test page note

Reading this reminds me of some of Brad Enslen and Kicks Condor's conversations about discovery on the net.

How can one leverage selfish behaviour to the benefit of all?

dch hgchgcvh hkhk

The amount of data we have from Cassini is crazy to think about. Quite a few nations were involved in Her mission, and the price tag is relatively low in terms of exploration craft in the past and present.

native to the web

Facebook does not allow third-party apps to display your newsfeed. This applies to Hootsuite. For this reason, you’ll always have to use Facebook natively. The same pretty much goes for Instagram.

defines local symbol f. local to file. without static, g is strong. 3rd line declares but doesnt defifne. right way to do things in c : 1. when you write func implemn/defn, you have 2 choices, you want func glbal enuf. if not global, local then declare fucn declare and declare it as static.

1. funcs should be static in filewhenever possible.

you can get away without using header files but you should not do that. every func declaration should be included in header file for a global. implicit func declar. if global func, pople sized, it should have declration in header file but trouble is that lang doesnt require it. cpp requries it therefore its not an extension of c but a diff language. make fucnkign header files. if header files, how does compiler look at functions calls. it looks for dunc declares. you cant overload func names, there is one or none. paramenter list in defn and declare. checks #para and types are compatible. what if compiler doesnt find declaration for func, it makes one up. and assumes return type int always. jeez. cimpiler leaves note for linker, relocation tag, this has to be mapped toa call. that can break down at link time if implicit declareation that compiler made up doesnt match the linker's?. never ignore implicit func declaration warning. PAY ATTENTION TO THESE. main is entry point. lol recursive main?! system calls main. have main call main.

Ejemplo de anotación: recomiendo visitar esta página web https://disfrutamiruta.com/tag/smorfia-napolitana/

As stated earlier in the article, the authors could only fairly compare the LSH and fly algorithms if each used the same number of mathematical operations.

They determined that if they used 20k random projections in the fly algorithm (where k is the length of the output tag, or hash) then the total number of operations for the fly and LSH algorithms would be equal.

For more detail, see the second paragraph under "Materials and Methods" in the Supplementary Materials document.

Common Core State Standards English Language Arts-Literacy, RST 11-12.6: Students should be able to explain why the authors provided this example in the paper, specifically addressing how it adds to the reader's understanding of the research.

http://www.corestandards.org/ELA-Literacy/RST/11-12/

AP Science Practices, Practice 1: The student can use representations and models to communicate scientific phenomena and solve scientific problems.

Looking at the model in Figure 1A, students should be able to provide a verbal explanation of what the diagram is showing and what each shape and symbol represents.

https://apcentral.collegeboard.org/courses/resources/science-practices

The authors looked at how the LSH algorithm performed when using two different methods to create the tag:

1. The tag is created from a random selection of Kenyon cells
2. The tag is created from the Kenyon cells with the highest firing rates (this is the "winner takes all" or WTA approach)

The result was that the LSH performed better with the WTA approach. Note that the authors measured performance using the mean average precision (see Fig. 2B)

Stevens outlines the three-layer architecture that makes up the fly's olfactory circuit.

He also presents the idea of a unique odor label, or "tag" that is comprised of a small set of neurons and helps the fly identify distinct odors.

First Testtext to pdf embedded via object tag.

This makes it sound like there is an HTML element with a tag name of <toc nav> rather than <nav epub:type="toc"> (which seems to be what's intended).

The landmarks example farther down is clearer--though the wording there of "the landmark nav element" is equally confusing.

There remains only a nav element, but of varying types.

The American dream has a price tag to rich for all citizens and ergo represents inequality amongst Americans

Owald and Waddell discovered that the fly olfactory circuit is able to recall previously-learned odors through the help of specialized dopamine neurons. After a fly smells an odor, these dopamine neurons trigger changes in parts of the olfactory circuit that cause the specific neurons associated with the odor (aka the "tag") to fire.

Reactivating the tag causes the fly to remember the odor, as well as the values/meanings/context associated with it. The fly can then exhibit the appropriate behavior based on its prior learning (e.g. avoidance if the odor is associated with danger or approach if the odor is associated with a reward).

How do you tag the lessons ?

Šis cilvēks nesaprot, kā darbojas aktīvisms (un pasaule :D), right? Ak nē, Papardes zieds uzskata, ka aborti nav jāaizliedz un ka abortu aizliegt gribētāji nav jauki cilvēki. Nokrāsojiet mani šokētu! :D

Pag, kurā dienā un konkrētā minūtē valstī bija šis maģiskais brīdis? Zinu! Tas notika tad, kad visas planētas sastājās rindā. :D ("Nerunā" neuztvēru 100% burtiski, sorry.)

Un arī nav mūsu valsts vienīgā un pa visu planētu, kā tajā senajā TV reklāmā. :D Polija, Krievija, konservatīvie politiskie spēki, kas atrodami it visur utt? Mm? Tāda sajūta, ka autors dzīvus cilvēkus sen nav saticis un īsti nesaprot, kā tie uztver pasauli.

No way! :D Cepums autoram, ka ir spējis pamanīt šo "slepeno" domu - ka nejaukās sekas no abortu aizliegšanas eksperte uzskata par sliktām (kur pilnībā viņai piekrītu) un negrib pieredzēt tādu nejaucību atkārtošanos.

No šit... A ko citu tad Papardes ziedam bija jādara, ņemot vērā šīs organizācijas mērķus? Tajos brīžos, kad tai rodas vēlme apspriest abortus, meklēt nepieredzējušus "ekspertus", kas uzskata, ka aborti ir grēks? :D

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When to use this code

is there a feature hidden somewhere that lets you see all the posts on dreamwidth that use a particular tag? if so, i haven't found it, which definitely makes content discovery difficult

I was referencing dreamwidth's syndication feature, which allows you to add anything with an RSS or Atom feed to your reading page. This turned out not to really work - reblogs mean that 95 percent of tumblrs are just way too high volume to work, though certain techniques can mitigate this (a feed for a specific tag on a tumblr blog can be uncovered by adding /rss to the end of the tag URL though automatic feed discovery redirects back to the home feed for some reason)

Instead of having a single large bundle file for your whole website, you can have multiple bundles for each page. This improves the load time of your website as you can tag bundle files to the various webpage of your site instead of one initial big download of script file.

For non module supporting browsers we can add another script tag with the nomodule attribute

modules have .mjs extension Not really important We can use .js as well The type attribute in our script tag is enough RECOMMENDED : We should use .mjs extension because during development it makes it easier to diffrentiate

Only the latest browsers are module-supporting. For these browsers you can use a script tag with a type attribute set to module.

Tales from Basques in the United States: Santiago Arrillaga, an atypical Basque immigrant

I have to say publicly that this sentence makes absolutely no sense as crossref and figshare are not comparable assigning authorities. CrossRef is a registration agency of the International DOI Foundation; FigShare is not. Based upon the example given, the assigning-authority for a DOI would only ever be the set of agencies that assign DOIs. FigShare is not one of those agencies. See it is not on the list: https://www.doi.org/registration_agencies.html

A stretch of DNA sequence that has the ability to be translated into protein (exons only). An ORF usually begins with a start codon (ATG) and ends with a stop codon (TAA, TAG or TGA).

