier work. For example: Extrapituitary action of gonadotropin-releasing hormone: direct inhibition ovarian steroidogenesis. Hsueh AJ, Erickson GF. Science. 1979 May 25;204(4395):854-5.

Gonadotropin-releasing hormone analogue binds to luteal cells and inhibits progesterone production. Clayton RN, Harwood JP, Catt KJ. Nature. 1979 Nov 1;282(5734):90-2.

Extrapituitary actions of gonadotropin-releasing hormone. Hsueh AJ, Jones PB. Endocr Rev. 1981 Fall;2(4):437-61. Review.

On 2014 Jan 07, Brett Snodgrass commented:

Dear Author,

Thank you for the excellent report. Please provide your kind attention to the difference between the vessels of Wearn and Thebesius. Approximately fifty percent of the medical literature applies the term "Thebesian veins" to the "vessels of Wearn."

Arteriosinusoidal vessels described by Wearn connect to myocardial sinusoids. The myocardial sinusoids are often pathologically altered and readily apparent on microscopy in pulmonary atresia with intact ventricular septum.

Dr. Wearn's work work detailing the difference between the Thebesian veins and the vessels of Wearn (which he referred to as arterioluminal vessels). http://bit.ly/JTWearn

The myocardial sinusoids are not phantom as they connect to the arteriosinusoidal vessels. The vessels of Wearn include the arteriosinusoidal and the arterioluminal vessels of the heart. Wearn's distinguished Harvey lecture of 1940 again reported the difference between the "Thebesian veins" and the "AL & AS" vessels (vessels of Wearn). However, this was soon obscured in the medical literature. My hypothesis is that with a lack of a pronoun such as the "vessels of Wearn," the vessels were described as Thebesian veins. Related references: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933738/ http://www.ncbi.nlm.nih.gov/pubmed/22704295 http://www.ncbi.nlm.nih.gov/pubmed/23332812 https://twitter.com/BrettSnodgrass1/status/419324443068874752

I appreciate comments, suggestions, and constructive criticism. Please feel free to E-mail me: brettsnodgrass@hotmail.com Alternatively, you may message me through Twitter https://twitter.com/BrettSnodgrass1

Thank you kindly.

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 NCT000004592. We believe the correct ID, which we have found by hand searching, is UMIN000004592.

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 2017 May 22, Christine Carson commented:

Full text of this Letter available in Swedish at: http://www.lakartidningen.se/07engine.php?articleId=18503

Full text of this Letter available in English from ResearchGate at: https://www.researchgate.net/profile/Christine_Carson/publication/236031981_Biverkningarnaar_overdrivna_Swedish_Tea_tree_oil_-_adverse_events_exaggerated/links/02e7e51cafbeed6002000000/Biverkningarnaaer-oeverdrivna-Swedish-Tea-tree-oil-adverse-events-exaggerated.pdf

On 2013 Oct 25, Gregory Francis commented:

I fear the science in this study does not back up the conclusions. An important part of the scientific process is to check whether the statistical data are consistent with the hypothesized theory. One way to do this is to compute the statistical power of the experiments. This analysis supposes that an experiment accurately measured the reported effect and then computes the probability that a randomly selected sample of data would satisfy the statistical criteria used to justify the scientific claims. For this study, these estimated probabilities are 0.56, 0.52, 0.48, and 0.57. That the values are close to one-half reflects the fact that the data were typically just to one side of the statistical criterion for finding an effect. Due to natural variation, data from some samples should fall on the other side of the criterion.

The power analysis suggests that, at best, the odds of showing the effect for each experiment is almost the equivalent of a coin flip. As such, the repeated success of the reported experiments is rather unbelievable (multiplying the power values gives 0.08). Scientists should doubt the veracity of the reported experiments; they are too good to be true.

A Excel file that computes the effect sizes used in the power analysis can be found at http://www1.psych.purdue.edu/~gfrancis/Publications/VohReddenRahinel2013.xls

On 2013 Nov 18, Gary Ward commented:

This paper describes a clever high-throughput assay to identify small molecules that disrupt the interaction of Plasmodium falciparum AMA1 and RON2, and can thereby block merozoite invasion of erythrocytes. The data provide promising proof-of-concept for the development of novel antimalarials that disrupt protein-protein interactions critical for invasion. This particular interaction is thought to happen extracellularly, within the blood, which could facilitate access of such drugs to their targets.

The three inhibitors described have IC50 values in the 20-30 uM range. The authors state that "small-molecule inhibitors will result in reduced or no emission signal depending on the strength of the inhibition". Our group was interested to know what the dynamic range of the AlphaScreen assay is and whether it can capture binding at both ends of the affinity spectrum (subnanomolar to millimolar).

A 1000-fold molar excess of inhibitor was required to disrupt RON2L-AMA1 interaction in the screen, perhaps because the compound is added after RON2L-AMA1 complex formation and must essentially displace the RON2L from AMA1. The assay may have been done this way purposely, to recapitulate what happens in the blood, i.e., secreted RON2 is probably not exposed on the surface of the red cell for long before it is bound by AMA1. It would nonetheless be interesting to know what happens to the IC50 if compound is added to AMA1 before the addition of RON2L.

Posted by Gary Ward on behalf of the University of Vermont Toxoplasma Journal Club (UVM ToxoJC); members include Jenna Foderaro, Anne Kelson, Shruthi Krishnamurthy, Jacqueline Leung, Pramod Rompikuntal, Luke Tilley & Gary Ward

On date unavailable, commented:

None

On 2014 Mar 22, Andrew R Kniss commented:

There are now two published criticisms of this work in New Phytologist, the journal that originally published the article.

• Letter from Gressel et al: http://onlinelibrary.wiley.com/enhanced/doi/10.1111/nph.12615/
• + author response to Gressel et al. http://onlinelibrary.wiley.com/enhanced/doi/10.1111/nph.12684/
• letter from Grunewald & Bury: http://onlinelibrary.wiley.com/enhanced/doi/10.1111/nph.12683/
• + author response to Grunewald & Bury: http://onlinelibrary.wiley.com/enhanced/doi/10.1111/nph.12748/

On 2013 Oct 23, Andrew R Kniss commented:

This is a very interesting paper, and it recieved wide media coverage after publication. There are some rather large oversights/deficiencies that should be noted. I have posted a rather long critique of this paper at my blog. There are several problems with the paper, all of which are described in detail at the link. In a nutshell:

• The breeding methods used did not ensure enough genetic uniformity between GE and non-GE hybrids to attribute differences to the transgene as they claim.
• The authors provided very little information about the the original transformation event, and how the transformed line differed from its parent line (even after presenting data suggesting the lines had different phenotypes).
• The authors do not adequately discuss other possibilities that may explain their results, particularly with respect to the promoter they used and the impact of closely linked genes.
• Closely related traits (tiller production, panicle production, seed production) are presented as independent evidence of an increase in fitness.
• None of the experiments presented in this study were repeated.

On 2013 Aug 10, Allison Stelling commented:

I find it a bit odd that the authors call for a national discussion on a very important topic in a journal that has put their words behind a paywall.

Biomedical science has a huge public impact, and the public needs to be aware how little we know about how to diagnose many diseases; let alone our lack of information on "cures". They are footing the majority of the bill and have a fundamental right to have the science explained to them. This is not a discussion that the researchers and "qualified experts" can keep within their own limited community; nor is it a one way lecture where interaction with the public is unidirectional. It's a conversation that needs to happen between medical doctors, the public taxpayers, and the scientific researchers.

On 2016 Aug 23, Ben Goldacre commented:

This trial has the wrong trial registry ID associated with it on PubMed: both in the XML on PubMed, and in the body of the text of the article. The ID given is NCT0005937. We believe the correct ID, which we have found by hand searching, is NCT00059371.

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

Many thanks,

Jessica Fleminger, Ben Goldacre*

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

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

On 2014 Sep 21, Annie De Groot MD commented:

I wonder if pathogens use this pathway to escape immune response? See our article at http://bit.ly/NTD_iVAX_2014

On 2014 Jan 31, Huiqi Qu commented:

This method is helpful to understand functional effects of some genetic variants.

On 2014 Apr 21, Morgan Price commented:

The annotation of DpigGOR10741-DpigGOR10744, which are important for hydrogen utilization and for growth in vivo by Desulfovibrio piger, as "hmc" is a bit misleading. This gene cluster is very similar to nhc (nine-heme cytochrome complex) of D. desulfuricans 27774 (Saraiva LM, 2001). While Hmc has subunits hmcABCDEF including a 16-heme periplasmic subunit (hmcA), nhc lacks the hmcE and hmcF genes, and the hmcA-like subunit is shorter and has nine hemes. Similarly, D. piger lacks hmcEF and has a shorter periplasmic subunit that is very similar to the nine-heme cytochrome (tree browser view).

On date unavailable, commented:

None

On 2017 Apr 23, Barbara L Fredrickson commented:

The following links provide the authors' responses:

• Cole SW, 2014
• Fredrickson BL, 2015
• Fredrickson BL, 2016

On 2017 Apr 11, Nicholas J L Brown commented:

The following articles comment directly on this article:

Brown et al., 2014: https://www.ncbi.nlm.nih.gov/pubmed/25157145 Walker, 2016: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068350/ Nickerson, 2017: http://www.collabra.org/article/10.1525/collabra.81/

On 2014 Dec 05, Gaetano Santulli commented:

Jiang and colleagues report that beta2-adrenergic receptor (B2AR) knockout (KO) mice exhibit a diabetic retinopathy phenotype. We recently demonstrated that B2AR KO mice display a progressive impairment in insulin release that leads to glucose intolerance (1). Our report, which seems to be mechanistically important in the pathophysiology of diabetic rethinopathy (if an animal is glucose intolerant a diabetic retinophaty phenotype appears to be more than obvious) is completely ignored by the Authors. A functional role for B2AR in the regulation of insulin secretion and thereby in the pathogenesis of diabetes had been suggested by the evidence of a decreased number of B2AR in type I diabetes patients (2, 3). Findings from other groups support such a mechanism (4-7). In addition, human polymorphisms in the B2AR gene have been associated with obesity and other metabolic disorders (8, 9). Moreover, the Authors state that B2AR KO mice exhibit attributes of retinal changes common in diabetes, despite normal glucose levels. However, they do not provide any experiment to show glucose (or insulin) levels, nor in basal conditions, nor after a challenge, nor during a clamp assay. Similarly, the Authors also state that in their previous studies they have shown that B1AR KO mice exhibit retinal changes similar to diabetic animals in spite of normal glucose levels, quoting a paper in which there is no experiment showing the claimed normal glucose levels, as well.

We believe that the Readers will appreciate a clarification on these studies by the Authors and the Editors.

Gaetano Santulli MD, PhD 1,2, and Guido Iaccarino MD, PhD 3,4

From the Departments of 1Translational Medical Sciences and 2Advanced Biomedical Sciences, Federico II University, Naples Italy; 3Department of Medicine and Surgery, University of Salerno, Salerno, Italy; 4Multimedica Research Hospital, Milan, Italy.

Essential References 1. Santulli G, Lombardi A, Sorriento D, Anastasio A, Del Giudice C, Formisano P, et al. Age-related impairment in insulin release: the essential role of beta(2)-adrenergic receptor. Diabetes. 2012;61(3):692-701. doi: 10.2337/db11-1027. PubMed PMID: 22315324; PubMed Central PMCID: PMC3282797; http://www.ncbi.nlm.nih.gov/pubmed/22315324

1. Schwab KO, Bartels H, Martin C, Leichtenschlag EM. Decreased beta 2-adrenoceptor density and decreased isoproterenol induced c-AMP increase in juvenile type I diabetes mellitus: an additional cause of severe hypoglycaemia in childhood diabetes? European journal of pediatrics. 1993;152(10):797-801. http://www.ncbi.nlm.nih.gov/pubmed/8223779. PubMed PMID: 8223779; http://www.ncbi.nlm.nih.gov/pubmed/8223779.
2. Noji T, Tashiro M, Yagi H, Nagashima K, Suzuki S, Kuroume T. Adaptive regulation of beta-adrenergic receptors in children with insulin dependent diabetes mellitus. Hormone and metabolic research. 1986;18(9):604-6. Epub 1986/09/01. doi: 10.1055/s-2007-1012385. PubMed PMID: 3023224; http://www.ncbi.nlm.nih.gov/pubmed/3023224.
3. Panagiotidis G, Stenstrom A, Lundquist I. Influence of beta 2-adrenoceptor stimulation and glucose on islet monoamine oxidase activity and insulin secretory response in the mouse. Pancreas. 1993;8(3):368-74. Epub 1993/05/01. http://www.ncbi.nlm.nih.gov/pubmed/8387193. PubMed PMID: 8387193; http://www.ncbi.nlm.nih.gov/pubmed/8387193.
4. Loubatieres A, Mariani MM, Sorel G, Savi L. The action of beta-adrenergic blocking and stimulating agents on insulin secretion. Characterization of the type of beta receptor. Diabetologia. 1971;7(3):127-32. http://www.ncbi.nlm.nih.gov/pubmed/4397807. PubMed PMID: 4397807; http://www.ncbi.nlm.nih.gov/pubmed/4397807.
5. Ahren B, Jarhult J, Lundquist I. Insulin secretion induced by glucose and by stimulation of beta 2 -adrenoceptors in the rat. Different sensitivity to somatostatin. Acta physiologica Scandinavica. 1981;112(4):421-6. http://www.ncbi.nlm.nih.gov/pubmed/6119001. PubMed PMID: 6119001; http://www.ncbi.nlm.nih.gov/pubmed/6119001.
6. Asensio C, Jimenez M, Kuhne F, Rohner-Jeanrenaud F, Muzzin P. The lack of beta-adrenoceptors results in enhanced insulin sensitivity in mice exhibiting increased adiposity and glucose intolerance. Diabetes. 2005;54(12):3490-5. Epub 2005/11/25. doi: 54/12/3490 [pii]. PubMed PMID: 16306366; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16306366.
7. Large V, Hellstrom L, Reynisdottir S, Lonnqvist F, Eriksson P, Lannfelt L, et al. Human beta-2 adrenoceptor gene polymorphisms are highly frequent in obesity and associate with altered adipocyte beta-2 adrenoceptor function. The Journal of clinical investigation. 1997;100(12):3005-13. Epub 1998/01/31. doi: 10.1172/JCI119854. PubMed PMID: 9399946; PubMed Central PMCID: PMC508512; http://www.ncbi.nlm.nih.gov/pubmed/9399946.
8. Thomsen M, Dahl M, Tybjaerg-Hansen A, Nordestgaard BG. beta2-adrenergic receptor Thr164Ile polymorphism, obesity, and diabetes: comparison with FTO, MC4R, and TMEM18 polymorphisms in more than 64,000 individuals. The Journal of clinical endocrinology and metabolism. 2012;97(6):E1074-9. doi: 10.1210/jc.2011-3282. PubMed PMID: 22466342; http://www.ncbi.nlm.nih.gov/pubmed/22466342

On 2014 Sep 24, Michael Schroda commented:

Excellent study, a big step forward toward the understanding of Hsp70 function in chloroplasts.

On 2017 Oct 31, Morten Oksvold commented:

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

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

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

On 2013 Oct 31, John Cannell commented:

The authors stated that markers of oxidative stress are present in autism spectrum disorder (ASD). I wonder if the authors are aware that the genes for the antioxidants superoxide dismutase and thioredoxin reductase are directly up-regulated by the secosteroid 1,25 di-hydroxy vitamin D3 (calcitriol). I believe both genes also harbor a vitamin D response element.

Peehl DM, Shinghal R, Nonn L, Seto E, Krishnan AV, Brooks JD, Feldman D. Molecular activity of 1,25‐dihydroxyvitamin D3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis. J. Steroid Biochem Mol. Biol 2004;92:131–141. PMID:15555907>

Calcitriol also directly up-regulates glutathione reductase and increases glutathione levels.

Jain SK, et alo. Vitamin D upregulates glutamate cysteine ligase and glutathione reductase, and GSH formation, and decreases ROS and MCP-1 and IL-8 secretion in high-glucose exposed U937 monocytes. Biochem Biophys Res Commun. 2013 Jul 19;437(1):7-11. doi: 10.1016/j.bbrc.2013.06.004. Jain SK, 2013

Also, supplemental vitamin D significantly reduces oxidative stress in humans.

Nikooyeh B, et al. Daily intake of vitamin D- or calcium-vitamin D-fortified Persian yogurt drink (doogh) attenuates diabetes-induced oxidative stress: evidence for antioxidative properties of vitamin D.J Hum Nutr Diet. 2013 Jul 5. doi: 10.1111/jhn.12142. Nikooyeh B, 2014

Asemi Z, et al. Vitamin D supplementation affects serum high-sensitivity C-reactive protein, insulin resistance, and biomarkers of oxidative stress in pregnant women. J Nutr. 2013 Sep;143(9):1432-8. doi: 10.3945/jn.113.177550. Asemi Z, 2013

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

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

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

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

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

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

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

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

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

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

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

70% of American toddlers do not take

On 2014 Jan 29, Amanda Capes-Davis commented:

Please be aware that KB is NOT an oral epithelial cell line. The cell line is known to be cross-contaminated with HeLa and so comes from cervical carcinoma. For a list of known cross-contaminated or misidentified cell lines, see http://iclac.org/databases/cross-contaminations/.