Männer mussten rituell rein sein, um das reine Brot zu essen (daher die Frage nach sexuellen Beziehungen => rituell unrein für 1 Tag (3. Mose 15:16))

The highlighted annotations with the airbnb tag were made to illustrate where Basic Javascript differs from the Airbnb Javascript Style Guide and typescript tag for differences from Typescript Deep Dive TIPs.

asdasd

https://via.hypothes.is/https://gendertrender.wordpress.com/tag/corrective-rape/

This is sort of the fun, I think, in maintaining things like listen and read posts on my site. While they're a useful archive for me, in some part I hope they might speed some discovery for folks who find them or search them by category/tag as well.

I could post somewhere, "Hey I listen to this podcast," or retweet a headline, but invariably in the morass of content out there, there isn't actually an indication that I invested my finite amount of time actually listening to or reading that thing. Perhaps I was just doing some social signaling to make myself seem more interesting or worldly? To me this is a lot of the value of these types of posts.

1. annotation test 1;
2. annotation 2;

Test.

Because the narrative jumps around, it would be interesting to map out each event chronologically and to tag it as to how it relates to the moonstone. Perhaps some kind of network / chronology?

On 2017 Jun 14, Youhe Gao commented:

I found two "overexpression"s in the paper. "Although the extent of bait overexpression is difficult to judge and varies across IP's, previous experimentation has shown that over-expression has little effect on identification of true interacting partners (Sowa et al., 2009)" "VAPBWT overexpression strongly increased the association of EGFP-LSG1 and OSBP with the ER (Figure 7E,G)" Personally, I am not sure if those are enough. In a system, increasing [A] or [B] will lead to more [AB]. As we know more about protein interaction now, this kind of systematic false positive should not be ignored any more. In cells, overexpression with tag may even change the location of the protein. That is why I think the next generation of massive protein interaction studies should start from in vivo crosslinking. I do not want to overemphasize the problem. Most of the protein interactions identified are probably true in cells. The amount of work done is very impressive and respected. I hope users who is using a particular interaction data as the only clue for their future experiment design, maybe they should start with an in vivo crosslinking as a conformation of that interaction. It may make them more confident to proceed.

On 2017 Jan 22, Eric Fauman commented:

I know nothing about cow genetics, but I have done some work on the genetics of metabolites in humans, so I was interested to see how the authors derived biological insights from this genetic study. In particular, I was intrigued by the suggestion in the abstract that they found evidence that genes involved in the synthesis of “milk components” are important for lactation persistence.

Unfortunately, the more I studied the paper the more problems I found that call this claim into question.

First off, the Q-Q plot is currently unavailable, but the text mentions there’s only a “slight deviation in the upper right tail”, which could mean there are no true significant signals.

To account for multiple testing, the authors decided to use a genome-wide association p-value cutoff of 0.95/44100 = 2.15e-5 instead of a more defensible 0.05/44100 = 1.1e-6.

Since their initial p-value cutoff yielded a relatively small number of significant SNPs, the authors used a much more lenient p-value cutoff of 5e-4 which presumably is well within the linear portion of the Q-Q plot.

The biggest problem with the enrichment analysis, however, is that they’ve neglected to account for genes drawn from a common locus. Often, paralogs of similar function are proximal in the genome. But typically we assume that a single SNP is affecting the function of only a single gene at a locus. So, for example, a SNP near the APOA4/APOA1/APOC3/APOA5 locus can tag all 4 genes, but it’s unfair to consider that 4 independent indications that “phospholipid efflux”, “reverse cholesterol transport”, “triglyceride homeostasis” and other pathways are “enriched” in this GWAS.

This issue, of overcounting pathways due to gene duplication, affects all their top findings, presumably rendering them non-significant. Besides lipid pathways, this issue also pertains to the “lactation” GO term, which was selected based on the genes GC, HK2, CSN2 and CSN3. GC, CSN2 and CSN3 are all co-located on Chromosome 6.

A perplexing claim in the paper is for the enrichment of the term “lipid metabolic process” (GO:0006629). According to the Ensembl Biomart, 912 Bos taurus genes fall into this category, or about 4% of the bovine protein coding genes (24616 according to Ensembl). So out of their set of 536 genes (flanking SNPs with P < 5e-4) we’d expect about 20 “lipid metabolic process” genes. And yet, this paper reports only 7. This might be significant, but for depletion, not enrichment.

Sample size is of course a huge issue in GWAS. While 3,800 cows is a large number, it appears this trait may require a substantially larger number of animals before it can yield biologically meaningful results.

On 2016 Sep 16, Hilda Bastian commented:

There are many important issues raised in this paper on which I strongly agree with John Ioannidis. There is a lot of research waste in meta-analyses and systematic reviews, and a flood of very low quality, and he points out the contributing factors clearly. However, there are some issues to be aware of in considering the analyses in this paper on the growth of these papers, and their growth in comparison with randomized and other clinical trials.

Although the author refers to PubMed's "tag" for systematic reviews, there is no tagging process for systematic reviews, as there is for meta-analyses and trials. Although "systematic review" is available as a choice under "article types", that option is a filtered search using Clinical Queries (PubMed Help), not a tagging of publication type. Comparing filtered results to tagged results is not comparing like with like in 2 critical ways.

Firstly, the proportion of non-systematic reviews in the filter is far higher than the proportion of non-meta-analyses and non-trials in the tagged results. And secondly, full tagging of publication types for MEDLINE/PubMed takes considerable time. When considering a recent year, the gulf between filtered and tagged results widens. For example, as of December 2015 when Ioannidis' searches were done, the tag identified 9,135 meta-analyses. Today (15 September 2016), the same search identifies 11,263. For the type randomized controlled trial, the number tagged increased from 23,133 in December to 29,118 today.

In the absence of tagging for systematic reviews, the more appropriate comparisons are using filters for both systematic reviews and trials as the base for trends, especially for a year as recent as 2014. Using the Clinical Queries filter for both systematic reviews and therapy trials (broad), for example, shows 34,126 for systematic reviews and 250,195 trials. Page and colleagues estimate there were perhaps 8,000 actual systematic reviews according to a fairly stringent definition (Page MJ, 2016) and the Centre for Reviews and Dissemination added just short of 9,000 systematic reviews to its database in 2014 (PubMed Health). So far, the Cochrane Collaboration has around 38,000 trials in its trials register for 2014 (searching on the word trial in CENTRAL externally).

The number of systematic reviews/meta-analyses has increased greatly, but not as dramatically as this paper's comparisons suggest, and the data do not tend to support the conclusion in the abstract here that "Currently, probably more systematic reviews of trials than new randomized trials are published annually".

Ioannidis suggests some bases for some reasonable duplication of systematic reviews - these are descriptive studies, with many subjective choices along the way. However, there is another critical reason that is not raised: the need for updates. This can be by the same group publishing a new version of a systematic review or by others. In areas with substantial questions and considerable ongoing research, multiple reviews are needed.