On 2016 Nov 15, Yuwei Fan commented:

Thermocycled for only 500 cycles seems to be insufficient to test aging effects.

On 2017 Jan 13, Yuwei Fan commented:

The description of CIELab a* and b* range in page 215 is incorrect. The range is ±100 or −128 to +127 (signed 8-bit integer). [https://en.wikipedia.org/wiki/Lab_color_space]. Delta-E equation in same page is wrong (or maybe a typo) apparently.

On 2017 Jan 11, Misha Koksharov commented:

It would be cool to also have data on evolution of decapod luciferases (Oplophorus, Systellaspis, etc). Unfortunately, only one is known so far. :( Although, there are plenty of them out there in the sea: http://dx.doi.org/10.1007/BF00347123 http://dx.doi.org/10.1016/j.ympev.2014.11.013

On 2013 Dec 16, Tarek Tawfik Amin commented:

This paper addresses a common problem in the developing countries, similar initiatives should be carried in other countries to make use of the available epidemiological data for the sake of comparison and trend exploration. i think the authors have used multiple data sources which strengthen their findings. best wishes and lots of respects.

On 2013 Dec 14, Mohsen Rostami commented:

I just read this paper and found it interesting! I think it will be highly cited by those who work in the related field! Reagrds

On 2013 Aug 08, Winston Timp commented:

Interesting paper highlighting a pathway which could grant predisposition to allergy. Perhaps more subtle effects on TGF-B signaling pathway (than mutation, e.g. epigenetic change) could lead to allergy in genotypically normal individuals.

On 2014 Mar 14, Jonathan Eisen commented:

The first author of this paper, Rachel Adams, wrote a post about the "story behind the paper" for the "microbiology of the Built Environment network" (microBEnet) blog. See How biomass can bias high-throughput surveys of microbial communities

On 2016 May 04, Lydia Maniatis commented:

The purpose of the issue of Visual Neuroscience in which this article appeared was to honor the achievements of Davida Teller and also to revisit her critical analysis of the casual use of 'linking propositions.' From the volume intro: “Davida provided critical thinking about criteria for, and approaches to, drawing inferences linking psychophysical phenomena and perception with underlying neural mechanisms (Teller, 1980, 1984; Teller and Pugh, 1983)....It is surprisingly routine today to see claims being made regarding the neural underpinnings of sensory and cognitive phenomena without articulation or even acknowledgment of implicit linking assumptions. With this special issue, Linking Hypotheses in Visual Neuroscience, we seek to...revisit this central challenge in visual neuroscience....”

Unfortunately, Maertens & Shapely's (2013) never make explicit their implicit linking propositions “Linking appearance to neural activity....” When analyzed, these assumptions contain all of the serious problems, including lack of face validity, flagged by Teller (1984). Additional problems include the vagueness of their use of the term “naturalistic,” which is oddly combined with the narrow tailoring of their conclusions to the specific and highly artificial stimuli employed in this study.

Below, I discuss the claims and offer critiques of Maertens & Shapley (2013):

Maertens & Shapely (2013): “The dependence of lightness perception on adjacent and nearby checks could be interpreted as a support for the idea that the neural computations of lightness are strongly influenced by local computations in the primary visual cortex, V1. Several previous studies have shown that responses to visual patterns in V1 resemble lightness perception qualitatively and quantitatively (Kinoshita & Komatsu, 2001; MacEvoy & Paradiso, 2001; Haynes et al., 2004; Paradiso et al., 2006).”

My response: The idea that a particular stimulus situation (“in the contexts we used...with the checkerboard displays”) specifically activates a particular [and fairly peripheral – see below] set of neurons which are then held to be directly responsible for the percept is untenable. How does the visual system decide, a priori, that this or that stimulus will be processed only by these particular neural populations, up to the point of consciousness, and what happens (with respect to perception) to that part of the process when it is supplemented by others? Here is what Teller (1984) had to say about this type of supposition:

First, she refers to the notion that “if psychophysical and physiological data can be manipulated in such a way that they can be plotted on meaningfully similar axes, such that the two graphs have similar shapes, then that physiological phenomenon is a major causal factor in producing that psychophysical phenomenon.” One of the problems with such a linking proposition, accordint to Teller, is “its peripherality: it states a causal or explanatory relationship between the activity of single cells at a peripheral level of the nervous system and a perceptual or behavioral phenomenon, without any accompanying proposal as to how this pattern maintains itself through the system. The proposition includes an implicit appeal to the “nothing mucks it up” proviso (Teller, 1980)....In the absence of any explicit treatment of these problems, [such proposals] would seem to amount to nothing more than a remote homunculus theory: the...stimulus sets up the pattern of activity...that “looks like” [the stimulus] and the homunculus peers down [at it].” Area 17, or V1, is referred to by Teller (1984) as a “relatively peripheral stage of neural processing. In that case, if the properties of Area 17 cells are to be used to explain the elements of our perceptions, a “nothing mucks it up” theory is needed to bridge the gap between the Area 17 cell and the still more central sites...”

Maertens and Shapely (2013) double down on this unviable notion: “Such an interpretation of our data does not preclude that there are significant influences of neural computations beyond V1. There are many phenomena of lightness perception that probably require an explanation in terms of longer distance interactions and higher-order interactions (reviewed in Gilchrist, 2006; Kingdom, 2011). However, in the contexts we used in our experiments, and with the checkerboard displays used (Fig. 1), the nearest-neighbor and next-nearest-neighbor interactions were sufficient to account for most [not all] of the lightness variation (Tables 2 and 3).”

I would add that, given that a small local change in a part of the visual field can alter the structure – including lightness effects – in the entire field, the idea that some stimuli activate only peripheral systems and local interactions and others more global ones is, if possible, even less plausible.

Even in the context of their experiments, the authors walk back their claims about the influence of local contrast, and, again, suggest that different neural populations underlie the perception of different stimuli:

“The data on lightness matches for the transparent condition suggest that more complicated neural computations were employed by the observers when they judged lightness through transparency.” First, I would note that there was no physical transparency in the stimuli – they were all computer generated on a single screen. Second, one must ask, again, at what stage, and why, does the visual system decide to shunt one computer-generated black and white stimulus to be addressed by one peripheral neural process, leading directly to the analogous percept, and another to a different, “more complicated” neural process? Are the activities of V1 supposed to be lost or ignored or altered in some cases, prioritized for conscious consumption in others, and conversely, the effects of higher-level populations shut down, altered, in some and not in others? The decision as to what process to prioritize for what stimulus would necessarily seem to be a high-level decision, because the low-level would not have the “authority” to decide not to send the issue upstairs.

In the end, the authors do nothing but go to a lot of trouble quantify well-known effects in the context of very specific stimuli, from which they draw conclusions that are purely ad hoc, strictly limited to these stimuli, and then construct an ad hoc and casual neural account lacking face validity. They furthermore fail to acknowledge the well-known effects of structure on lightness; by using checkerboards they avoid even basic figure-ground effects. This is a methodological choice which should have been explained and its consequences for drawing conclusions discussed.

The artificiality and of the stimuli makes it even more difficult to understand what the authors mean to imply by the term “naturalistic.” The assertion that stimuli contain a wide range of luminances obviously isn't enough. As mentioned above, the claim that they are in a position to make a generalization about “naturalistic” stimuli in general also conflicts with limiting of their claims to ““in the contexts we used...with the checkerboard displays...”

On 2014 Mar 06, David Keller commented:

An osteoporosis patient may express excessive anxiety about the risks of jaw osteonecrosis and atypical brittleness fractures ("porcelain bones"), rare harmful side-effects of prolonged bisphosphonate therapy, even though the patient may not yet be at risk. I agree that performing a DEXA scan every 2 to 3 years is probably excessive for medication-compliant patients with moderate risk of osteoporotic fracture, for whom such frequent scans are unlikely to result in a change in treatment. However, these scans can motivate a "change of treatment" if the patient has been surreptitiously skipping her bisphosphonate pills due to publicity about their potential side-effects, and she learns from her DEXA scan that her BMD has decreased as a result, placing her at even higher risk for a typical osteoporotic fracture.