I strongly agree with the concerns raised about conflicted systematic reviews. In addition to the issues of manufacturer conflicts, it is important not to underestimate the extent of other kinds of bias (see for example my comment here). Realistically, though, conflicted reviews will continue, building in a need for additional reviewers to tackle the same ground.

Systematic reviews have found important homes in clinical practice guidelines, health technology assessment, and reimbursement decision-making for both public and private health insurance. But underuse of high quality systematic reviews remains a more significant problem than is addressed here. Even when a systematic review does not identify a strong basis in favor of one option or another, that can still be valuable for decision making - especially in the face of conflicted claims of superiority (and wishful thinking). However, systematic reviews are still not being used enough - especially in shaping subsequent research (see for example Habre C, 2014).

I agree with Ioannidis that collaborations working prospectively to keep a body of evidence up-to-date is an important direction to go - and it is encouraging that the living cumulative network meta-analysis has arrived (Créquit P, 2016). That direction was also highlighted in Page and Moher's accompanying editorial (Page MJ, 2016). However, I'm not so sure how much of a solution this is going to be. The experience of the Cochrane Collaboration suggests this is even harder than it seems. And consider how excited people were back in 1995 at the groundbreaking publication of the protocol for prospective, collaborative meta-analysis of statin trials (Anonymous, 1995) - and the continuing controversy that swirls, tornado-like, around it today (Godlee, 2016).

We need higher standards, and skills in critiquing the claims of systematic reviews and meta-analyses need to spread. Meta-analysis factories are a serious problem. But I still think the most critical issues we face are making systematic reviews quicker and more efficient to do, and to use good ones more effectively and thoroughly than we do now (Chalmers I, 2009, Tsafnat G, 2014).

Disclosure: I work on projects related to systematic reviews at the NCBI (National Center for Biotechnology Information, U.S. National Library of Medicine), including some aspects that relate to the inclusion of systematic reviews in PubMed. I co-authored a paper related to issues raised here several years ago (Bastian H, 2010), and was one of the founding members of the Cochrane Collaboration.

On 2016 Jun 02, Michael Tatham commented:

Is SUMO5 a pseudogene?

There are known to be many SUMO pseudogenes in humans (http://www.ncbi.nlm.nih.gov/pubmed/12383504). A fair position when confronted with a claim that a new SUMO paralog has been discovered is to assume it is a non-expressed pseudogene until otherwise convincing evidence is provided. This paper lacks one piece of critical evidence supporting the idea that SUMO5 really exists as a protein, and that is the presence of endogenous protein.

When BLAST searched, the nucleotide sequence of SUMO5 (originally termed SUMO13 according to the authors’ GenBank entry: FJ042790.1), returns a top hit of “Homo sapiens SUMO1 pseudogene 1 (SUMO1P1), non-coding RNA Sequence ID: ref|NR_002189.3|”. The only difference is a single nucleotide T23 (in SUMO13/SUMO5), which is C in SUMO1P1. This may be a primer synthesis error or a DNA sequencing error.

To put beyond reasonable doubt that SUMO5 is not a pseudogene at least two pieces of new experimental evidence showing SUMO5 is expressed in cells is required. I can think of three good ways to do this:

(1) Mass-spectrometric evidence of a peptide unique to SUMO5.

(2) Cross-reaction of a SUMO5-specific antibody with an endogenous protein.

(3) Editing of the genome to insert an epitope tag into the endogenous SUMO5 gene, with the intention of detecting the protein using an antibody specific to the tag.

All three of these pieces of evidence will be strengthened by parallel studies comparing cells with and without SUMO5-specific knock-down.

RTPCR experiments intending to detect mRNA are particularly uninformative given that DNA contamination often leads to false-positives. This is especially true for SUMO5 given the fact the gene is intronless, a notable characteristic of pseudogenes.

On 2016 Jul 27, Duke RNA Biology Journal Club commented:

This is a summary of a journal club discussion:

This is one of four articles using similar imaging techniques to study translation in living cells published at the same time. These publications add to the growing number of techniques used to image translation such as mature fluorescent proteins Yu J, 2006, TRICK Halstead JM, 2015, and RNA-binding protein/mRNA co-fluorescence Wu B, 2015. The technique presented in this article is similar to the last except that it uses Suntag to image the nascent chain and PP7 aptamers on the mRNA. The colocalization of the two represents active translation in polysomes.

The technique is novel because co-localization is detected as the protein is translated. This brings fluorescent V4-peptide antibodies into concentrated foci at a single point, and can thus be used to follow multiple rounds of translation. Because of this, only detection of the translated protein is needed and indeed, past the first figure, the mRNA fluorescence is not shown. Fast changes in translation can be detected as shown using the ATF4 ORF construct translational response to stress shown in Figure 4 with the possibility of extending the time of tracking to hours by anchoring the mRNA Yan X, 2016 or using fast 3D imaging techniques. One unusual observation the authors made was the vast heterogeneity of transcript translation within a single cell; at any given time only a subset of the transcripts undergo translation and translation rates may vary depending on as yet unknown factors. A related observation is the diffusion of polysomes within the cell: polysomes translating cytosolic transcripts have slower diffusion rates in the perinuclear region of the cell compared to the cytoplasm. This could be due to the restrictive architecture of a membranous area but the exact mechanism remains unknown. A second surprising observation indicates mRNAs that have begun translation and are associated with polysomes can be transported in dendrites, contrary to earlier reports Besse F, 2008. However, the authors cannot detect if translation is temporarily stalled during transport.

While this technique makes substantial findings in the area of single transcript translation behavior, there are limitations. All in all, these images are dots that respond to translation inhibitors, meaning the resolution is not good enough to detect codon resolution and should be coupled with other techniques to verify observations and determine their mechanism. Additionally, since detection of the nascent chain wouldn’t be detected until the majority of the V4 peptides were translated, initiation would be overlooked; however, TRICK is an existing technique for studying the first round of translation.Our main criticism with this technique is the extensive construct engineering that must be performed which raises concerns over disturbing the mRNA and protein functions from both the PP7 aptamers, the Suntag peptides and an ornithine decarboxylase tag to facilitate rapid degradation of the protein. These engineering steps add over 2 kb to the original gene. Additionally, an antibody against the Suntag and a fluorescent PP7 coat protein must be expressed in the cytosol. While the constructs studied did not cause harm to the cell, each construct of interest must be tested individually. Along this line, while there is the possibility to multiplex by changing the aptamer loop or peptide-antibody combination, it would be difficult to multiplex above two individual transcripts. Thus large-scale studies involving individual translation dynamics of mRNA subsets would remain time consuming and technically challenging.

A quick comparison with the three other papers show agreements among all of them Iwasaki S, 2016 however, there is a great opportunity to learn by reading the papers to compare experimental approaches of three groups. We look forward to see what novel findings this technique uncovers as it becomes adopted in different laboratories.

On 2016 Jun 21, Evelina Tutucci commented:

We have also recently discussed Nelles et al. Nelles DA, 2016. Since we are interested in developing new techniques for studying gene expression and mRNA localization at the single molecule level, a potential tag-less system to detect mRNAs in fixed and live cells would be a further advance. As pointed out by the Duke RNA Biology journal club we think that Nelles et al. represents an attempt to apply the Cas9 System to detect endogenous mRNA molecules. Unfortunately, no evidence is presented to demonstrate that this system is ready to be used to study gene expression at the single molecule level, as the MS2-MCP system allows. The RNA letter by Garcia and Parker Garcia JF, 2015 showed that in S. cerevisiae the binding of the MS2 coat protein to the MS2-loops diminished tagged mRNA degradation by the cytoplasmic exonuclease Xrn1. However, these observations were not extended to higher eukaryotes. Previous work from our lab described the generation of the beta-actin-MS2 mouse, whereby all the endogenous beta-actin mRNAs were tagged with 24 MS2 loops in the 3’UTR (Lionnet T, 2011, Park HY, 2014). This mouse is viable and no phenotypic defects are observed. In addition, control experiments were performed to show that the co-expression of the MS2 coat protein in the beta-actin-MS2 mouse allowed correct mRNA degradation and expression (Supplementary figure 1b, Lionnet T. et al 2011). Furthermore, multi-color FISH (Supplementary figure 6, Lionnet T. et al 2011) showed substantial co-localization between the ORF FISH probes and MS2 FISH probes, demonstrating the validity of this model. We think that the observations by Garcia and Parker are restricted to yeast because of the short half-life of their mRNAs, wherein the degradation of the MS2 becomes rate-limiting. Based on our extensive use of the MS2-MCP system, we think that higher eukaryotes may have more time to degrade the high affinity complexes formed between MS2-MCP, providing validation for this system to study multiple aspects of gene expression. In conclusion, we think that the MS2-MCP system remains to date the best method to follow mRNAs at the single molecule level in living cells. For the use of the MS2-MCP system in S. cerevisiae we have taken the necessary steps to improve it for the study of rapidly degrading mRNAs and are preparing this work for publication.<br> Evelina Tutucci and Maria Vera, Singerlab

On 2016 Apr 18, Gwangseong Kim commented:

In two recent articles [1, 2] two techniques for removing or inactivating blood borne pathogens were introduced. The initial experiments were performed in vitro under simplified conditions. First, the primary achievement of the PDT work deserves clarification [1]. PDT is a powerful therapeutic modality, but its clinical application has been hampered by the inability of light to penetrate deep layers of the tissue, which is mainly due to hemoglobins in the blood readily absorbing photons. Utilizing a millimeter- diameter transparent tube for extracorporeal blood circulation allows PDT to function well despite the presence of hemoglobins in blood. Another point that deserves clarification is that the tube capturing device is not a microfluidic device [2]. This technique can be adapted using existing medical tubing without the need for complicated microfluidics and micro-fabrication. The device is a medical tube that has been chemically modified using simple steps to adapt the internal surface for cell capturing. We would like to take this opportunity to respond to concerns brought up in [3]. We start off by addressing concern (1), which speculates about the possibility of overheating during the use of near IR light. Our control data (Fig.3 and Fig.4 of [1]), confirmed that controls illuminated without photosensitizer-antibody conjugates did not undergo cell death, whereas those with photosensitizer-antibody conjugates underwent significant cell death under identical conditions. Thus it is clear from our data that temperature did not affect the outcome. It has been shown that 660 nm irradiation is safe and effective [4-6]. Moving on to concern (2) part (a) that brings up the problem of using the CD-44 antigen as a target. Limitations of antibody specificity are common knowledge and not unique to CD-44, but to all antibodies. To our knowledge, a targeting method that exclusively binds only to cancer cells does not yet exist, making the use of such a compound an unreasonable standard for publication. We used CD-44 antibody to demonstrate feasibility. As targeting methodologies advance and better selectivity to target cells becomes available, this technique will have improved selectivity. Our experiments were designed to avoid non-specific damage to other cells by pre-staining pure cancer cells with the photosensitizer-antibody conjugates and subsequently removing extra free conjugates before spiking into blood (described in detail in [1]). This elimination of the possibility of side effects due to undesired binding to other blood cells and excess free photosensitizer-antibody conjugates precluded the need for a toxicity study, particularly because we were at the proof-of-principle stage. Part (b) of concern (2) suggests that we may have caused non-specific damage to non-cancerous cells by ROS' convection in the blood stream. We believe that this is highly unlikely. One of the authors has been conducting research focusing on ROS and PDT for years, in collaboration with other researchers [7-15]. This research demonstrated that PDT is extremely selective to targeted cells [13]. Part (c) of concern (2) states that we should have used additional cytotoxicity assays, such as Annexin V, TUNEL, and MTT. However, because none of these techniques are cell-type specific, they would be useless for the particular objective they were suggested. Once our line of investigation reaches a more mature stage, we plan to undertake more useful studies, such as applying separate fluorescent tags, or radio labels, in addition to a cell viability assay and analyzing cell death with a cell sorting technology, such as FACS, MACS, density gradient centrifugation, etc. Concern (3) is that the capturing work [2] lacked purity confirmation concerning non-specific capturing of blood cells. Though purity confirmation is critical in diagnostic testing, our work was strictly limited to in vitro conditions, using spiked pure PC-3 cells as a model. To visualize and quantify PC-3 cells in the presence of whole blood, PC-3 cells were pre-labeled using a fluorescence tag (Calcein AM) and the extra free dye was subsequently removed before spiking PC-3 cells into blood. Because only PC-3 cells can have fluorescence in the blood mixture, and because quantification was based on fluorescing cells, false-positive results from other blood cells can be reasonably excluded. Furthermore, if other blood cells were captured but not identified by our detection method our data would then indicate that the simple tube captured cancer cells despite being blocked by other blood cells. If our technique were applied to CTC diagnosis, independent isolation procedures could be used to ensure the purity of captured cells. In contrast, if used for removal or killing, the purity of captured cells would not be as critical, provided that CTCs are effectively removed. If, by chance, capturing is hampered by accumulation of non-specific binding in filtering the entire blood volume, this issue can be addressed with strategies such as scaling up the tube and carefully determining the tube dimensions, flow rate, frequency of tube replacements, etc. Finally, concern (4), points out that the experimental conditions were not translatable to clinical applications. Part (a) regards scaling up the system to show high throughput. The concept of extracorporeal blood processing of the entire blood volume has been used for years in cases such as hemodialysis. We already are working on optimizing the technique for larger blood volume processing. Part (b) of concern (4) discusses the static no-flow condition as being unrealistic. This issue was brought up during the review process, and we provided with our results showing data under constant flow conditions by peristaltic pump (to be published in future publication). The reviewers agreed that the use of a no-flow condition as a conservative approach during a proof-of-concept stage was appropriate. Despite its preliminary nature, we believe that our work communicates novel ideas, an important objective of research and publication. Given the number of research articles dealing with diagnostics and microfluidics, perhaps a further point of confusion came about by thinking of our work in those terms. We want to clarify that diagnostics were not the primary objective in our work. Furthermore, as it becomes evident by this response our experimental design was carefully devised to minimized unnecessary interferences. We hope that this response mitigates any confusion and addresses the concerns raised. The entire response appears in the PLOS1 comment section under response: http://www.plosone.org/article/comments/info:doi/10.1371/journal.pone.0127219. Feel free to contact us for further clarifications.