To facilitate discussion, I respectfully request the person who found this comment "not helpful" to state their reason

On 2014 Dec 10, Egon Willighagen commented:

The supplementary figures and tables are also available from FigShare: http://figshare.com/articles/_NanoMiner_8212_Integrative_Human_Transcriptomics_Data_Resource_for_Nanoparticle_Research_/744959

On 2017 Mar 15, Michelle Fiander commented:

Reproducibility and transparent reporting are among the hallmarks of a methodologically sound systematic review. One aspect of reproducibility and sound reporting (per PRISMA) is the presentation of at least one database search strategy. In this review, one strategy (for OVID Medline) is reported and the authors say the strategy was written by an "expert librarian" whose initials are provided--but who is not an author on the review. I mention this because the strategy looks more like a work in progress than an "expert" or finished product.

There are quite a few problems with the Medline search strategy in terms of its logic, search terms, syntax and organization, and the outcome is that the strategy does not identify 19 of the 31 included studies. There are a number of reasons for this, the main one of which is the absence of a reasonable set of synonyms for the concept of 'electronic syndromic surveillance. It is always difficult to develop search strategies for concepts not represented by consistent keywords or controlled vocabulary (such as MeSH), but the goal of an expert searcher is to tease out the concept after becoming familiar with the subject matter by examining and reading few relevant discussions/studies and or checking definitions. This does not appear to have happened for this review,and it begs the question as to how the evidence base for this review was assembled, and if it was objective or biased.

On 2014 Feb 07, Paul Brookes commented:

A correction was recently issued for this paper... http://www.plosbiology.org/annotation/listThread.action?root=78137

The correction is a good start, but is deficient on several levels. I have outlined these in detail at a blog post on my lab' website (http://www.psblab.org/?p=268), but here's the short version...

1) The splicing issue raised WRT Figure 4A has not been addressed at all. 2) The phrase "inadvertently used the wrong blots" is inadequate, since in some cases it was not re-use of blots that was problematic, it was apparent re-use of single bands within blots. How exactly does one use the wrong band in a blot? 3) The correction images provided are askew - they appear to have been scanned in from paper copies and misaligned slightly during this process. There could be any number of reasons why files were not provided in a more direct manner (i.e. straight from the software they were generated in, without inserting a print-scan step). Regardless, the provision of apparent paper scans does nothing to enhance my confidence in these data. 4) The correction contains zero explanation for why it took 7 months to achieve.

I look forward to hearing from either the authors or the journal, about these remaining unresolved issues.

On 2013 Oct 22, Paul Brookes commented:

FYI, the follow up on this...

It has been known about on PubPeer for some time, as well as tweeted to the NYT author who wrote an article all about [http://well.blogs.nytimes.com/2013/07/17/exercise-in-a-pill-the-search-continues/]("Exercise in a Pill"), and blogged about on In the Pipeline.

The journal knows this, and was emailed several times over the past 3 months, with minimal responses ("we're still investigating" etc.) My most recent emails last week (13 weeks since the original comment was removed) have been left unanswered. This raises the question as to exactly how one goes about dealing with a paper that contains problematic data? These internal journal commenting systems and blogs and other publicity don't appear to have a very rapid impact on correcting the literature, so let's hope maybe PubMed Commons will be a bit faster?

Also, I apologize for a stupid mistake made very early on in using this PubMed Commons system - in between logging in via eRA commons and pasting my original comment across from PubPeer, I screwed up and tagged the wrong paper (albeit one with an almost identical title), then I got flustered when the comment was removed, when in fact it was just PubMedCommons staff doing their job! Put it down to a combination of me being stupid and caffeine deprived. Anyway, now the comment is in its right place, thanks to an eagle-eyed editor!

On 2013 Oct 22, Paul Brookes commented:

The following comment was left on the PLoS Biology site on July 18th, and promptly removed.

Fig. 1A, LCAD panel, lanes 2-4, appear identical to Fig. 1B LCAD panel lanes 1-3. Slightly rotated and with different exposure. This is despite these samples allegedly originating from different experimental treatments. Lots of shared features including the shapes and relative orientations of the bands, and the "kink" in the right most band, the relative shapes and orientations of the bands, and the "streak" emitting from the top right corner of the 3rd band.

Fig. 1A, PGC-1a panel (top one) appears identical to the PGC1a panel in Fig. 1B. They are simply different exposures of the same image, despite allegedly originating from different experimental treatments. Lots of shared features including the white spot in the lower part of the right-most band. The same appears true for the "SUO" blot (3rd from the top) - some "noise" spots added in panel A, but there's no doubt these are the same bands.

Fig. 4A (and elsewhere), some blots are used as loading controls for other blots, but cannot possibly have originated from the same gels, because some panels are spliced (as indicated by lines), and others are not. Sometimes it is permissible to run your samples on separate gels at the same time, in the exact same order, and then do the phospho vs. total blots separately and re-unite the data at the end. Here we are asked to believe the gels were run separately and with the samples in different order, then some of the blots were spliced (presumably to remove unwanted samples) but the others were not. This is not adherent to the usual standards of data presentation for this type of experiment.

Fig. 6B. The CYTO C panel appears to be simply a darker exposure of the one above it (COX IV). Lots of shared features, most notably the bubble above the right lane.

Fig. 3A, compare the band in the right lane of the cyto C panel (2nd blot from the top), with the band in the right lane of the CYTO C blot in Figure 4C. They are both of a shape that is too similar to be coincidental.

There are numerous other problems here.... the entire paper contains 85 (!!!) panels of western blotting data, every single one of which is "letter-boxed" to show only the band of interest, and none of which are annotated with molecular weight markers. In addition, despite the common origin of most of the samples, there is variability in the properties of the bands. For example in Fig. 4A, the phospho-ACC blot has 2 bands but the total ACC blot only has one band. Why? In Fig. 5B the ATPase blot has 3 bands, but only a single band in Fig. 3B. In several other cases, enhancing the contrast of the western blots reveals that adjacent bands have completely different color histograms - some are grayscale while others are color. As such, it is difficult to believe that these bands originated from the same scanned blot images (which is a prerequisite for being able to splice together blots).

Note... these are NOT allegations of any type of misconduct. I'm just pointing out what appears to be a very sloppy attitude toward data presentation, which seems to have resulted in a number of the "wrong" western blot images ending up in the published paper. Hey, with 85 almost identical looking images to keep track of, there were bound to be a few that slipped through the net! I'm sure these can easily be attributed to mistakes during electronic figure preparation, and corrected in a manner that in no way affects the conclusions of the paper.

On 2015 Jan 01, Alexander Minella commented:

Figure 7A, upper panel, in the Pubmed Central version of this paper contains a formatting error that depicts inverted GATA-1 occupancy results for control and experimental states. The publisher’s PDF version displays the transcription factor occupancy data correctly.

On 2015 Dec 18, Ahmed Adeel commented:

The figures for the therapeutic efficacy of AS/SP in this article need to be clarified. The ABSTRACT says: "PCR corrected per-protocol efficacy was 93.7%." Then in the DISCUSSION it is 90.5%:Page5 column2, they say “In the present study the in vivo per-protocol PCR corrected efficacy of AS/SP over 28 days is 90.5%.” The paper does not show how the figure of 90.5% was calculated.

Do they mean 90.5% rather than 93.7% .They make reference to a previous study in which they reported the figure of 93% for cure rate with AS/SP in a 2004-2005 study .They say (in 2013 paper ) (page 5,column 2 line8):

“..while we have reported a base-line PCR corrected efficacy of 93% [57]. "

According to its title the paper is about "declining artesunate/sulphadoxine-pyrimethamine efficacy...". However, the figure of “90.5 %" is more in line with a decline than "93.7%", because 93.7% is not declining from 93 %. Moreover, in this paper they say: “Approximately 10% of patients exhibited recrudescing parasites as confirmed by msp1 and msp2 genotyping during the study.” Approximately 10% is closer to 90.5% than 93.7%.

On 2013 Nov 09, Claudiu Bandea commented:

What is a virus?

With 1 µm in length and 0.5 µm in diameter, and a genome coding for more than 2500 putative proteins, the intriguing parasites isolated in Acanthamoeba cultures by Philippe et al. (1) dwarf numerous cellular microorganisms, including many eukaryotic microbes. To confirm microscopic observations that these parasitic organisms, labeled Pandoraviruses, are indeed viruses, Philippe et al., used negative defining criteria based on absence of components for three “basic cellular functions”: (i) production of adenosine 5’-triphosphate, (ii) binary fission, and (iii) translational machinery.

However, many cellular microorganisms lack the components for production of adenosine 5’-triphosphate (2, 3), and some, such as yeast (4), produce multiple internal spores by de novo assembly of cellular membranes, rather than by binary fission. Moreover, some cellular microorganisms (2) don’t encode a full set of translational components, and many viruses produce translational components (e. g. tRNAs, amino acid-tRNA ligases, eIF4E translation initiation factor). Therefore, the negative criteria used by Philippe et al. for defining viruses, which were also emphasized in the accompanying article (5) and in ‘About the Cover’ (Science, 19 July 2013), do not differentiate between viruses and cellular microorganisms.

Are Pandoraviruses viruses? Addressing this question might require a fundamental re-evaluation of the conventional view about the nature of viruses (6-9), not only in light of the vast amounts of data and knowledge about their composition, life cycle and evolution, but also in context of the expanding data and knowledge about the diversity of cellular microorganisms (2,3). One of the fundamental features that differentiate viruses from parasitic or symbiotic cellular organisms is that, during their intracellular development, viruses have their genome and other specific components more or less ‘free’ or dispersed within the host cell, whereas intracellular bacterial, archaeal and eukaryotic microorganisms maintain a cellular membrane and cellular organization throughout their life cycle.

This view on the fundamental nature of viruses is consistent with a radical evolutionary model proposing that viral lineages originated from parasitic cellular species that started their intracellular development by fusing with their host cell (6, 8). By discarding their cellular membrane within their particular environment, the host cell, these novel parasites increased their access to host’s resources, including the translation machinery. Nevertheless, after synthesizing their specific molecules and replicating their genome using the resources found in their intracellular environment, the parasites produced spore-like transmissible forms, which started a new life cycle by fusing with other host cells.

Although the life cycle of many extant viruses, including Poxviruses and Mimivirus, fully support the fusion model on the origin of viral lineages (8), Pandoraviruses represent overwhelming evidence. There is also strong evidence, including the absence of ribosomes, that some previously isolated parasitic microorganisms, such as KC5/2 (10) and KLaHel endocytobionts (11), are complex viral organisms. However, as previously discussed (8), there are many other complex parasitic species that are genuine viral organisms although they still produce ribosomes or ribosomal remnants.

The absence of a cellular membrane within the host cell has presented the viral lineages with unique evolutionary opportunities, not readily available for their relatives that maintained a cellular membrane during their intracellular development. However, similar to all intracellular parasitic or symbiotic cellular species, which have been evolving towards a smaller genome (2, 3), the viral lineages have diversified by reductive evolution into a myriad of viruses with smaller genome and diverse life cycles (6, 8). One of the most remarkable implications of the fusion model is that numerous cellular species have evolved into viral lineages throughout the history of life and that this process is still active.

This radical evolutionary theory on the nature and origin of viral lineages also addresses one of the most persisting and intriguing issue in biology, an issue that has puzzled scientists and philosophers for more than a century: are viruses alive? If viral lineages originated from cellular microorganisms as proposed in the fusion model, then, there are few remaining arguments against their living status and their rightful place on the Tree of Life (8).

References

(1) Philippe N. et al., Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. Science 341, 281, (2013). Philippe N, 2013

(2) Keeling PJ, Corradi N. Shrink it or lose it: balancing loss of function with shrinking genomes in the microsporidia. Virulence 2, 67, (2011). Keeling PJ, 2011

(3) McCutcheon JP, Moran NA. Extreme genome reduction in symbiotic bacteria. Nature reviews. Microbiology 10, 13, (2011). McCutcheon JP, 2011

(4) Neiman AM. Sporulation in the budding yeast Saccharomyces cerevisiae. Genetics 189, 737, (2011). Neiman AM, 2011

(5) Pennisi E. Microbiology. Ever-bigger viruses shake tree of life. Science 341, 226, (2013). Pennisi E, 2013

(6) Bandea CI. A new theory on the origin and the nature of viruses. Journal of Theoretical Biology 105, 591, (1983). Bândea CI, 1983

(7) Claverie JM. Viruses take center stage in cellular evolution. Genome Biol. 7, 110, (2006). Claverie JM, 2006

(8) Bandea CI. The origin and evolution of viruses as molecular organisms. Nature Precedings: http://precedings.nature.com/documents/3886/version/1; (2009).

(9) Forterre P. Giant viruses: conflicts in revisiting the virus concept. Intervirology 53, 362, (2010). Forterre P, 2010

(10) Hoffmann R, Michel R, Müller KD, Schmid EN. Archaea-like endocytobiotic organisms isolated from Acanthamoeba sp. (Gr II). Endocytobiosis & Cell Res.12, 185, (1998). http://zs.thulb.uni-jena.de/servlets/MCRFileNodeServlet/jportal_derivate_00100794/ECR_12_1997_185-188_Hoffmann.pdf

(11) Scheid P, Zoller L, Pressmar S, Richard G, Michel R. An extraordinary endocytobiont in Acanthamoeba sp. isolated from a patient with keratitis. Parasitol. Res.102, 945, (2008). Scheid P, 2008

On 2014 Nov 25, Hongkui Deng commented:

We are wondering how you decided to search for an Oct4 substitute using the SKM viruses instead of initially screening using the small molecule cocktail that you would ultimately use the substitute with? Or did you do this and just not report this in the paper?

[reply] When we started to screen for Oct4 replacers, we didn’t know at that time which small molecules would ultimately be used in chemical reprogramming.

Can you provide an explanation for how come GFP controlled by the Oct4 promoter was expressed but Oct4 protein was not expressed (or at least visualized) after VC6TF induction? (see Figure S3)

[reply] In our experiments, we used pOct4-GFP (GOF18) transgenic mice, but not endogenous knockin reporter mice. It is possible that the transgenic reporter may not accurately indicate the endogenous expression of Oct4, which was also reported in other studies (Cell. 2010 143:617-27; Cell Stem Cell. 2008; 3:603).

We would like to know how come you used two the different DOX inducible systems described in the methods section for the screen of late reprogramming molecules? Did you use a second system to replicate the results or were they used on separate molecules for screening? Were the results reported in Figure S4 derived from data using both systems?

[reply] In the initial screen, we used a viral transduction system to introduce the DOX-inducible expression of Oct4 (Maherali et al., Cell Stem Cell, 2008) in OG-MEFs. To make the Oct4 expression level stable from batch to batch, we then used the transgenic mice with inducible Oct4 expression (Hochedlingeret al., Cell, 2005). These two Oct4-inducible systems were used to screen different small molecule libraries.

We also are wondering how come you used a DOX inducible system at all for this screen instead of FSK to simulate early reprogramming? Or did you do this and just not report the outcome of that screen?

[reply] Before we found DZNep, we didn’t know whether a single additional small molecule was sufficient to induce CiPSCs in together with these small molecules. At that time, we only intended to screen for small-molecule candidates that facilitate late stage reprogramming.

What final concentrations and durations were used after optimization? We cannot find this in the paper, and it seems like this should be reported in order to allow others to reproduce the results?

[reply] We presented detailed data about concentrations titration in Figure S7A and listed the concentrations that we used in Table S1B. The optimized duration was also shown in figure S7B-C. In the main text, limited by the space, we did not state the optimized concentrations and durations of the small molecules. But the results were clearly presented in these figures.

Have you performed any experiments to prove that FSK acts at the Oct4 promoter? Versus perhaps at other steps, including even just stabilizing GFP protein expression from a leaky promoter?

[reply] We found FSK was required in stimulating Sall4, as shown in figure S24B. There is no evidence that FSK acts directly at the Oct4 promoter, as FSK could not induce pOct4-GFP positive cells directly.

How exactly do you calculate the 0.2% reprogramming efficiency? Calculating efficiency based on the number of cells originally plated may not be accurate if the cells have been passaged several times during long-term culture for reprogramming?

[reply] The efficiency was calculated according to Yamanaka’s method, by which the number of cell colonies was counted and divided by the number of cells plated (Takasashi et. al., 2007). In addition, I recommend a review paper which carefully discussed how to calculate the reprogramming efficiency: “Guidelines and Techniques for the Generation of Induced Pluripotent Stem Cells”, Nimet Maherali and Konrad Hochedlinger, Cell Stem Cell, 2008. We followed these principles: (1) calculation of plating efficiency, which can be done via cell counts or single-cell plating; (2) eliminating the count of sister clones through single-cell plating; and (3) use of a reliable and stringent method to identify and quantify iPSC colonies. In our study, we only calculated the numbers of CiPSC colonies, characterized by 2i-competent, ESC-like in morphology, and GFP-positive.

Have you tested these small molecule compounds yet on any human somatic cells for reprogramming?

[reply] We have tested these small molecules on human fibroblasts, and found that they were not sufficient to induce pluripotency. Maybe some different small molecule combinations are needed for human cells.