1. Kim G, Gaitas A. PloS One. 2014;10(5):e0127219-e.
2. Gaitas A, Kim G. PLoS One. 2015;10(7):e0133194. doi: 0.1371/journal.pone.0133194.
3. Marshall JR, King MR. DOI: 101007/s12195-015-0418-3. 2015;First online.
4. Ferraresi C, et al. Photonics and Lasers in Medicine. 2012;1(4):267-86.
5. Avci P, et al. Seminars in cutaneous medicine and surgery; 2013.
6. Jalian HR, Sakamoto FH. Lasers and Light Source Treatment for the Skin. 2014:43.
7. Ross B, et al. Biomedical Optics, 2004
8. Kim G, et al. Journal of biomedical optics. 2007;12(4):044020--8.
9. Kim G, et al Analytical chemistry. 2010;82(6):2165-9.
10. Hah HJ, et al. Macromolecular bioscience. 2011;11(1):90-9.
11. Qin M, et al. Photochemical & Photobiological Sciences. 2011;10(5):832-41.
12. Wang S, et al. et al. Lasers in surgery and medicine. 2011;43(7):686-95.
13. Avula UMR, et al.Heart Rhythm. 2012;9(9):1504-9.
14. Kim G, et al. R. Oxidative Stress and Nanotechnology, 2013. p. 101-14.
15. Lou X, et al. E. Lab on a Chip. 2014;14(5):892-901.

On 2016 Nov 29, James C Coyne commented:

This study makes some dubious claims that should be subject to independent scrutiny and re- evaluation. It was published in APA journal, which requires sharing of data upon request. However, as I detail and document below, the author responded to a request for just a few variables with an invoice for $450 and a demand that an independent researcher sign a contract not to depart from some arbitrary limits on reanalysis. This sort of behavior threatens routine data sharing. It is deplorable that the American Psychological Association does not support their members to exercise their right to data. See the blog post below for documentation.

https://jcoynester.wordpress.com/2016/11/29/a-quixotic-quest-to-obtain-a-dataset-on-media-violence-with-an-unexpected-price-tag/

On 2015 Sep 26, Eric Fauman commented:

I applaud the authors for identifying novel genetic associations with metabolites but I disagree with their interpretations and conclusions in several regards.

As tempting as it is to use eQTL data to assign causal genes to SNPs it is frequently seen that SNPs tag expression of unrelated genes as often as they tag the true causal gene for the given trait.

In this study the most obvious example is at rs2066938 where the authors report eQTL associations with 5 egenes (RNF10, MLEC, UNC1198B, CAMKK and COQ5), but not ACADS which is almost certainly the true causal gene, as the authors acknowledge in the text.

At the ARG1 and CRAT loci, other genes have stronger eQTL signals so here too the eQTL data is incomplete.

The ALMS1/NAT8 locus is less clear, but previous authors have assigned this locus to NAT8 given the association with N-acetylornithine and NAT8's presumed acetylation function. The biochemical linkage of N-acetylornithine and arginine in the urea cycle suggests that NAT8 is also the causal gene for this paper. If NAT8 is truly the causal gene, the eQTL data missed it at this locus.

A striking example of the over-reliance on eQTL data in this paper is at the "PPP1R16A" locus which associates with the ratio of aspartic acid to alanine. This SNP is in fact just upstream of GPT which encodes glutamic-pyruvic transaminase, also known as alanine transaminase. GPT is a far more plausible causal gene even though it is not one of the 10 egenes listed for this SNP. Interestingly, there is a coding variant in GPT in reasonable LD with the lead SNP (rs1063739, r2=0.77).

In fact 6 of the loci are linked to coding variants in the most probable causal gene (NAT8, GPT, ACADS, SLC22A16, MCCC1 and CPS1).

Again, it's great to see new SNP-metabolite associations still emerging. However any GWAS interpretation must make use of all biological lines of evidence and not rely only on one or two types of data or analysis.

On 2015 Sep 23, Angelo Gaitas commented:

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

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

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

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

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

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

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

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

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

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

On 2017 Jul 09, Sunil Verma commented:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Ref & Suppl Information

On 2015 Sep 09, Bill Cayley commented:

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

On 2015 Sep 23, Angelo Gaitas commented:

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

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

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

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

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

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

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

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

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

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

On 2017 Jul 09, Jeffrey Ross-Ibarra commented:

In our manuscript exploring the population genetics of local adaptation (Tiffin and Ross-Ibarra 2014) we included a discussion about the potential uses of reduced representation data (e.g. RAD-seq, GBS). To provide a sense of the probability of using reduced representation data to identify targets of selection, we included a figure showing the probability of having a SNP included in a region of the genome in which diversity had been severely reduced due to a recent selective sweep. Unfortunately this figure is not correct; an error in the code inadvertently used centimorgans as morgans, causing the recombination rate to be off by a factor of 100.

To correct this we have generated a new figure (see http://rpubs.com/rossibarra/257207; raw code is available at https://gist.github.com/rossibarra/be44cc3b3796f45840d942ad11c01ba1) that corrects this error and presents a more realistic model. Our previous model assumed SNPs were distributed evenly across the genome and the presence of a single SNP near a sweep was sufficient for detection. Instead, here we explicitly model sequence “tags” coming from RAD-seq or GBS, and incorporate information about the variation in diversity expected among tags in neutral regions of the genome. The figure clearly shows that with dense marker coverage and strong selection, the probability of detecting reductions in diversity due to recent selective sweeps from new beneficial mutations can be relatively high. We emphasize, however, that the purpose of the figure is solely to develop an intuition of the likelihood of detecting a recent selective sweep. The many simplifying assumptions made in generating the figure (no recent demographic change, both sequence tags and recombination occur uniformly along the genome, selection is on a novel beneficial mutation with additive effect that has recently swept to fixation), as well as the specific mutation rates, sample size, sequence length, and recombination rates assumed will all affect the actual probability of a tag being included in a selective sweep. Moreover, this figure does not touch on many other relevant issue such as multiple testing, complex demography, background selection, or other modes of positive selection (e.g. from standing variation, balancing selection, or selection on polygenic traits).

We have submitted a correction to the journal.

We thank Eric Johnson for drawing our attention to the error, and Eric Johnson, Kathleen Lotterhos, and Graham Coop for kindly reviewing previous versions of the code and assumptions we have used in generating this new figure.

On 2014 Oct 27, David Colquhoun commented:

For all the reasons given by Hilda Bastian (and a few more, like P = 0.04 provides lousy evidence) it astonishes me that this study should have been trumpeted as though it represented a great advance. That's the responsibility of Nature Neuroscience (and, ultimately, of the authors).

I wonder whether what happens is as follows. Authors do big fMRI study. Glamour journal refuses to publish without functional information. Authors tag on a small human study. Paper gets published. Hyped up press releases issued that refer mostly to the add on. Journal and authors are happy. But science is not advanced.

I certainly got this impression in another recent fMRI paper in Science. Brain stimulation was claimed to improve memory (P = 0.043)

I guess these examples are quite encouraging for those who think that expensive glamour journals have had their day. Open access and open comments are the way forward.

On 2014 Oct 21, George McNamara commented:

This is a nice paper. The abstract refers to using 24 epitope tags (24mer), much of the paper uses a 10mer. Just doing GFP is boring. When I came up with the "Tattletales" (TALE-FPn ... I came up with the idea before sgRNA:Cas9 became popular), I immediately realized that multimerizing FP biosensors. The current paper is the same as my what I refer to as "Binary Tattletales", as in: 1. TALE-(linker-epitope tag)n 2. "binder"-(linker-FP)m with Tattletales being T-cells -- TALE FPs/Biosensors. Since I moved to MD Anderson Cancer Center, the first T now refers to "T-cells and Tumor cells". Likewise T-bow refers to rainbow T-cells and Tumor cells for promoter bashing and otherwise multicolor dots labeling cells (rainbow in homage of course to Brainbow mice etc, and especially to real rainbows). For more on Tattletales, Binary Tattletales, and T-Bow, see http://works.bepress.com/gmcnamara/63 http://works.bepress.com/gmcnamara/42

Giving credit where credit is due: The authors really should have cited the first mammalian cell paper localizing a lot of FPs in one spot (they came 'close' with a Gordon 1997 Cell paper on GFP:LacO in E.coli, but the Tanenbaum paper is all mammalian cells): Robinett et al 1996 JCB http://www.ncbi.nlm.nih.gov/pubmed/8991083 http://jcb.rupress.org/content/135/6/1685.long See their figure 4A. Straight, Robinett et al also published a yeast paper in 1996, http://www.ncbi.nlm.nih.gov/pubmed/8994824 and it would have been useful to cite that.

The PDF download at http://works.bepress.com/gmcnamara/63 has a table of 130 FP biosensors (if you are Laconic about ATeam and Fire, too bad) and an extensive reference list with ZF-FP, TALE-FP, Cas9-FP (the latter from the Weissman group), and more (PUF's and PPR's are RNA binding protein families with structural similarities to TALEs). My favorite name -- besides Tattletales and T-Bow, of course -- is "TALE-Lights" from Yuan, Shermoen, O'Farrell 2014, http://www.ncbi.nlm.nih.gov/pubmed/24556431

On 2014 Oct 06, Leonid Teytelman commented:

Dear Authors,

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

On 2014 May 03, Stefanie Butland commented:

All interaction data from this paper are freely available at IntAct http://www.ebi.ac.uk/intact/query/24705354 and are featured as Dataset of the Month for May 2014. These include 312 binary interactions from yeast two-hybrid, anti tag coimmunoprecipitation, fluorescence microscopy and luminescence based mammalian interactome mapping (LUMIER) experiments.

We submitted our data directly to IntAct through the IMEx Consortium as part of the publication process. I encourage others to consider this route to making your data available for re-use and re-mixing as the expert biocuration service provided by IntAct was smooth, accurate, and required very little of our time. A very positive experience.

On 2014 May 14, David Keller commented:

Still waiting for anyone to answer my criticisms of this USPSTF report

When I first read the 2014 USPSTF update on vitamins for disease prevention, I expected, based on the headlines, to find evidence that there is no reason to take a multivitamin. Instead, I became convinced by the data presented by the USPSTF that the evidence of benefits versus harms favors men over 50 taking a multivitamin to prevent cancer and possibly reduce overall mortality. At the very least, the USPSTF would be fully justified in recommending that men over 50 who do not consume a diet rich in vegetables and fruits should consider adding a multivitamin. For some reason, most editorials and comments have completely ignored the significant reductions in cancer for men randomized to multivitamins in the 2 studies cited by USPSTF, and the significant reduction in overall mortality for men randomized to the high dose multivitamin tested in the French study. Instead, we saw headlines and editorials stating or implying that we now have proof that multivitamins are useless. I request that an expert in this area reply to my comments, giving good reasons why you are not convinced by the data we have, and what it would take to convince you. If you are knowledgeable in this area, and especially if you are a member of the USPSTF, I would greatly appreciate your pointing out where my thinking on this issue is wrong or even debatable. For details, data and references, please see my Open Letter to the USPSTF on the following PubMed Commons web page:

http://www.ncbi.nlm.nih.gov/pubmed/24566474#cm24566474_4093

Lastly, it is not helpful to tag a comment as "not helpful" without specifying why. PubMed Commons should foster meaningful debate, not merely anonymous unexplained contradiction of each other.

On 2014 Jan 04, Dorothy V M Bishop commented:

I was pleased to see that Professor Farthing took the opportunity to tackle the subject of research misconduct in his lecture. He cogently notes the nature of the problem and makes suggestions to deal with it. I thought his analysis was generally on-target, but I was concerned about his second suggested solution: enhanced monitoring and audit, and his failure to consider an additional approach, which is to change the incentive structure for researchers. The following points are taken from a blogpost I wrote on these topics (http://deevybee.blogspot.co.uk/2013/06/research-fraud-more-scrutiny-by.html).

I agree we need to think about how to fix science, and that many of our current practices lead to non-replicable findings. I just don't think more scrutiny by administrators is the solution.

So what would I do? The answers fall into three main categories: incentives, publication practices, and research methods.

Incentives: Currently, we have a situation where research stardom, assessed by REF criteria, is all-important. Farthing notes that RAE/REF criteria have been devised to stress quality rather than quantity of research, which is a good thing, but it is still the case that too much emphasis goes on the prestige of journals (see http://deevybee.blogspot.co.uk/2013/01/journal-impact-factors-and-ref-2014.html).

Instead of valuing papers in top journals, we should be valuing research replicability. This would entail a massive change in our culture, but a start has already been made in my discipline of psychology :see http://www.nature.com/news/psychologists-strike-a-blow-for-reproducibility-1.14232.

Publication practices: the top journals prioritize exciting results over methodological rigour. There is therefore a strong temptation to do post hoc analyses of data until an exciting result emerges. I agree with Farthing that pre-registration of research projects is a good way of dealing with this. I'm pleased to say that here too, psychology is leading the way in extending research registration beyond the domain of clinical trials: http://blogs.lse.ac.uk/impactofsocialsciences/tag/registered-reports/

Research methods: we need better training of scientists to become more aware of the limitations of the methods that they use. Too often statistical training is a dry and inaccessible discipline. All scientists should be taught how to generate random datasets: nothing is quite as good at instilling a proper understanding of p-values as seeing the apparent patterns in data that will inevitably arise if you look hard enough at some random numbers. In addition, not enough researchers receive training in best practices for ensuring quality of data entry, or in exploratory data analysis to check the numbers are coherent and meet assumptions of the analytic approach.