Did you evaluate any off-target effects of these compounds?

[reply] For the two novel small molecules FSK and DZNep, we have revealed their potential targets in CiPSC induction (Fig. S17 and S18). We did not further evaluated their off-target effects.

We cannot seem to find the real-time PCR and immunofluorescence results data referenced in figure S21. We see what appears to be the results of a genotyping microarray experiment?

[reply] The original sentences in our paper are: “To better understand the pluripotency-inducing properties of these small molecules, we profiled the global gene expression during chemical reprogramming and observed the sequential activation of certain key pluripotency genes, which was validated by real-time PCR and immunofluorescence (fig. S21). The expression levels of two pluripotency-related genes, Sall4 and Sox2, were most significantly induced in the early phase in response to VC6TF, as was the expression of several extra-embryonic endoderm (XEN) markers Gata4, Gata6, and Sox17 (Fig. 4, D to F, and figs.S22 to S24).”

In the sentence referenced in figure 21, we only intended to show the global gene expression profiling data during chemical reprogramming and the initial observation of the sequential activation of certain key pluripotency genes. Actually, “which was validated by real-time PCR and immunofluorescence” was an attributive clause that modifies the initial observations, and is redundant with the next sentence, where the detailed validation results by real-time PCR and immunofluorescence was stated, as referenced to Fig. 4, D to F, and figs.S22 to S24. As the two sentences were written together, we thought the figure citations are not necessary to be duplicated in these two sentences.

On 2013 Oct 23, Gholson J Lyon commented:

We reviewed this paper for our lab journal club and we were left with a few questions. We decided to post these questions here in case others have the same questions and/or in case the authors feel like responding to any of them. We posted the same thing to PubPeer.

1. We are wondering how you decided to search for an Oct4 substitute using the SKM viruses instead of initially screening using the small molecule cocktail that you would ultimately use the substitute with? Or did you do this and just not report this in the paper?
2. Can you provide an explanation for how come GFP controlled by the Oct4 promoter was expressed but Oct4 protein was not expressed (or at least visualized) after VC6TF induction? (see Figure S3)
3. We would like to know how come you used two the different DOX inducible systems described in the methods section for the screen of late reprogramming molecules? Did you use a second system to replicate the results or were they used on separate molecules for screening? Were the results reported in Figure S4 derived from data using both systems?
4. We also are wondering how come you used a DOX inducible system at all for this screen instead of FSK to simulate early reprogramming? Or did you do this and just not report the outcome of that screen?
5. What final concentrations and durations were used after optimization? We cannot find this in the paper, and it seems like this should be reported in order to allow others to reproduce the results?
6. Have you performed any experiments to prove that FSK acts at the Oct4 promoter? Versus perhaps at other steps, including even just stabilizing GFP protein expression from a leaky promoter?
7. How exactly do you calculate the 0.2% reprogramming efficiency? Calculating efficiency based on the number of cells originally plated may not be accurate if the cells have been passaged several times during long-term culture for reprogramming?
8. Have you tested these small molecule compounds yet on any human somatic cells for reprogramming?
9. Did you evaluate any off-target effects of these compounds?
10. We cannot seem to find the real-time PCR and immunofluorescence results data referenced in figure S21. We see what appears to be the results of a genotyping microarray experiment?

Anyway, we thought we would post our questions and comments here, in case others in the world read this paper and have similar questions. We would of course welcome answers to any of our questions from the authors.

On 2013 Nov 08, Seth Bordenstei commented:

Blog posts and papers on phylosymbiosis, evolution, speciation, and the hologenome.

http://www.sciencedirect.com/science/article/pii/S0944200613000718

http://symbionticism.blogspot.com/2013/10/guest-blog-post-on-story-behind-new.html

http://symbionticism.blogspot.com/2013_09_01_archive.html

http://symbionticism.blogspot.com/2013/06/talk-from-evolution-2013-snowbird-utah.html

http://symbionticism.blogspot.com/2013/10/in-defense-of-hologenome.html

http://symbionticism.blogspot.com/2013/11/the-gravity-of-symbiosis.html

On 2013 Oct 23, Philip Jones commented:

(I am an author.)

The full dataset for this RCT can be found at

http://dx.doi.org/10.6084/m9.figshare.682404

which includes the native Stata dataset, the Stata .do file necessary to replicate all of the analyses in the manuscript, and the Stata command necessary to calculate the difference in medians used in the bootstrap command.

On 2013 Dec 09, John Cannell commented:

The authors report that immune system dysregulation is common in autism spectrum disorder (ASD). The question is why does this immune dysregulation occur and what has caused such dysregulation to skyrocket in recent decades?

Vitamin D deficiency produces very similar immune dysregulation to what the authors reported.

Prietl B, 2013

Kamen DL, 2010

Yang CY, 2013

Baeke F, 2010

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

Gong ZL, 2014

Meguid NA, 2010

Mostafa GA, 2012

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

DeLuca GC, 2013

Eyles DW, 2013

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

Cannell JJ, 2010

Cannell JJ, 2008

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

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

Kočovská E, 2012

As the authors point out, immune dysregulation is common in ASD. The question is why now and what is causing it?

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2013 Oct 31, John Cannell commented:

I congratulate the authors on a fine review. I wonder if they are aware that vitamin D deficiency has been implicated in virtually all the immune abnormalities they review.

Prietl B, 2013

Kamen DL, 2010

Yang CY, 2013

Baeke F, 2010

It goes to show how little communication is occurring among scientists in different fields. Each scientist seems to be immersed in his or her own research interest but seemingly oblivious to the larger body of research.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2013 Jul 30, Allison Stelling commented:

I am confused by one of the author's statements at the end of the paper (page 8):

"Infrared dyes and Raman spectroscopy have emerged as leading optical technologies, providing excellent selectivity in many different cases of solid tumors (27–30). However, they provide only limited biological or chemical information, and in vivo data suggest that it lacks the sensitivity and specificity of REIMS (31)."

I am familiar with both vibrational spectroscopic and mass spectroscopic methods for the analysis of tissues. If anything, vibrational (Raman and IR) spectra yield more information than mass spectra; as the techniques are a direct and non-invasive measure of chemical bonds. Mass spectra- as the name implies- give only molecular weight information, and do so in a manner that destroys the sample and permits no further analysis. My recent paper (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3604012/) demonstrates that infrared spectroscopy in particular is quite suitable for intraoperative measurements, is likely more cost-effective and is a goodly bit smaller than the mass spectrometer used in this work.

It would have been interesting to see the results from an animal model for both healthy and tumor tissue, as such a model might help train the classification programs and aid in data interpretation.

That being said, this is a quite interesting paper that illuminates the power of traditional analytical chemistry methods for rapid tumor diagnostics. I am mildly skeptical of the results shown in table 2, as the authors show sensitivity and specificity levels over a sampling of, in some cases, n = 2 or even 1 patients. However, I completely understand that these measurements are difficult to orchestrate and this is interesting preliminary work.

I would have liked to see a plot of variance within individuals with the same diagnosis- the presence of low molecular weight chemical species must surely have a fair amount of variance between patients (or even within the same patient), as the expression of such species depends on individual genotypes and their interrelationships with the chemical micro/nano environment. (Basically, I'd like to be able to see on the plots which spectra came from which patients, as many spectra were acquired from individual patients. The total numbers of spectra and total number of patients are listed in tables 1 and 2, but it would be nice know in each case how many measurements were taken and have this visualized with error bars in the plots shown in figures 3, 4, and 5- or even listed in table S1.) -Dr. Stelling

On 2014 Feb 03, S Sundar commented:

Radium-223: Radiobiological conundrum and confounding factors.

Radium 223 is an alpha emitter with limited tissue penetration as evidenced by minimal myelosuppression in the study.(1) But this bone targeted radioisotope seems to have a major effect on bone secondaries while having minimal effect on the bone marrow. This radiobiological conundrum means that potential confounding factors on the observed survival benefit need to be excluded.

One significant confounding factor is the liberal use of effective systemic therapies such as anti-androgens, estrogens and steroids in the study. Concurrent use of these effective agents was not controlled in the study and patients were not stratified for use of these agents.

For instance, dexamethasone and diethylstilbesterol, are widely used in Europe. These therapies are active systemic agents with a median overall survival of 19.4 months in a phase 3 study.(2) Since the median overall survival was only 14 months in Radium 223 study arm, the potential imbalances in the use of these concurrently administered therapies could have significantly biased the overall survival results.