Finally, before any new regulation is introduced, there should be a cold-blooded cost-benefit analysis that considers, among other things, the cost of the regulation both in terms of the salaries of people who implement it, and the time and other costs to those affected by it. My concern is that among the 'other costs' is something rather nebulous that could easily get missed. Quite simply, doing good research takes time and mental space of the researchers. Most researchers are geeks who like nothing better than staring at data and thinking about complicated problems. If you require them to spend time satisfying bureaucratic requirements, this saps the spirit and reduces creativity.

I think we can learn much from the way ethics regulations have panned out. When a new system was first introduced in response to the Alder Hey scandal, I'm sure many thought it was a good idea. It has taken several years for the full impact to be appreciated. The problems are documented in a report by the Academy of Medical Sciences, which noted "Urgent changes are required to the regulation and governance of health research in the UK because unnecessary delays, bureaucracy and complexity are stifling medical advances, without additional benefits to patient safety"

On 2013 Jul 04, John Overington commented:

A standard for database tag structures would be really useful in general - 1ABC is a PDB code, but it's also many other things, so PDB1ABC would be more general and useful. However, as a database provider this paper highlighted several features that I didn't know and will now explore - e.g. the NLM JATS DTD.

On 2015 Sep 24, Bill Cayley commented:

A good example of when "less" is "more" in Obstetric care: https://lessismoreebm.wordpress.com/tag/obstetric-gynegologic/

On 2016 Jan 21, Sebastian Lourido commented:

The conditional dimerizable Cre recombinase (DiCre) has been a powerful technique for conditional genome engineering in Toxoplasma, as first established in this article, and elaborated later (see Pieperhoff, et al. 2015. PLoS One). It has worked well in our hands for a variety of applications. Recently, we discovered that the reporter construct used in this study was cloned down stream and in frame of a Ty-tag (EVHTNQDPLD), such that the KillerRed expressed prior to recombination contains and N-terminal Ty-tag. This observation does not affect any of the experiments presented in the article. However, it might be important to note for future investigators planning further manipulations of the existing DiCre strains or reporter constructs.

On 2014 Jan 08, Tom Kindlon commented:

Early diagnosis of CFS/ME has been shown to lead to a better prognosis

It was interesting to see the various views expressed by GPs in this paper[1]. However I think a couple of useful points could have been added. There is much discussion in the paper about whether a label of CFS/ME is useful or not. The authors refer to NICE guidelines which "emphasise the importance of a definitive diagnosis"[2]. However, I think it would have been useful to add some direct evidence on this issue.

For example, research published by the Centres for Disease Control and Prevention (CDC) which found that an earlier diagnosis led to a better prognosis[3]. This prompted the CDC to launch a two-pronged awareness drive aimed at both health professionals and the general public - the tag line for the latter was, "Get informed. Get diagnosed. Get help."[4].

A UK study found that the longer the interval between a patient falling ill and getting a diagnosis, the greater the likelihood that they would become severely affected. [5]

The authors mention the issue of CFS/ME being managed in primary care. It is important for GPs to know that GPs encouraging patients to do a graded exercise programme is associated with a higher rate of adverse reactions. For example, a survey which asked patients about their experiences of treatments over the previous three years found that 45% reported being made worse by a graded exercise therapy (GET) programme overseen by their GP, compared to 31% who reported being made worse by a GET under a NHS specialist and 29% of those who did a GET in other circumstances[6]. The NICE guidelines do not recommend that a GP oversee such an approach[2].

References:

[1] Chew-Graham C, Dowrick C, Wearden A, Richardson V, Peters S. Making the diagnosis of Chronic Fatigue Syndrome/Myalgic Encephalitis in primary care: a qualitative study. BMC Fam Pract. 2010 Feb 23;11:16.

[2] NICE CG 53 Chronic fatigue syndrome/Myalgic encephalomyelitis (or encephalopathy) guideline.

[3] Nisenbaum R, Jones JF, Unger ER, Reyes M and Reeves WC. A population-based study of the clinical course of chronic fatigue syndrome. Health and Quality of Life Outcomes 2003;1:49-58.

[4] CDC Chronic Fatigue Syndrome Awareness Campaign. http://cdc.gov/cfs/awareness.htm [Last accessed: 31 March, 2010]

[5] Pheby D and Saffron L. Risk factors for severe ME/CFS. Biology and Medicine (2009); 1 (4):50-74. http://biolmedonline.com/Articles/vol1_4_50-74.pdf [Last accessed: 31 March, 2010]

[6] Action for M.E. and AYME Survey 2008 Results http://afme.wordpress.com/5-treatments-and-symptoms/ [Last accessed: 31 March, 2010]

On 2015 Apr 18, Dorothy V M Bishop commented:

As a psychologist interested in the genetics of lateralization, I frequently come across this paper, which is cited as evidence for early genetic influences on brain asymmetry. As of today, 160 citations are shown in Web of Science.

When I read the paper a couple of years ago, I found some details that did not seem to support the conclusions of the authors. These are described in a blogpost: http://deevybee.blogspot.co.uk/2012/12/genes-brains-and-lateralisation-how.html

I will summarise the main issue below, but I was interested to note that, since that time, other papers have appeared, using larger datasets, which have stressed the remarkable symmetry of early gene expression, notably:

Johnson, M. B., et al (2009). Functional and evolutionary insights into human brain development through global transcriptome analysis. Neuron, 62(4), 494-509. doi: 10.1016/j.neuron.2009.03.027

and

Pletikos, M., Sousa, A. M. M., Sedmak, G., Meyer, K. A., Zhu, Y., Cheng, F., . . . Sestan, N. (2014). Temporal specification and bilaterality of human neocortical topographic gene expression. Neuron, 81(2), 321-332. doi: 10.1016/j.neuron.2013.11.018

Here is a brief account of the main issue I raised about the study. Please see the blogpost for more details.

Sun et al used a method called Serial Analysis of Gene Expression (SAGE) which compares gene expression in different tissues or – as in this case – in corresponding left and right regions of the embryonic brain. The analysis looks for specific sequences of 10 DNA base-pairs, or tags, which index particular genes. SAGE output consists of simple tables, giving the identity of each tag, its count (a measure of cellular gene expression) and an identifier and more detailed description of the corresponding gene. These tables are available for left and right sides for three brain regions (frontal, perisylvian and occipital) for 12- and 14-week old brains, and for perisylvian only for a 19-week-old brain. The perisylvian region is of particular interest because it is the brain region that will develop into the planum temporale, which has been linked with language development. One brain at each age was used to create the set of SAGE tags.

To verify asymmetrically expressed genes the authors performed chi square tests. The chi square involves testing whether the distribution of expression on left and right is significantly different from the distribution of left vs. right expression across all tags in this brain region – which is close to 50%. In the left-right perisylvian region of a 12-week-old embryonic human brain, there were 49 genes with chi square greater than 6.63 (p < .01): 21 were more highly expressed on the left and 28 more highly expressed on the right. But for each region the authors considered several thousand tags. My analysis indicated that the number of asymmetrically expressed genes appeared to be lower than you would expect by chance – entirely consistent with the conclusions of Pletikos et al.