Furthermore, a significant usage of these systemic hormonal agents in the study arms would imply that Radium 223 ideally should not be used as mono therapy in clinical practice. Abiraterone and Enzalutamide have recently been approved for castration refractory prostate cancer. Widespread use of Radium 223 along with these newer agents, which is likely to occur in routine clinical practice, is not without any biological rationale. . There is an urgent need for combination therapy trials with Radium 223 and newer hormonal agents

References: 1. Parker C, Nilsson S, Heinrich D, Helle SI, O'Sullivan JM, Fosså SD, et al. Alpha emitter radium-223 and survival in metastatic prostate cancer. N Engl J Med. 2013 Jul 18;369(3):213-23. 2. Shamash J, Powles T, Sarker SJ, Protheroe A, Mithal N, Mills R, et al. A multi-centre randomised phase III trial of Dexamethasone vs Dexamethasone and diethylstilbestrol in castration-resistant prostate cancer: immediate vs deferred Diethylstilbestrol. Br J Cancer. 2011 Feb 15;104(4):620-8.

On 2014 Feb 03, S Sundar commented:

None

On 2013 Oct 29, John Cannell commented:

I congratulate the authors on an important work but it shows how little communication is occurring among autism scientists. Each scientist seems to be immersed in his or her own research interest but oblivious to the larger body of autism research.

Vitamin D's effect on the seizure threshold is robust.

Siegel A, 1984

Holló A, 2014

Furthermore, a recent study found supplemental vitamin D reduced seizures in children with severe epilepsy.

Holló A, 2012

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

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

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

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

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

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

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

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

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

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

70% of American toddlers do not take the American Pediatric Association's recommended vitamin D supplement of 400 IU/day.

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

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

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

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

John J Cannell, MD

Vitamin D Council

http://www.vitamindcouncil.org

On 2015 Oct 13, Bill Cayley commented:

An example showing that sometimes for care of atrial fibrillation, "less" is better: https://lessismoreebm.wordpress.com/?s=atrial

On 2013 Aug 06, Allison Stelling commented:

This was a wonderful overview of the literature regarding the expression of collagen domains in gliomas. I feel that the microenvironment experienced by the cells has been neglected a bit in the literature, but it is important to recall that natural selection works via the interactions between cells and their nanoscale, biochemical environment. These environments influence the expression of genes through a myriad of mechanisms- from protein protein interactions, to small molecules that activate signaling cascades via (for example) phosphorylation events. The chemical pressures experienced by tumor cells in particular during treatment can lead to selection for drug resistant phenotypes- and, this resistance likely must be considered on an individual, "personal genomics/phenotype" level. Gliomas in particular are what is know within the medical community as "highly malignant", and recent studies highlighted in this review point to these cells as manufacturing their own extracellular collagens to facilitate infiltration of healthly tissue. Given how responsive and adaptable tumor cells can be, I would not find it surprising if these molecules played a role in "converting" or "infecting" healthy cells into tumor cells.

This review does a fine job of going over the biochemistry of the "major collagen players" discovered thus far in tumors. I particularly enjoyed the section on the effects of the physical properties of the matrix on glioma cell growth and motility. There is much fundamental work to be done in this area, and gaining physical models and insight on the molecular level about the complex microenvironment these malignant cells inhabit will improve our understanding of these tumors and greatly aid in the design of effective therapies.

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 NCT0069999. We believe the correct ID, which we have found by hand searching, is NCT00699998.

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

Many thanks,

Jessica Fleminger, Ben Goldacre*

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

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

On 2014 Nov 30, Michael Wood commented:

As the first author of this study, I'd like to address a misleading headline that's been making the rounds lately: the idea that this study says that people who believe 9/11 conspiracy theories are better-adjusted than those who do not. This grossly misinterprets our results: this study says nothing about mental health, and its results do not justify any conclusions about one group of people being more or less "sane" than another.

The main basis for this misinterpretation appears to be the observed difference in hostility between conspiracist (pro-conspiracy-theory) and conventionalist (anti-conspiracy-theory) comments. On average, conventionalist comments tended to be somewhat more hostile. In the paper, we interpret this difference as the product of a fairly specific social situation in which the two rival opinion-based groups use different strategies of social influence according to their relative popularity, rather than as an inherent psychological difference. In fact, previous research by Marina Abalakina-Paap and colleagues has shown that dispositional hostility is positively, not negatively, correlated with beliefs in conspiracy theories - in other words, people who believe more conspiracy theories tend to be more hostile. However, that finding doesn't necessarily justify the conclusion that conventionalists are better-adjusted than conspiracists. Either of these conclusions relies on the unstated premise that hostility is never good or justified, and that less hostility is always better. This is at least an arguable assumption, and there's certainly no evidence for it here.

In general, I would urge anyone who found this paper via the "sanity" article to please think critically about headlines in the future. It is tempting to believe without question self-serving headlines that validate your prejudices and beliefs, but that's precisely when critical thinking is most important.

On 2015 Aug 10, Hilda Bastian commented:

This is an excellent overview of the research on the impact of celebrity and public figure announcements around cancer. The conceptual model proposed for studying impact on behavioral and disease outcomes is an important contribution, but I think it would benefit by being extended in several ways.

The issue of potentially deepening inequalities in cancer (Lorenc T, 2013) is so critical here, that equity needs to be considered at the outcome end of the picture: incorporating demographics and SES as a mediator/moderator isn't enough. Nor is age the only critical socioeconomic factor about the celebrity/public figure that should be taken into account. The authors point to some notable omissions among the cases that have drawn researcher interest. One of the most striking omissions, though, is the lack of study of non-Caucasian celebrities and public figures (for example Robin Roberts and Donna Summer on the timeline in this article).

Also striking is the extent to which existing stigma around some cancers is reinforced, both in which cancers are publicly discussed by celebrities, and which are studied by researchers. Are we doing, at the community level (and in the researcher community), what we do in private life as well - reinforcing stigma and poor knowledge of critical diseases in our lives (Qureshi N, 2009)? Take colorectal cancer for example. Whether it's the cases in the timeline (which included only Farrah Fawcett) or the included studies (which included only Ronald Reagan), the under-representation of such a stigmatized condition points to a critical issue for research in this field. Impact on stigma would be a valuable addition to the outcomes in the conceptual model, to emphasize the importance of this dimension of belief.

In general, it would be good if the potential for adverse effects was more explicit in the model. Critically, impact on over-diagnosis and screening/testing-related harm needs to be included - a key issue the paper discusses, for example, after Kylie Minogue's cancer (Kelaher M, 2008, Twine C, 2006). Accuracy in personal risk assessment, similarly, is an important outcome that is an important outcome to consider.

Focusing on behavioral and disease outcomes in the model leaves out the impact on resources, and ways systems can best respond to these unpredictable events. That was a major issue after Angelina Jolie's announcement (Evans DG, 2014).

It would be helpful to understand the impact of famous family members' announcements and pleas around cancer, as well: Katie Couric's public intervention (Cram P, 2003), for example, are relevant to this field.

Finally, the model of considering these events only in terms of cancer prevention as the end interest, risks missing potentially important impacts of these cultural events. They contribute in the complex ways we think about and deal with life-threatening illness, life, and death (Førde OH, 1998). The lack of studies that address these broader issues is striking, too.

Note: Rick Nolan noted in a comment on my blog that Dan Fogelberg's is wrongly attributed to pancreatic cancer in this article: he died of prostate cancer. I had discussed these issues, and the studies on Angelina Jolie that occurred after these reviewers completed their search, in this blog post.

On 2013 Nov 27, R Aldridge commented:

At a time when there is considerable interest in the potential for the fluoroquinolones to shorten treatment for drug-sensitive tuberculosis, the trial conducted by the National Institute for Research in Tuberculosis (NIRT) in Chennai[1] could represent an important contribution to our understanding of whether this is achievable. Unfortunately, we have serious concerns about the conduct and analysis of this study which makes the interpretation of the results challenging.

Because moxifloxacin was not available at the start of the study, patients were only enrolled into the gatifloxacin and control regimens during the first 12 months. Thereafter, an attempt was made to equalise the numbers in each treatment arm by changing the allocation ratio to 2:1:1 in favour of the moxifloxacin arm. However, this meant that probably only around half of the 170 patients on the control arm were enrolled concurrently with patients allocated to the moxifloxacin arm. In addition the gatifloxacin arm was terminated 10 months before the moxifloxacin and control arms. No attempt appears to have been made to compare regimens during concurrent enrolment periods of the study in what would be a properly randomised comparison, in order to examine whether the conclusions would have been altered. Known or unknown differences in patient management or between patients enrolled in the different periods of enrolment could have an impact on the differences between regimens in the analyses presented.

The Data Safety and Monitoring Board (DSMB), whose members and independence are not noted in the report or the study protocol, made a recommendation in February 2006 to terminate the gatifloxacin arm of the study and in October 2006 to terminate the moxifloxacin arm. No information is given either on the guidelines under which the DSMB made this decision, or the results that were available to the DSMB in each case; only that that the decision was due to ‘high TB recurrence rates in these two arms compared to the control arm’[1]. However, most of the data on recurrence (from 24 months follow-up) would have accrued after the study had terminated. The final reported results comparing moxifloxacin with the control arm would suggest there was minimal difference between the outcomes of these two regimens, and that there was no evidence for a difference in the recurrence rate between these two arms (P=0.38). These results suggest the termination of the study is very likely to have been premature. Early termination introduces bias and, as noted in Pocock and White in relation to another trial: ‘the premature pulling out of the trial on insufficiently convincing evidence inhibits the ability of clinical science to resolve an important therapeutic issue’[2].

The culture results at two months are of particular interest since they suggest that 10% more patients receiving moxifloxacin converted at that time compared to the control arm. The authors fail to comment on what this means for the predictive ability of two month culture conversion for successful treatment shortening, a subject recently addressed by other publications in this journal.[3,4]

The results of this study might appear to support those of the OFLOTUB randomised controlled trial, presented at the recent conference of the IUATLD in Paris, which failed to show a shortened daily gatifloxacin based arm was non-inferior to the standard six month daily regimen. However, as we have pointed out, there are several serious limitations in the NIRT trial report, in particular the premature closure of enrolment, which need to be recognised when assessing the conclusions of the study and any future meta-analyses which includes these data.

Rob Aldridge on behalf of the North London TB Journal Club (http://northlondontb.org/). North London TB Journal Club meets monthly; it is organised by the London School of Hygiene and Tropical Medicine, University College London and MRC Clinical Trials Unit. The points above represent concerns raised during a discussion of this paper at a meeting of the Journal Club on 12th November 2013.

1. Jawahar MS, Banurekha VV, Paramasivan CN, Rahman F, Ramachandran R, et al. (2013) Randomized clinical trial of thrice-weekly 4-month moxifloxacin or gatifloxacin containing regimens in the treatment of new sputum positive pulmonary tuberculosis patients. PLoS One 8: e67030.
2. Pocock S, White I (1999) Trials stopped early: too good to be true? Lancet 353: 943-944.
3. Phillips PP, Fielding K, Nunn AJ (2013) An Evaluation of Culture Results during Treatment for Tuberculosis as Surrogate Endpoints for Treatment Failure and Relapse. PLoS One 8: e63840.
4. Wallis RS, Wang C, Meyer D, Thomas N (2013) Month 2 Culture Status and Treatment Duration as Predictors of Tuberculosis Relapse Risk in a Meta-Regression Model. PLoS One 8: e71116.

On 2013 Oct 23, Stephen Turner commented:

This paper presents a new method for species identification and strain attribution using next-generation sequencing data from a cultured pathogen or mixed environmental sample. The paper presented compelling results showing that this new method performed markedly better than a 'naive' mapping approach and other leading read classification methods based on taxonomic binning or compositional analysis. Importantly, the method was shown to work well, even when the species being sequenced was not present in the reference database; sequence data from this organism were assigned to the closest phylogenetic near-neighbor. Finally, the method was also shown to work well when multiple species were present, faithfully recapitulating the actual abundances of organisms present in the sample.

On 2015 Jul 14, Bill Cayley commented:

A good example of when "less" is "more": https://lessismoreebm.wordpress.com/2015/07/14/effect-of-timing-of-umbilical-cord-clamping-of-term-infants-on-maternal-and-neonatal-outcomes/

On 2013 Oct 31, John Cannell commented:

The authors found markers oxidative stress is present in autism spectrum disorder (ASD). I wonder if the authors are aware that the genes for the antioxidants superoxide dismutase and thioredoxin reductase are directly up-regulated by the secosteroid 1,25 di-hydroxy vitamin D3 (calcitriol). I believe both genes also harbor a vitamin D response element.

Peehl DM, Shinghal R, Nonn L, Seto E, Krishnan AV, Brooks JD, Feldman D. Molecular activity of 1,25‐dihydroxyvitamin D3 in primary cultures of human prostatic epithelial cells revealed by cDNA microarray analysis. J. Steroid Biochem Mol. Biol 2004;92:131–141. PMID:15555907>

Calcitriol also directly up-regulates glutathione reductase and increases glutathione levels.

Jain SK, et alo. Vitamin D upregulates glutamate cysteine ligase and glutathione reductase, and GSH formation, and decreases ROS and MCP-1 and IL-8 secretion in high-glucose exposed U937 monocytes. Biochem Biophys Res Commun. 2013 Jul 19;437(1):7-11. doi: 10.1016/j.bbrc.2013.06.004. Jain SK, 2013

Also, supplemental vitamin D significantly reduces oxidative stress in humans.

Nikooyeh B, et al. Daily intake of vitamin D- or calcium-vitamin D-fortified Persian yogurt drink (doogh) attenuates diabetes-induced oxidative stress: evidence for antioxidative properties of vitamin D.J Hum Nutr Diet. 2013 Jul 5. doi: 10.1111/jhn.12142. Nikooyeh B, 2014

Asemi Z, et al. Vitamin D supplementation affects serum high-sensitivity C-reactive protein, insulin resistance, and biomarkers of oxidative stress in pregnant women. J Nutr. 2013 Sep;143(9):1432-8. doi: 10.3945/jn.113.177550. Asemi Z, 2013

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

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

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

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

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

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

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

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

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

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

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

70% of American toddlers do not take the Ameri

On 2014 Feb 22, Christopher Southan commented:

Additional BACE1/BACE2 patents are reviewd in http://www.ncbi.nlm.nih.gov/pubmed/23506624

On 2014 Dec 10, Kath Wright commented:

Other search filters are available from the InterTASC Information Specialists' Sub-Group Search Filter Resource at https://sites.google.com/a/york.ac.uk/issg-search-filters-resource/home

On 2013 Oct 23, Kenneth Daily commented:

The topic of this paper is very interesting - if there are long-term viruses that rapidly evolve along other more stable viruses. However, there are a few misstatements and omissions that would help strengthen this paper.

The authors write:

"Sequence reads from each sample were first assembled individually using MetaIDBA (16)."

The reference for this statement (Peng Y, 2012) refers to IDBA-UD, however.

Could the authors comment on which tool was used for metagenome assembly?

The authors write:

"The correlation coefficient between replicate samples from the same time point was at least 0.99, indicating a high degree of reproducibility (Fig. S1)."

In the supplemental methods, a section titled "Reproducibility of Contig Detection" gives the correlation coefficients from Figure S1.

1. The minimum correlation coefficient given is 0.9743. Hence, the above statement does not seem to be correct. I do not dispute that this is still a high degree of reproducibility across the two sample replicates.

2. The "day" given in this list does not correspond to the day in Figure S1; it seems to match the sample name given in the SRA submitted data (example: BLS020824_d13-1 and BLS020825_d13-2 would correspond with 13 in this table).

The methods in this paper are difficult to reproduce. No metadata about each of the samples (most importantly, the day on which they were taken) is present in the data supplied to SRA, BioSample, or BioProject, except for sample SRS413841.

Details on the versions and parameters for running the various tools are not included (for MetaIDBA, promer, Bowtie2, Glimmer, MetaPhlAn, PILER-CR, Blastn, and the "custom scripts"). Given the complexity and number of steps of the analysis and that most of the tools are openly available, some sort of pipeline of the procedures run on this sequencing data would assure reproducibility.

On 2014 Mar 06, David Keller commented:

Patient safety trumps cost containment

Professor Horowitz is to be congratulated for correctly diagnosing his sister's transient global amnesia (TGA) over the telephone, and for predicting the benign and self-limited course it would take. In my 16 years of practice as a general internist, plus my years of residency training and medical school, I did not encounter a single patient with this condition. Therefore, I sympathize with the internist who was called in to the ER to evaluate this patient. Given an elderly woman who, at first, exhibited total amnesia, including complete disorientation regarding her own identity and surroundings, I would have ordered the same thorough workup that her admitting physician did, unless an experienced neurologist examined her in person and was confident enough of a benign diagnosis to write her discharge orders. If the admitting internist could not obtain a neurological consultation, and did not feel comfortable sending the patient home based on his own findings, then I cannot blame him for ordering an extensive workup to rule out atherosclerotic vascular disease or embolism as the cause of these dramatic symptoms in an elderly patient. When in doubt, patient safety should trump cost containment.

To facilitate discussion, I respectfully request the person who found this comment "not helpful" to state their reason