Unless my analysis is mistaken, it would seem this paper should not be cited as evidence for asymmetric fetal gene expression, as it actually shows the opposite.

On 2015 Oct 07, Bill Cayley commented:

One of several examples showing that sometimes in obstetric care "less" is "more": https://lessismoreebm.wordpress.com/tag/obstetric-gynegologic/

On 2014 Feb 12, Diana Frame commented:

A note for researchers, MESH indexing terms on this article erroneously tag it as non-small cell lung cancer (NSCLC). It should be under Small Cell Lung Carcinoma[MeSH].

A meta tag to put into the header to support RSS discovery.

If we choose to add an astrology/astronomy tag

Not sure what to tag

Not sure what to tag this

as we have seen this works pretty well...

Display Advertising ist immer nur so erfolgreich wie es die Zielgruppen-Definition zulässt. catchyou® sorgt dafür, dass die richtigen Werbebotschaften die richtigen Leute zur richtigen Zeit erreichen. Deshalb erreichen wir auch Click-Through-Rates, die bis um den Faktor 10 höher sind als der Durchschnitt. Wobei wir Display-Werbung auch und gerade im Retargeting einsetzen. Unsere kontinuierliche Optimierung der Kampagnen nach individuellen Leistungskennzahlen sorgt dafür, dass die Kampagnen von Tag zu Tag besser werden.

When writing PHP, if you have a file that is solely PHP, it is recommended that you do not add a closing PHP tag (?>). Doing so then having a bunch of whitespace after it can create some pretty weird errors. I lost a day of working time to this issue. Take it from an experienced PHP developer, save yourself lots of trouble and don't put the closing PHP tag.

This is my blog URL: https://fcpayan.wixsite.com/portfolio/blog

So all I do is tag it with #inte5340 and that will be it? Does it work with the Wix website?

I love that this exists!

example tag: <link rel="canonical" href="http://where/your/old/page/is.html" />

.

.

Isn't that an opportunity to put some formalism & equations and offer some spacing again ? Just suggesting (phrases are clear)

What about the tag "Freedom"?

After determining that neither the SM tag nor Fab would disrupt cellular processes, the authors wanted to determine how soon Fab would mark KDM5B. To do this, the authors repeated their first trial but imaged at 6 hours instead of 24.

The two tags did not appear to affect the synthesis or location of the mRNA or the ribosomes.

Preliminary testing was conducted twenty-four hours after the plasmid was transiently transferred to ensure that the methods of coupling the FLAG SM tag (that can be labeled with the anti-fluorescein antibody, anti-FLAG Fab) and the MS2 stem-loop (that can be identified with a MS2 coat protein) would not inhibit transcription, translation, or the movement of SM-KDM5B mRNAs throughout the cell.

The results are highlighted in Fig. 1C with the SM-KDM5B protein in green and the MCP-labeled mRNAs in red.

Antibodies "against" the FLAG-tag (SM).

A specific sequence of amino acids that can be added to proteins to "tag" them. Antibodies have been developed that have high affinity for the tag, so it is a popular choice in this kind of visualization experiment.

We welcome you to share thoughts and ideas as we come together and reflect on so many years of #clmooc Just remember to tag posts clmooc, plus any other tag you want

Abstract

annotation and image test

can heavier nucleotide bases act as a tag?

Here's the feed with youth writing about Black Lives Matter on YVL.

auf einem normalen Browser-Fenster, wenn ich einen Tag wähle, kommt den Text "Wähle die Dauer deiner Massage" schon auf. Allerdings wäre super wenn man nicht deswegen wieder unterscrollen muss. Konkret: bitte wann möglich, Platz beim Kalendar-Control sparen, in dem die Kreisen ein bisschen schmäler machen und zwischen Zeilen Abstände reduzieren?

is the tag properly nested in this example?

Doing what you love, isolates and degrades other workforces and elevates others and more so, the ones of higher economic class. One should be paid fair dues as per their work and have good working conditions. She wants people to realize that they deserve goods jobs and they should never settle for less in the name of doing what they love.

The article argues different theories regarding doing what is right, with the bigger question being is it wrong for people to strive to be able to do what they love. But, at the same time, what is the limit of people seeking what they love without overstepping the boundaries?

Friction is why balls don't bounce forever (even if gravity existed)

The artwork/photo is very interesting. To be honest, I am not sure whether your "tag" or headline is as clear as it might be, in terms of relating to your project. Could you also provide more information regarding the contact in the lower right hand corner? I'm not sure what "Futures" means.

Can we change the video tag line?

I know he does not mean this literally, but with one kid in college and another on the way, I only see this literally. The cost of college in the US is staggering, and a huge barrier that no politician in the US takes on.

Open Calais

Li et al. develop and perform ChIA-PET (Chromatin Interaction Analysis by Paried-End-Tag sequencing), a method to identify regions of chromatin that interact with each other at higher resolution than previous methods.

Using this method in MCF7 cells (a breast cancer cell line), they identify interactions between the location of several variants of interest to Crawford et al. with the promoter of the gene DDB1.

The authors look for what part of the cell the MFSD12 protein they're interested in is located. To find the protein, they first "tag" it at the C terminus with an HA epitope.

The C terminus is the end of the protein. Tags are often put on the end of the protein because in some cases they are less likely to interfere with expression or function if they're at the end.

HA stands for human influenza hemagglutinin, which is a protein on the surface of human cells. A particular part of it, the HA epitope, can be used to tag proteins in cells because it's small, so it's unlikely to interfere with protein function or location.

An epitope is a part of a protein that is recognized by an antibody, which can be bound to fluorescent molecules so it can be visualized by a technique called immunofluorescence microscopy.

add a tag

http://aknextphase.com/tag/felix-teran/

I'm just wondering how privacy is ensured and how information is restricted only to members of the class, especially considering the the hash tag function. As I'm not much of an active twitter user, how does one ensure this?

根据.cc 源文件中的注释来理解：tag 是区分不同 request 的标识，当服务端返回请求的结果时，通过同样的 tag 值来区分结果是哪一个请求的。

所以，tag 应该用 UUID 来实现？

Commentaire

Excellent way to add users and tags to the Fediverse (the federation of free networks) -- through Friendica's RSS functionality.

is it possible to tag the origina commit on Git with rm2.1-wd01 or something similar, please?

Clearly no one understands how our systems of record work.

These images all make one think of festive summers and relaxing vacations. These words all work together to help create that sort of mood. Just one of these thing on its own wouldn't have created such a powerful image.

Aquí vienen muchos datos físicos útiles

Lo repitieron

Bien, son AISLANTES no materiales principales, ya me quedó claro.

I'm keeping a running list of other works that I've come across that respond to the CARE Framework in the Zotero Open Knowledge Practices library under the tag "careframework". Reply to this annotation to add more (or you can contact me with suggestions or to join the Zotero OKP group).

Note: in Zotero you can open Library Settings at the upper right to show other columns in the list, like date (of publication), and then sort by exposed columns.

Remove beta tag

test

maybe this could be a bit bigger than the text below. Also maybe we should discuss this tag line in a staff meeting to co-develop something we are all on board with.

part of critique

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